Patent Application
20240301373
The contents of the electronic sequence listing (M065670531US03-SEQ-EAS.xml; Size: 6,974,866 bytes, and Date of Creation: Aug. 15, 2023) is herein incorporated by reference in its entirety.
The present invention relates generally to methods and products of using programmable RNA-guided DNA endonucleases for genome-editing.
Prokaryotic and eukaryotic genomes are replete with diverse transposons, a broad class of mobile genetic elements (MGE). Transposons of the highly abundant IS200/605 family encode a pair of genes: TnpA, which codes for a DDE class transposase responsible for single-strand ‘peel and paste’ transposition, and TnpB, which has an unknown role in the transposition mechanism (Kapitonov et al. 2015; He et al. 2013). TnpB contains a RuvC-like nuclease domain (RNase H fold) that is specifically related to the homologous nuclease domain of the type V CRISPR effector Cas12 (Zetsche et al. 2015; Fonfara et al. 2016), specifically the Cas12f systems (Harrington et al. 2018), suggesting a direct evolutionary path from TnpB to Cas12 (Karevelis et al. 2021; Bao and Jurka 2013, Altae-Tran et al. 2021). This relationship is supported by phylogenetic analysis of the RuvC-like domains, which indicates independent origins of Cas12s of different type V subtypes from distinct groups of TnpBs. Bioinformatic analysis demonstrated that, along with IscB, IsrB, and IshB nucleases, TnpBs are components of obligate mobile element-guided activity (OMEGA) systems, which encode the guide wRNA nearby the nuclease gene, often overlapping the coding region. Biochemical and cellular validation demonstrated ωRNA-TnpB complex forms an RNA-guided DNA endonuclease system (Karevelis et al. 2021; Altae-Tran et al. 2021).
RuvC-containing proteins are not limited to prokaryotic systems: a set of TnpB homologs, Fanzors, are present in eukaryotes (Bao and Jurka 2013). Mirroring the diversity of TnpBs in bacteria and archaea, Fanzor nucleases have been identified in diverse eukaryotic lineages, including metazoans, fungi, algae, amorphea, and double-stranded (ds)DNA viruses. Identified Fanzors fall into two major groups: 1) Fanzor1 nucleases are associated with eukaryotic transposons, including Mariners, IS4-like elements, Sola, Helitron, and MuDr, and occur predominantly in diverse eukaryotes; 2) Fanzor2 nucleases are found in IS607-like transposons and are present in large dsDNA viral genomes. Despite the similarities between TnpB and Fanzors, Fanzors have not been surveyed comprehensively throughout eukaryotic diversity, and they have not been demonstrated to be active nucleases in either biochemical or cellular contexts.
The present disclosure reports a comprehensive census of RNA-guided nucleases in eukaryotic and viral genomes, discovering a broad class of nucleases termed Fanzors. Fanzor diversity was used herein to perform phylogenetic analysis revealing their evolution from prokaryotic origins and to validate activity through biochemical and cellular experiments, demonstrating the programmable RNA-guided endonuclease activity of the Fanzor. The invention relates, in one aspect, to the discovery that Fanzors comprise programmable RNA-guided endonuclease activity that can be harnessed for genome editing in human cells, highlighting the utility of the widespread eukaryotic RNA-guided nucleases for biotechnology applications. The invention relates, in some aspects, to the discovery that Fanzor programmable RNA-guided endonuclease activity can be harnessed for genome editing in any type of organism (e.g., eukaryotic, prokaryotic, and/or fungi).
Accordingly, aspects of the present disclosure provide compositions non-naturally occurring, engineered composition comprising: (a) a Fanzor polypeptide comprising an RuvC domain; and (b) a fRNA molecule comprising a scaffold and a reprogrammable target spacer sequence, wherein the fRNA molecule is capable of forming a complex with the Fanzor polypeptide and directing the Fanzor polypeptide to a target polynucleotide sequence.
In some embodiments, the RuvC domain further comprises a RuvC-I subdomain, a RuvC-II subdomain, and a RuvC-I subdomain, wherein the RuvC-subdomain is a rearranged RuvC-II subdomain.
In some embodiments, the Fanzor polypeptide comprises about 200 to about 2212 amino acids.
In some embodiments, the reprogrammable target spacer sequence comprises about 12 to about 22 nucleotides.
In some embodiments, the scaffold comprises about 21 to about 1487 nucleotides.
In some embodiments, the complex binds a target adjacent motif (TAM) sequence 5′ of the target polynucleotide sequence. In some embodiments, the TAM sequence comprises GGG. In some embodiments, the TAM sequence comprises TTTT. In some embodiments, the TAM sequence comprises TAT. In some embodiments, the TAM sequence comprises TTG. In some embodiments, the TAM sequence comprises TTTA. In some embodiments, the TAM sequence comprises TA. In some embodiments, the TAM sequence comprises TTA. In some embodiments, the TAM sequence comprises TGAC.
In some embodiments, the target polynucleotide is DNA.
In some embodiments, the Fanzor polypeptide is selected from a sequence listed in Table 1. In some embodiments, the Fanzor polypeptide shares at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with a Fanzor polypeptide listed in Table 1.
In some embodiments, the Fanzor polypeptide is selected from a sequence listed in Table 4. In some embodiments, the Fanzor polypeptide shares at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with a Fanzor polypeptide listed in Table 4.
In some embodiments, (a) the Fanzor polypeptide is a Fanzor polypeptide; and (b) the fRNA molecule is an fRNA molecule. In some embodiments, the Fanzor polypeptide is a Fanzor 1 polypeptide. In some embodiments, the Fanzor polypeptide is a Fanzor2 polypeptide. In some embodiments, the Fanzor polypeptide further comprises a nuclear localization signal (NLS).
In some embodiments, the Fanzor polypeptide further comprises a helix-turn-helix (HTH) domain.
Further aspects of the present disclosure relate to compositions comprising one or more vectors comprising (a) a nucleic acid sequence encoding a Fanzor polypeptide comprising an RuvC domain; and (b) a nucleic acid sequence encoding a fRNA molecule comprising a scaffold and a reprogrammable target spacer sequence, wherein the fRNA molecule is capable of forming a complex with the Fanzor polypeptide and directing the Fanzor polypeptide to a target polynucleotide sequence. In some embodiments, (a) and (b) are comprised by one vector. In some embodiments, (a) and (b) are comprised by more than one vector.
In some embodiments, the composition further comprises one or more of a donor template comprising a donor sequence, optionally for use in homology-directed repair (HDR), a linear insert sequence, optionally for use in non-homologous end joining-based insertion, a reverse transcriptase, optionally for use in prime editing, a recombinase, optionally for use for integration, a transposase, optionally for use for integration, an integrase, optionally for use for integration, a deaminase, optionally for use of base-editing, a transcriptional activator, optionally for use of targeted gene activation, a transcriptional repressor, optionally for use of targeted gene repression, and/or a transposon, optionally for RNA guided transposition.
In some embodiments, the linear insert sequence comprises DNA. In some embodiments, the linear insert sequence comprises RNA. In some embodiments, the linear insert sequence comprises mRNA. In some embodiments, the linear insert is comprised by a viral vector, optionally wherein the viral vector is Adeno-associated viral (AAV) vector, a virus, optionally wherein the virus is an Adenovirus, a lentivirus, a herpes simplex virus, and/or a lipid nanoparticle.
In some embodiments, the integration comprises programmable addition via site-specific targeting elements (PASTE).
In some embodiments, the transposon is a eukaryotic transposon, optionally wherein the eukaryotic transposon is CMC, Copia, ERV, Gypsy, hAT, helitron, Zator, Sola, LINE, Tc1-Mariner, Novosib, Crypton, or EnSpm.
Further aspects of the present disclosure relate to engineered cells comprising (a) a Fanzor polypeptide comprising an RuvC domain; and (b) a fRNA molecule comprising a scaffold and a reprogrammable target spacer sequence, wherein the fRNA molecule is capable of forming a complex with the Fanzor polypeptide and directing the Fanzor polypeptide to a target polynucleotide sequence.
In some embodiments, the engineered cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the engineered cell is a non-mammalian, animal cell. In some embodiments, the engineered cell is a plant cell. In some embodiments, the engineered cell is a bacterial cell. In some embodiments, the engineered cell is a fungal cell. In some embodiments, the engineered cell is a yeast cell.
In some embodiments, the engineered cell further comprises one or more of a donor template comprising a donor sequence, optionally for use in homology-directed repair (HDR), a linear insert sequence, optionally for use in non-homologous end joining-based insertion, a reverse transcriptase, optionally for use in prime editing, a recombinase, optionally for use for integration, a transposase, optionally for use for integration, an integrase, optionally for use for integration, a deaminase, optionally for use of base-editing, a transcriptional activator, optionally for use of targeted gene activation, a transcriptional repressor, optionally for use of targeted gene repression, and/or a transposon, optionally for RNA guided transposition.
In some embodiments, the linear insert sequence comprises DNA. In some embodiments, the linear insert sequence comprises RNA. In some embodiments, the linear insert sequence comprises mRNA. In some embodiments, the linear insert is comprised by a viral vector, optionally wherein the viral vector is Adeno-associated viral (AAV) vector, a virus, optionally wherein the virus is an Adenovirus, a lentivirus, a herpes simplex virus; and/or a lipid nanoparticle.
In some embodiments, the integration comprises programmable addition via site-specific targeting elements (PASTE).
In some embodiments, the transposon is a eukaryotic transposon, optionally wherein the eukaryotic transposon is CMC, Copia, ERV, Gypsy, hAT, helitron, Zator, Sola, LINE, Tc1-Mariner, Novosib, Crypton, or EnSpm.
Further aspects of the present disclosure relate to methods of modifying a target polynucleotide sequence in a cell, comprising delivering to the cell (a) a nucleic acid encoding a Fanzor polypeptide comprising an RuvC domain; and (b) a nucleic acid encoding a fRNA molecule comprising a scaffold and a reprogrammable target spacer sequence, wherein the fRNA molecule is capable of forming a complex with the Fanzor polypeptide and directing the Fanzor polypeptide to a target polynucleotide sequence.
In some embodiments, the modifying comprises cleavage of the target polynucleotide sequence. In some embodiments, the cleavage occurs within the target polynucleotide near the 3′ end of the target polynucleotide sequence. In some embodiments, the cleavage occurs about −6 to about +3 nucleotides relative to the 3′ end of the target polynucleotide sequence.
In some embodiments, the cleavage occurs with the TAM sequence. In some embodiments, the target polynucleotide sequence is DNA.
In some embodiments, one or more mutations comprising substitutions, deletions, and insertions are introduced into the target polynucleotide sequence.
In some embodiments, (a) and (b) are delivered to the cell together. In some embodiments, (a) and (b) are delivered to the cell separately. In some embodiments, the delivering to a cell occurs (a) in vivo; (b) ex vi); or (c) in vitro.
In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the cell is a non-mammalian, animal cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is a bacterial cell. In some embodiments, the cell is a fungal cell. In some embodiments, the cell is a yeast cell. In some embodiments the cell is a rodent cell. In some embodiments, the cell is a primate cell.
Further aspects of the present disclosure relate to compositions comprising a stabilized Fanzor polypeptide comprising an RuvC domain, comprising one or more mutations relative to wildtype Fanzor polypeptide wherein the mutations stabilize the Fanzor polypeptide. Further aspects of the present disclosure relate to methods of modifying a target polynucleotide sequence in a cell, comprising (a) delivering to the cell a stabilized Fanzor polypeptide comprising an RuvC domain and further comprising one or more mutations relative to a wildtype Fanzor polypeptide wherein the mutations stabilize the Fanzor polypeptide; and (b) separately delivering to the cell a fRNA molecule.
Further aspects of the present disclosure relate to method of modifying a target polynucleotide sequence in a mammal in vivo, comprising delivering to the mammal (a) a nucleic acid encoding a Fanzor polypeptide comprising an RuvC domain; and (b) a nucleic acid encoding a fRNA molecule comprising a scaffold and a reprogrammable target spacer sequence, wherein the fRNA molecule is capable of forming a complex with the Fanzor polypeptide and directing the Fanzor polypeptide to a target polynucleotide sequence.
Further aspects of the present disclosure relate to methods of modifying a target polynucleotide sequence in a mammal in vivo or in a mammalian cell ex vivo, comprising delivering to the mammal or the mammalian cell a composition of the present disclosure. In some embodiments, the mammal is a human, a primate, or a rodent, optionally a mouse; or the mammalian cell is a human cell, a primate cell, or a rodent cell, optionally a mouse cell. Further aspects of the present disclosure relate to method of modifying a target polynucleotide sequence in a plant in vivo, comprising delivering to the plant (a) a nucleic acid encoding a Fanzor polypeptide comprising an RuvC domain; and (b) a nucleic acid encoding a fRNA molecule comprising a scaffold and a reprogrammable target spacer sequence, wherein the fRNA molecule is capable of forming a complex with the Fanzor polypeptide and directing the Fanzor polypeptide to a target polynucleotide sequence.
Further aspects of the present disclosure relate to methods of modifying a target polynucleotide sequence in a plant in vivo, comprising delivering to the plant a composition of the present disclosure.
Further aspects of the present disclosure relate to method of modifying a target polynucleotide sequence in a fungi in vivo, comprising delivering to the fungi (a) a nucleic acid encoding a Fanzor polypeptide comprising an RuvC domain; and (b) a nucleic acid encoding a fRNA molecule comprising a scaffold and a reprogrammable target spacer sequence, wherein the fRNA molecule is capable of forming a complex with the Fanzor polypeptide and directing the Fanzor polypeptide to a target polynucleotide sequence.
Further aspects of the present disclosure relate to methods of modifying a target polynucleotide sequence in a fungi in vivo, comprising delivering to the fungi a composition of the present disclosure.
Further aspects of the present disclosure relate to method of modifying a target polynucleotide sequence in a virus, comprising delivering to the virus (a) a nucleic acid encoding a Fanzor polypeptide comprising an RuvC domain; and (b) a nucleic acid encoding a fRNA molecule comprising a scaffold and a reprogrammable target spacer sequence, wherein the fRNA molecule is capable of forming a complex with the Fanzor polypeptide and directing the Fanzor polypeptide to a target polynucleotide sequence.
Further aspects of the present disclosure relate to methods of modifying a target polynucleotide sequence in a virus, comprising delivering to the virus a composition of the present disclosure.
Further aspects of the present disclosure relate to method of modifying a target polynucleotide sequence in a bacteria, comprising delivering to the bacteria (a) a nucleic acid encoding a Fanzor polypeptide comprising an RuvC domain, and (b) a nucleic acid encoding a fRNA molecule comprising a scaffold and a reprogrammable target spacer sequence, wherein the fRNA molecule is capable of forming a complex with the Fanzor polypeptide and directing the Fanzor polypeptide to a target polynucleotide sequence.
Further aspects of the present disclosure relate to methods of modifying a target polynucleotide sequence in a bacteria, comprising delivering to the bacteria a composition of the present disclosure.
Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing”, “involving”, and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. The figures are illustrative only and are not required for enablement of the invention disclosed herein.
RNA-programmed nucleases serve diverse functions in prokaryotic systems, yet their prevalence and role in eukaryotic genomes are unclear. Searching for putative RNA-guided nucleases in genomes of diverse eukaryotes and their viruses, the present disclosure identifies numerous predicted nucleases homologous to the prokaryotic family of RNA-guided TnpB nucleases. Reconstruction of the evolutionary trajectory of these nucleases, which are referred to herein as Fanzor(s), uncovers at least two potential routes for their diversification. Surprisingly, biochemical and cellular evidence described herein shows that Fanzor families, which include the previously discovered Fanzor systems, employ non-coding RNAs encoded adjacent to the nuclease for RNA-guided cleavage of double-stranded DNA. Fanzor nucleases contain a re-arranged catalytic site inside the split RuvC domain, similar to a distinct subset of TnpB ancestors, yet lack collateral cleavage activity. In their adaptation and spread in eukaryotic lineages, Fanzor nucleases acquired N-terminal nuclear localization signals necessary for nuclear translocation, and Fanzor ORFs acquired introns, suggesting extensive spread and evolution within eukaryotes and their viruses. The present disclosure provides that Fanzor systems can be harnessed for genome editing in human cells, highlighting the potential of these widespread eukaryotic RNA-guided nucleases for biotechnology applications.
RNA-guided nucleases are prominent in prokaryotes, with roles in both adaptive immunity, such as CRISPR systems, and putative RNA-guided transposition or mobility, such as OMEGA systems (Karevelis et al. 2021; Altae-Tran et al. 2021). It is shown herein that the previously uncharacterized eukaryotic homologs of the OMEGA effector TnpB, previously termed Fanzors, are RNA-guided, programmable DNA nucleases. Additionally, the metagenomic analysis described herein permitted discovery of thousands of additional RuvC-containing nucleases in eukaryotes and their viruses, which are collectively referred to herein Fanzor systems (Table 1 and Table 4). As used herein, the term “Fanzor nuclease(s)” is interchangeable with “Fanzor polypeptide(s)” and “Fanzor protein(s)”.
The phylogenetic analysis shown herein confirmed that the two previously identified families of Fanzors (Fanzors1 and Fanzor2) are distantly related. The Fanzor1 family, as well as diverse other Fanzor families, are present in numerous eukaryotes, including animals, plants, fungi and diverse protists whereas the Fanzor2 family is more narrowly represented in giant viruses of the family Mimiviridae. These two subsets of Fanzor systems most likely entered eukaryotes via distinct mechanisms in separate events. From evolutionary distances of different Fanzor families (
Fanzor nuclease association with transposases reported herein suggests a role for their RNA-guided nuclease activity in transposition. This role could be performed through a variety of mechanisms, including 1) precise excision of the transposon from the genome via self-homing, 2) passive homing of the transposon to new alleles via leveraging nuclease-induced DSBs and DNA repair mechanisms, such as homologous recombination, and 3) active homing of the transposon via RNA guided DNA binding or cleavage for direct targeting of transposase activity. The latter mechanism would be analogous to the CRISPR-associated Tn7-like transposons (CASTs) that undergo RNA-guided transposition mediated by CRISPR effectors that were captured by these transposons on multiple occasions, in conjunction with transposase components (Strecker et al. 2019; Klompe et al. 2019). Furthermore, given that Fanzor-containing transposons harbor associated genes with diverse functions, and different groups of Fanzor contain different N-terminal domains, Fanzor might perform additional functions that remain to be investigated.
The biochemical characterization of the Fanzor nucleases of the present disclosure revealed both similarities with the homologous TnpB and CRISPR-Cas12 nucleases and several important distinctions. Similar to TnpB and Cas12, Fanzor nucleases generate double-stranded breaks through a single RuvC domain and cleave the target DNA near the 3′ end of the target. However, unlike TnpB and Cas12 enzymes, which have strong collateral activity against free DNA and RNA species nearby, Fanzor proteins have a rearranged glutamic acid and do not have collateral activity. Accordingly, TnpB systems with similarly mutated and rearranged catalytic sites also do not display collateral activity, despite having targeted double-stranded DNA cleavage activity. As opposed to the more T rich sequence constraints of TnpB and Cas12 nucleases, the Fanzor TAM preference is diverse, with GC rich preference for Fanzor2 like nucleases. Importantly, the TAM preference seems to align with the insertion site sequence supporting the role of Fanzor systems in transposition. Finally, the fRNA of Fanzor overlaps with the transposon IRR, much like TnpB's ωRNA, but it extends farther downstream of the Fanzor ORF, in contrast to the ωRNAs that ends within the 3′ regions of the TnpB ORF as the noncoding region is significantly longer in the Fanzor MGE. Thus, although the Fanzor nucleases originated from TnpB systems, the properties of these eukaryotic RNA-guided nucleases are surprisingly and notably different from those of the prokaryotic ones.
It is demonstrated herein that Fanzor nucleases can be applied for genome editing with detectable cleavage and indel generation activity in human cells. While the Fanzor nucleases are compact (˜500 amino acids), which could facilitate delivery, and their eukaryotic origins might help to reduce the immunogenicity of these nucleases in humans, additional engineering is needed to improve the activity of these systems in human cells, as has been accomplished for other miniature nucleases like Cas12f systems. See, e.g., Bigelyte et al. 2021; Wu et al. 2021; Xu et al. 2021; Kim et al. 2021. The broad distribution of Fanzor nucleases among diverse eukaryotic lineages and associated viruses suggests many more currently unknown RNA-guided systems could exist in eukaryotes, serving as a rich resource for future characterization and development of new biotechnologies.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2nd edition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4th edition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F. M. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (M. J. MacPherson, B. D. Hames, and G. R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2nd edition 2013 (E. A. Greenfield ed.); Animal Cell Culture (1987) (R. I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2nd edition (2011).
As used herein, the singular forms “a”, “an,” and “the” include both singular and plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells.
As used herein, the term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.
As used herein, the term “about” or “approximately” refers to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of +/−10% or less, +/−5% or less, +/−1% or less, +/−0.5% or less, and +/−0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosure. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.
In some aspects, the present disclosure relates to non-naturally occurring, engineered compositions comprising a Fanzor polypeptide encoding a Fanzor nuclease. Fanzor polypeptides comprise a single RuvC domain. The single RuvC domain is further comprised of three subdomains: a RuvC-I subdomain, a RuvC-II subdomain, and a RuvC-III subdomain. In some embodiments, the RuvC-II subdomain of a Fanzor polypeptide is a rearranged RuvC-II subdomain. As used herein, a “rearranged RuvC-II subdomain” refers to a domain within a RuvC-containing nuclease (e.g., a Fanzor nuclease) further comprising a loss of the canonical glutamic acid in the RuvC-II subdomain and an alternative conserved glutamate approximately residues away. As described herein, all Fanzor members and the rearranged TnpB orthologs, contained an alternative conserved glutamate approximately 45 residues away (
In some embodiments, a Fanzor polypeptide comprises an amino acid sequence identified by any one of the sequences provided herein (see e.g., Table 1, SEQ ID NOs: 1, 95-5029, and Table 4, SEQ ID NOs: 1-3, 5-7, and 9-16, or having an amino acid sequence at least at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity (including all values in between) with a Fanzor polypeptide listed in Table 1 or Table 4 (SEQ ID NOs: 1-3, 5-7, 9-16 and 95-5029).
As used herein, the term “percent identity” refers to a relationship between two nucleic acid sequences or two amino acid sequences, as determined by sequence comparison (alignment). In some embodiments, identity is determined across the entire length of a sequence. In some embodiments, identity is determined over a region of a sequence.
Identity of sequences can be readily calculated by those having ordinary skill in the art. In some embodiments, the percent identity of two sequences is determined using the algorithm of Karlin and Altschul 1990 Proc. Natl. Acad. Sci. U.S.A. 87:2264-68, modified as in Karlin and Altschul 1993 Proc. Natl. Acad. Sci. U.S.A. 90:5873-77. This algorithm is incorporated into the NBLAST® and XBLAST® programs (version 2.0) of Altschul et al. 1990 J. Mol. Biol. 215:403-10. BLAST® protein searches can be performed, for example, with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the invention. Where gaps exist between two sequences, Gapped BLAST® can be utilized, for example, as described in Altschul et al. 1997 Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST® and Gapped BLAST® programs, the default parameters of the respective programs (e.g., XBLAST® and NBLAST®) can be used, or the parameters can be adjusted appropriately as would be understood by one of ordinary skill in the art.
In some embodiments, a Fanzor polypeptide comprises about 200 to about 2212 amino acids (including all values in between). In some embodiments, a Fanzor polypeptide comprises about 200 amino acids. In some embodiments, a Fanzor polypeptide comprises about 500 amino acids. In some embodiments, a Fanzor polypeptide comprises about 1000 amino acids. In some embodiments, a Fanzor polypeptide comprises about 1500 amino acids. In some embodiments, a Fanzor polypeptide comprises about 2000 amino acids. In some embodiments, a Fanzor polypeptide comprises about 2212 amino acids.
In some embodiments, loci surrounding a nucleotide sequence encoding a Fanzor nuclease comprises a conserved non-coding sequence. In some embodiments, the conserved non-coding sequence extends at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 base pairs (including all values in between) past the end of a Fanzor open reading frame (ORF).
In some embodiments, directed evolution may be used to design modified Fanzor proteins capable of genome editing. In some embodiments, the directed evolution is performed using phage-assisted continuous evolution (PACE). In some embodiments, the directed evolution is performed using phage-assisted non-continuous evolution (PANCE). PACE technology has been described, for example, in International PCT Application, PCT/US 2009/056194, filed Sep. 8, 2009, published as WO 2010/028347 on Mar. 11, 2010; International PCT Application, PCT/US2011/066747, filed Dec. 22, 2011, published as WO 2012/088381 on Jun. 28, 2012; U.S. Pat. No. 9,023,594, issued May 5, 2015; U.S. Pat. No. 9,771,574, issued Sep. 26, 2017; U.S. Pat. No. 9,394,537, issued Jul. 19, 2016; International PCT Application, PCT/US2015/012022, filed Jan. 20, 2015, published as WO 2015/134121 on Sep. 11, 2015; U.S. Pat. No. 10,179,911, issued Jan. 15, 2019. U.S. Pat. No. 10,179,911, issued Jan. 15, 2019; International PCT Application, PCT/US2016/027795, filed Apr. 15, 2016, published as WO 2016/168631 on Oct. 20, 2016, and International Patent Publication WO 2019/023680, published Jan. 31, 2019, the entire contents of each of which are incorporated herein by reference. In some embodiments, directed evolution is implemented using a protein folding neural network, e.g., based on a published approach or on software such as AlphaFold2. In some embodiments, the Fanzor proteins obtained by methods of directed evolution are physically synthesized.
In some embodiments, the modified Fanzor protein has improved editing efficiency relative to a control Fanzor protein. In some embodiments, the improved editing efficiency is detected in mammalian cells. In some embodiments, the improved editing efficiency can be measured by an indel formation rate. In some embodiments, the indel formation rate is at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, including all values in between. In some embodiments, the modified Fanzor protein comprises one or more mutations of amino acid residues in the catalytic core (e.g., the catalytic RuvC domains) and/or of amino acid residues that contact the polynucleotide target relative to the wild type Fanzor protein. Non-limiting examples of mutations include one or more amino acid residues in a modified Fanzor protein mutated to arginine, lysine, and/or histidine relative to a wild type Fanzor protein. In some embodiments, the modified Fanzor protein comprises a mutation to arginine relative to the wild type Fanzor protein. In other embodiments, the modified Fanzor protein comprises one or more mutations to arginine relative to the wild type Fanzor protein. In some embodiments, the modified Fanzor protein comprises a mutation to lysine relative to the wild type Fanzor protein. In other embodiments, the modified Fanzor protein comprises one or more mutations to lysine relative to the wild type Fanzor protein. In some embodiments, the modified Fanzor protein comprises a mutation to histidine relative to the wild type Fanzor protein. In other embodiments, the modified Fanzor protein comprises one or more mutations to histidine relative to the wild type Fanzor protein. In some embodiments, the modified Fanzor protein contains one or more mutations to arginine, lysine, and/or histidine relative to the wild type Fanzor protein.
In some embodiments, the conserved non-coding sequence encodes a nuclease-associated RNA. In some embodiments, the nuclease-associated RNA is a Fanzor (“fRNA”) molecule. In some embodiments, the fRNA molecule is capable of directing binding and cleavage activity (e.g., guiding) of a Fanzor nuclease to a specific sequence (e.g., a target polypeptide sequence). In some embodiments, a fRNA is a guide RNA or gRNA. In some embodiments, the fRNA molecule comprises a scaffold. In some embodiments, the scaffold comprises about 21 to about 1487 nucleotides (including all values in between). In some embodiments, the scaffold comprises about 21 nucleotides. In some embodiments, the scaffold comprises about 50 nucleotides. In some embodiments, the scaffold comprises about 100 nucleotides. In some embodiments, the scaffold comprises about 150 nucleotides. In some embodiments, the scaffold comprises about 200 nucleotides. In some embodiments, the scaffold comprises about 250 nucleotides. In some embodiments, the scaffold comprises about 300 nucleotides. In some embodiments, the scaffold comprises about 350 nucleotides. In some embodiments, the scaffold comprises about 400 nucleotides. In some embodiments, the scaffold comprises about 450 nucleotides. In some embodiments, the scaffold comprises about 500 nucleotides. In some embodiments, the scaffold comprises about 550 nucleotides. In some embodiments, the scaffold comprises about 600 nucleotides. In some embodiments, the scaffold comprises about 650 nucleotides. In some embodiments, the scaffold comprises about 700 nucleotides. In some embodiments, the scaffold comprises about 750 nucleotides. In some embodiments, the scaffold comprises about 800 nucleotides. In some embodiments, the scaffold comprises about 850 nucleotides. In some embodiments, the scaffold comprises about 900 nucleotides. In some embodiments, the scaffold comprises about 950 nucleotides. In some embodiments, the scaffold comprises about 1000 nucleotides. In some embodiments, the scaffold comprises about 1050 nucleotides. In some embodiments, the scaffold comprises about 1150 nucleotides. In some embodiments, the scaffold comprises about 1200 nucleotides. In some embodiments, the scaffold comprises about 1250 nucleotides. In some embodiments, the scaffold comprises about 1300 nucleotides. In some embodiments, the scaffold comprises about 1350 nucleotides. In some embodiments, the scaffold comprises about 1400 nucleotides. In some embodiments, the scaffold comprises about 1487 nucleotides.
In some embodiments, the fRNA molecule comprises a reprogrammable target spacer sequence. In some embodiments, the reprogrammable target spacer sequence comprises about 12 to about 22 nucleotides (including all values inbetween). In some embodiments, the reprogrammable target spacer sequence comprises about 12 nucleotides. In some embodiments, the reprogrammable target spacer sequence comprises about 13 nucleotides. In some embodiments, the reprogrammable target spacer sequence comprises about 14 nucleotides. In some embodiments, the reprogrammable target spacer sequence comprises about 15 nucleotides. In some embodiments, the reprogrammable target spacer sequence comprises about 16 nucleotides. In some embodiments, the reprogrammable target spacer sequence comprises about 17 nucleotides. In some embodiments, the reprogrammable target spacer sequence comprises about 18 nucleotides. In some embodiments, the reprogrammable target spacer sequence comprises about 19 nucleotides. In some embodiments, the reprogrammable target spacer sequence comprises about 20 nucleotides. In some embodiments, the reprogrammable target spacer sequence comprises about 21 nucleotides. In some embodiments, the reprogrammable target spacer sequence comprises about 22 nucleotides.
In some embodiments, the fRNA molecule comprises a scaffold and a reprogrammable target spacer sequence. In some embodiments, the fRNA molecule comprises a scaffold about 21 to about 1487 nucleotides and a reprogrammable target spacer sequence comprises about 12 to about 22 nucleotides.
In some embodiments, the fRNA molecule is capable of forming a complex with the Fanzor polypeptide (e.g. a “Fanzor complex”) and directing the Fanzor polypeptide to a target polynucleotide sequence. The target polynucleotide of a complex (e.g., a Fanzor complex) can be any polynucleotide endogenous or exogenous to the eukaryotic cell. For example, the target polynucleotide can be a polynucleotide residing in the nucleus of the eukaryotic cell. The target polynucleotide can be a sequence coding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or a junk DNA). In some embodiments, the complex (e.g., a Fanzor complex) binds a target adjacent motif (TAM) sequence (e.g., a short sequence recognized by the complex). In some embodiments, the complex (e.g., a Fanzor complex) binds a TAM sequence 5′ of the target polynucleotide sequence. In some embodiments, the TAM sequence comprises GGG. In some embodiments, the TAM sequence comprises TTTT. In some embodiments, the TAM sequence comprises TAT. In some embodiments, the TAM sequence comprises TTG. In some embodiments, the TAM sequence comprises TTTA. In some embodiments, the TAM sequence comprises TA. In some embodiments, the TAM sequence comprises TTA. In some embodiments, the TAM sequence comprises TGAC. A person of skill in the art would be able to identify further TAM sequences for use with a given Fanzor polypeptide. It is also contemplated herein that TAM interacting domain may be engineered by techniques known in the art to allow programming of specificity, improvement of target site P1 recognition fidelity, and increased the versatility of the Fanzor nuclease genome engineering platform described herein. It is further contemplated that Fanzor nuclease may be engineered to alter their TAM specificity.
Examples of target polynucleotide sequences include, but are not limited to, a sequence associated with a signaling biochemical pathway, e.g., a signaling biochemical pathway-associated gene or polynucleotide. Further non limiting examples of target polynucleotide sequences include a disease associated gene or polynucleotide. A “disease-associated” gene or polynucleotide refers to any gene or polynucleotide which is yielding transcription or translation products at an abnormal level or in an abnormal form in cells derived from a disease-affected tissues compared with tissues or cells of a non-disease control. It may be a gene that becomes expressed at an abnormally high level, it may be a gene that becomes expressed at an abnormally low level, where the altered expression correlates with the occurrence and/or progression of the disease. A disease-associated gene also refers to a gene possessing mutation(s) or genetic variation that is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease. The transcribed or translated products may be known or unknown, and may be at a normal or abnormal level.
In some embodiments, a Fanzor polypeptide in a Fanzor polypeptide. In some embodiments, the Fanzor polypeptide is a Fanzor1 polypeptide. In some embodiments, the Fanzor polypeptide is a Fanzor2 polypeptide. In some embodiments, the RNA molecule associated with a Fanzor polypeptide is a fRNA. In some embodiments, a fRNA molecule is a fRNA molecule.
As described herein, in some embodiments, a Fanzor polypeptide may comprise additional domains other than the RuvC domain. In some embodiments, a Fanzor polypeptide comprises a nuclear localization signal (NLS). In some embodiments, a Fanzor polypeptide comprises a helix-turn-helix (HTH) domain.
In some embodiments, one or more vectors may comprise a nucleic acid sequence encoding a polypeptide described herein (e.g., a Fanzor polypeptide). As such, aspects of the present disclosure relate to one or more vectors for the expression of (a) a nucleic acid sequence encoding a Fanzor polypeptide; and (b) a nucleic acid sequence encoding a fRNA molecule comprising a scaffold and a reprogrammable target spacer sequence. In some embodiments, a vector may comprise both (a) a nucleic acid sequence encoding a Fanzor polypeptide; and (b) a nucleic acid sequence encoding a fRNA molecule. In some embodiments, a vector may comprise a nucleic acid sequence encoding a Fanzor polypeptide; and a second vector may comprise a nucleic acid sequence encoding a fRNA molecule.
The term “vector” or “expression vector” or “construct” means any molecular vehicle, such as a plasmid, phage, transposon, recombinant viral genome, cosmid, chromosome, artificial chromosome, virus, viral particle, viral vector (e.g., lentiviral vector or AAV vector), virion, etc. which can transfer gene sequences (e.g., a nucleic acid encoding a Fanzor polypeptide and/or a nucleic acid sequence encoding a fRNA molecule) into a cell or between cells.
In some embodiments, the vector may be maintained in high levels in a cell using a selection method such as involving an antibiotic resistance gene. In some embodiments, the vector may comprise a partitioning sequence which ensures stable inheritance of the vector. In some embodiments, the vector is a high copy number vector. In some embodiments, the vector becomes integrated into the chromosome of a cell.
Generally, a vector is capable of replication when associated with the proper control elements. In general, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g. retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses (AAVs)). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as “expression vectors.” Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory elements) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vilro transcription/translation system or in a host cell when the vector is introduced into the host cell). With regards to recombination and cloning methods, mention is made of U.S. patent application Ser. No. 10/815,730, published Sep. 2, 2004 as US 2004-0171156 A1, the contents of which are herein incorporated by reference in their entirety.
The vectors can include the regulatory elements, (e.g., promoters). The vectors can comprise Fanzor nuclease encoding sequences, and/or fRNA(s). In a single vector there can be a promoter for a Fanzor nuclease encoding sequence and an fRNA. In multiple vectors, there can be a first vector comprising a promoter for a Fanzor nuclease encoding sequence and a second vector comprising a promoter for a fRNA. A non-limiting example of a suitable vector is AAV, and a non-limiting example of a suitable promoter is a U6 promoter. Accordingly, from the knowledge in the art and the teachings in this disclosure the skilled person can readily make and use vectors), e.g., a single vector, expressing multiple RNAs or guides under the control or operatively or functionally linked to one or more promoters—especially as to the numbers of RNAs or guides discussed herein, without any undue experimentation.
The Fanzor nuclease encoding sequences and/or fRNA, can be functionally or operatively linked to regulatory elements. In some embodiments, the regulatory elements drive expression of the Fanzor nuclease and the fRNA. Promoters can be constitutive promoters and/or conditional promoters and/or inducible promoters and/or tissue specific promoters. Exemplary promoters include RNA polymerases, pol I, pol H, pol U1, T7, U6, HI, retroviral Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, the EFla promoter, the U6 promoter, and the pCAG promoter. An advantageous promoter is the pCAG promoter. Other promoters known in the art are also contemplated for use herein.
In addition to a Fanzor polypeptide and a nucleic acid sequence encoding an fRNA molecule, compositions of the present disclosure may comprise additional components useful for gene-editing. As non-limiting examples, compositions of the present disclosure may comprise one or more of a donor template (e.g. exogenous template) comprising a donor sequence, a linear insert sequence, a reverse transcriptase, a recombinase, a transposase, an integrase, a deaminase, a transcriptional activator, a transcriptional repressor, and/or a transposon. In some embodiments, a composition of the present disclosure comprises a donor template (e.g., exogenous template) comprising a donor sequence. In some embodiments, the donor template comprising a donor sequence is optionally for use in homology-directed repair (HDR). In some embodiments, compositions optionally for use in homology-directed repair further comprises introducing specific sequences or genes at targeted genomic locations. Reference is made to PCT Publication No. WO2008/021207, the entire contents of which is incorporated herein by reference. In some embodiments, a composition of the present disclosure comprises a linear insert sequence. A linear insert sequence as described herein comprises, for example, DNA, RNA, or mRNA. In some embodiments, a linear insert sequence is DNA. In some embodiments, a linear insert sequence is RNA. In some embodiments, a linear insert sequence is mRNA. In some embodiments, a linear insert sequence is comprised by a viral vector, optionally wherein the viral vector is Adeno-associated viral (AAV) vector, a virus, optionally wherein the virus is an Adenovirus, a lentivirus, a herpes simplex virus; and/or a lipid nanoparticle (LNP). In some embodiments, a LNP comprises one or more components of the compositions of the present disclosure. In some embodiments, the linear insert sequence is optionally for use in non-homologous end joining-based insertion. Reference is made to US Patent Publication No. US2022/0000933A1, the entire contents of which is incorporated herein by reference. In some embodiments, a composition of the present disclosure comprises a reverse transcriptase. In some embodiments, a reverse transcriptase is optionally for use in prime editing. Reference is made to U.S. Pat. No. 11,447,770, the entire contents of which is incorporated herein by reference. In some embodiments, a composition of the present disclosure comprises a recombinase, optionally for use for integration. Reference is made to U.S. Pat. No. 11,572,556, the entire contents of which is incorporated herein by reference. In some embodiments, a composition of the present disclosure comprises a transposase, optionally for use for integration. In some embodiments, the transposase naturally occurs with Fanzor systems. In some embodiments, the transposase is any one of Table 1. Non-limiting examples of transposes include Ty3, Novosib, Copia, CMC, Tc1_Mariner, hAT, Helitron, LINE, Zator, ERV, Sola, Crypton, EnSpm, IS607, Gin, and piggybac. Reference is made to PCT Publication No. WO2021030756A1, the entire contents of which is incorporated herein by reference. In some embodiments, a composition of the present disclosure comprises an integrase, optionally for use for integration. Reference is made to PCT Application No. PCT/2023/070031 and U.S. application Ser. No. 18/048,238, the entire contents of each which is incorporated herein by reference. In some embodiments, compositions optionally for use for integration further comprises programmable addition via site-specific targeting elements (PASTE). Reference is made to U.S. Pat. No. 11,572,556, the entire contents of which is incorporated herein by reference. In some embodiments, a composition of the present disclosure comprises a deaminase, optionally for use of base-editing.
In some embodiments, compositions optionally for the use of base-editing are capable of acting on single-stranded DNA. In some embodiments, compositions optionally for the use of base-editing are capable of acting on double-stranded DNA. In some embodiments, compositions optionally for the use of base-editing are capable of acting on RNA. In some embodiments, the deaminase is a cytidine deaminase. In some embodiments, compositions optionally for use of base-editing further comprises changing cytosine to thymine. In some embodiments, compositions optionally for use of base-editing further comprises changing cytosine to thymine without double-stranded breaks. In some embodiments, the deaminase is an adenine deaminase. In some embodiments, compositions optionally for use of base-editing further comprises changing adenine to guanine. In some embodiments, compositions optionally for use of base-editing further comprises changing adenine to guanine without double-stranded breaks.
In some embodiments, a composition of the present disclosure comprises a transcriptional activator, optionally for use of targeted gene activation. In some embodiments, compositions optionally for the use of targeted gene activation recruit transcriptional domains. Non-limiting examples of transcriptional domains include the transactivation domain of a zinc-finger protein, transcription activator-like effector, the Herpes simplex viral protein 16 (VP16), multiple tandem copies of VP16, such as VP64 or VP160, p65, and HSF1. Other t In some embodiments, a composition of the present disclosure comprises a transcriptional repressor, optionally for use of targeted gene repression. Non-limiting examples of transcriptional repressors include Kruppel-associated box (KRAB), Sin3 interaction domain (SID), Enhancer of Zeste Homolog2 (EZH2), histone deacetylases, and TETI. In some embodiments, the transcriptional repressor is a methyltransferase. In some embodiments, the methyltransferase is DNMT3A. In some embodiments, the methyltransferase is an enzyme that enhances the activity of DNMT3A. In some embodiments, the methyltransferase is DNMT3L. In some embodiments, the transcriptional repressor is a histone modifier. Non-limiting examples of histone modifiers include p300, LSD1, and heterochromatin protein 1 (HP1).
In some embodiments, a composition of the present disclosure comprises an epigenetic modification domain, optionally for use of epigenetic editing. In some embodiments, the epigenetic editing further comprises modifying histone modifications. In some embodiments, the epigenetic editing further comprises modifying DNA methylation patterns. In some embodiments, the epigenetic editing upregulates gene expression. In some embodiments, the epigenetic editing downregulates gene expression. Non-limiting examples of epigenetic modification domains include histone acetyltransferase p300, histone demethylase (LSD1), histone methyltransferases, such as DOT1L and PRDM9, and DNA methyltransferase DNMT3A.
In some embodiments, a composition of the present disclosure comprises a transposon, optionally for RNA guided transposition. Non-limiting examples of eukaryotic transposons include CMC, Copia, ERV, Gypsy, hAT, helitron, Zator, Sola, LINE, Tc1-Mariner, Novosib, Crypton, and EnSpm. Other eukaryotic transposons known in the art are contemplated for use herein. Reference is also made to PCT Publication No. WO2022/087494 and PCT Publication No. WO2022/159892, the entire contents of each, which is incorporated herein by reference. Compositions of the present disclosure further comprising other components known in the art for use in gene-editing are also contemplated herein. Further aspects of the disclosure comprise engineered cells comprising the Fanzor polypeptides and fRNA molecules described herein. In some embodiments, engineered cells comprise mammalian cells. Non-limiting examples of engineered cells include human cells, and any non-human eukaryote or animal or mammal as herein discussed, e.g., rodent, mouse, rat, rabbit, dog, livestock, or non-human mammal or primate. In some embodiments, the engineered cell is a rodent cell. In some embodiments, the engineered cell is a human cell. Other mammalian cell types are contemplated for use herein. In some embodiments, engineered cells of the disclosure may be isolated from human cells or tissues, plants and/or seeds, or non-human animals. It is contemplated herein that in some embodiments, host cells and/or cell lines are generated from the engineered cells of the disclosure comprising Fanzor nucleases and fRNAs described herein. It is further contemplated that host cells and/or cell lines modified by the Fanzor nucleases and fRNAs described herein include isolated stem cells and progeny thereof.
Further aspects of the disclosure provide methods of modifying a target polynucleotide sequence in a cell comprising delivering to the cell the Fanzor polypeptides and fRNA molecules described herein. In some embodiments, delivery of the Fanzor polypeptides and fRNA molecules form a complex (e.g., a Fanzor complex) for modifying a target DNA or RNA (single or double stranded, linear or supercoiled). The Fanzor complex of the invention have a wide variety of utility including modifying (e.g., deleting, inserting, translocating, inactivating, activating) a target DNA or RNA in a multiplicity of cell types. As such, the nucleic acid-targeting complex of the invention has a broad spectrum of applications in, e.g., gene therapy, drug screening, disease diagnosis, and prognosis. An exemplary nucleic acid-targeting complex comprises a DNA or RNA-targeting effector protein complexed with a co-RNA or guide RNA (gRNA) hybridized to a target polynucleotide sequence within the target locus of interest.
In some embodiments, modifying a target polynucleotide sequence comprises cleavage (e.g., a single or a double strand break) of the target polynucleotide sequence. In some embodiments, the target polynucleotide sequence is DNA. In some embodiments, one or more mutations comprising substitutions, deletions, and insertions are introduced into the target polynucleotide sequence. In some embodiments, the one or more mutations introduces frameshift mutations. In some embodiments, the cleavage creates a single-stranded break. In some embodiments, the single-stranded break reduces off-target effects. In some embodiments, the single-stranded breaks are used in pairs to create staggered double-stranded breaks. In some embodiments, the one or more mutations introduces a point mutation. In some embodiments, the one or more mutations are introduced without double-stranded breaks. In some embodiments, the one or more mutations are introduced without donor DNA. In some embodiments, the cleavage occurs proximal to the 3′ end of the target polynucleotide sequence. In some embodiments, the cleavage occurs in a specific location relative to the 3′ end of the target polynucleotide sequence. In some embodiments, a Fanzor nuclease modifies a target polynucleotide sequence by cleaving between about −6 to about +3 nucleotides relative to the 3′ end of the target polynucleotide sequence. In some embodiments, a Fanzor nuclease modifies a target polynucleotide sequence by cleaving −6 nucleotides relative to the 3′ end of the target polynucleotide sequence. In some embodiments, a Fanzor nuclease modifies a target polynucleotide sequence by cleaving −5 nucleotides relative to the 3′ end of the target polynucleotide sequence. In some embodiments, a Fanzor nuclease modifies a target polynucleotide sequence by cleaving −4 nucleotides relative to the 3′ end of the target polynucleotide sequence. In some embodiments, a Fanzor nuclease modifies a target polynucleotide sequence by cleaving −3 nucleotides relative to the 3′ end of the target polynucleotide sequence. In some embodiments, a Fanzor nuclease modifies a target polynucleotide sequence by cleaving −2 nucleotides relative to the 3′ end of the target polynucleotide sequence. In some embodiments, a Fanzor nuclease modifies a target polynucleotide sequence by cleaving −1 nucleotides relative to the 3′ end of the target polynucleotide sequence. In some embodiments, a Fanzor nuclease modifies a target polynucleotide sequence by cleaving 0 nucleotides relative to the 3′ end of the target polynucleotide sequence (e.g., cleaving at the 3′ end of the target polynucleotide sequence). In some embodiments, a Fanzor nuclease modifies a target polynucleotide sequence by cleaving +1 nucleotides relative to the 3′ end of the target polynucleotide sequence. In some embodiments, a Fanzor nuclease modifies a target polynucleotide sequence by cleaving +2 nucleotides relative to the 3′ end of the target polynucleotide sequence. In some embodiments, a Fanzor nuclease modifies a target polynucleotide sequence by cleaving +3 nucleotides relative to the 3′ end of the target polynucleotide sequence.
In some embodiments, the Fanzor nuclease modifies a target polynucleotide sequence by cleaving within the TAM sequence.
The methods of according to the invention as described herein comprehend modifying a target polynucleotide sequence, comprising contacting a sample that comprises the target polynucleotide sequence with the composition, vectors, polynucleotides comprising Fanzor nucleases and fRNA molecules described herein wherein contacting results in modification of a target polynucleotide sequence or modification of the amount or expression of a gene and/or gene product. In some embodiments, the expression of the targeted gene and/or gene product is increased by the method relative to an unmodified control. In some embodiments, the expression of the targeted gene and/or gene product is increased by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, p at least 90%, at least 95%, 100% relative to an unmodified control. In some embodiments, the expression of the targeted gene and/or gene product is increased at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 10-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least 100-fold relative to an unmodified control. In some embodiments, the expression of the targeted gene and/or gene product is reduced by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 100% relative to an unmodified control. In some embodiments, the expression of the targeted gene and/or gene product is reduced at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 10-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least 100-fold relative to an unmodified control. In some embodiments, the expression of the targeted gene and/or gene product is reduced by the method. In some embodiments, expression of the targeted gene may be completely eliminated, or may be considered eliminated as remnant expression levels of the targeted gene fall below the detection limit of methods known in the art that are used to quantify, detect, or monitor expression levels of genes.
The compositions and methods according to the invention as described herein comprehend inducing one or more nucleotide modifications in a eukaryotic cell (e.g., in a target polynucleotide sequence within a cell). In some embodiments, one or more modifications in a eukaryotic cell occurs in vitro, i.e. in an isolated eukaryotic cell, including but not limited to, a human cell) as herein discussed comprising delivering to cell a vector as herein discussed. In other embodiments, one or more modifications in a eukaryotic cell occurs in vivo. The mutation(s) can include the introduction, deletion, or substitution of one or more nucleotides at each target sequence of cell(s) via the guide RNA(s) or fRNA(s). The mutations can include the introduction, deletion, or substitution of a range of nucleotides (e.g., at each target sequence of said cell(s) via the guide(s) RNA(s) or fRNA(s). The mutations can include the introduction, deletion, or substitution of 1-100 nucleotides at each target sequence of said cell(s) via the guide RNA(s) or fRNA(s). The mutations can include the introduction, deletion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides at each target sequence of said cell(s) via the guide RNA(s) or fRNA(s). The mutations can include removing, adding, or rearranging large chromosomal segments at each target sequence of said cell(s) via the guide RNA(s) or fRNA(s). In some embodiments, the fRNA includes a primer binding site. In some embodiments the primer binding site (PBS) binds to exposed DNA. In some embodiments, the primer binding site binds to exposed DNA generated by Fanzor cleavage. In some embodiments, the fRNA further includes a reverse transcriptase (RT) region. In some embodiments, the RT region is complementary to the genome. In some embodiments, the mutation is introduced between the RT and PBS sites.
The nucleic acid molecule encoding a Fanzor nuclease may be codon optimized for expression in a particular host species. A codon optimized sequence includes a sequence optimized for expression in a different eukaryote relative to the eukaryote of origin for a Fanzor nuclease. As a non-limiting example, the nucleic acid molecule encoding a Fanzor nuclease from Chlamydomonas reinhardtii may be codon-optimized for expression in humans, or for another eukaryote, animal or mammal as herein. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp/codon and these tables can be adapted in a number of ways. See Nakamura, Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA), are also available. In some embodiments, one or more codons (e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a Fanzor nuclease correspond to the most frequently used codon for a particular amino acid. Other methods of codon optimization known in the art are contemplated for use herein.
The methods of modifying a target polynucleotide sequence in a cell according to the invention as described herein may comprise a Fanzor nuclease and a fRNA to be delivered together (e.g., by the same vector) or delivered separately (e.g. as separate vectors). A Fanzor nuclease of the present disclosure may be unstable without co-delivery of the fRNA molecule (e.g., when a Fanzor nuclease and the fRNA molecule are delivered by separate vectors). In some embodiments, the Fanzor nuclease is stable in the presence of the fRNA molecule. In some embodiments, the Fanzor nuclease is stable in the absence of the fRNA molecule. In some embodiments, the Fanzor polypeptide encoding the Fanzor nuclease (e.g., the Fanzor nuclease encoding sequence) is modified to increase stability. In some embodiments, the modifications include, but are not limited to, one or more mutations relative to the wildtype Fanzor polypeptide wherein the one or more mutations result in a Fanzor polypeptide that has increased stability in the absence of the fRNA relative to an unmodified Fanzor polypeptide. An exemplary modification is the fusion of a stabilizing domain to a Fanzor polypeptide to increase stability. Non-limiting examples of stabilizing domains that can be fused with a Fanzor nuclease of the present disclosure include a small ubiquitin-like modifier (SUMO) tag, glutathione-S-transferase (GST) tag, and/or superfolder green fluorescent protein (sfGFP). Other modifications known in the art for increasing the stabilization of a polypeptide, and/or ofa nuclease, are contemplated herein.
The compositions described herein may be used in various nucleic acids-targeting applications, altering or modifying synthesis of a gene product, such as a protein, nucleic acids cleavage, nucleic acids editing, nucleic acids splicing; trafficking of target nucleic acids, tracing of target nucleic acids, isolation of target nucleic acids, visualization of target nucleic acids, etc. Aspects of the invention also encompass methods and uses of the compositions and systems described herein in genome engineering, e.g. for altering or manipulating the expression of one or more genes or the one or more gene products, in prokaryotic or eukaryotic cells, in vitro, in vivo or ex vivo. In some examples, the target polynucleotides are target sequences within genomic DNA, including nuclear genomic DNA, mitochondrial DNA, or chloroplast DNA. In some embodiments, the target sequence is a viral polynucleotide. In some embodiments, the viral polynucleotide is integrated within a host genome. Aspects of the invention also encompass methods and uses of the compositions and systems described herein for multiplexed editing. In some embodiments, the multiplexed editing targets 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sites. In some embodiments, the target polynucleotide is a gene related to disease resistance or pest control. In some embodiments, the genome engineering is directed towards modifying crop traits. Non-limiting examples of crop trait modifications include improved yield, improved taste, and improved nutritional value. In some embodiments, the genome engineering is directed towards bioenergy production. In some embodiments, the genome engineering is directed towards modifying organisms to optimize the production of biofuels. Non-limiting examples of organisms that can be modified to optimize the production of biofuels include algae, bacteria, yeast, microalgae, sugarcane, corn, switchgrass, miscanthus, sorghum, soybean, canola, jatropha, Trichoderma, Aspergillus, and macroalgae. In some embodiments, the genome engineering is directed towards bioremediation. In some embodiments, the genome engineering is directed towards modifying microbes to degrade environmental pollutants. Non-limiting examples or microbes that can be modified to degrade environmental pollutants include Brevibacterium epidermis EZ-K02. Microbacterium oleivorans, Irpex lacteus, Bacillus subtilis HUK15, Anaeromyxobacter sp Fw109-5, Bacillus, Corprothermobacter, Rhodobacter, Pseudomonas, Achromobacter, Desfiilitobacter, Desulfosporosinus, T78. Methanobacterium, Methanosaeta, Proteobacteria, Firmicutes, Naegleria, Vorticella, Arabidopsis, Asarum, Populus, Koribacter, Acidomicrobium, Bradyrhizobiu, Burkholderia, Solibacter, Singulisphaera, Desulfomonile, Rhodcococus, Bordatella, Chromobacter, Variovorax, Thiobacillus sp., Pseudoxanthomonas sp., Aleanivorax sp., Acinetobacter venetianus RAG-1, Dehalococcoides mccartyi, Actinobacter, Mycobacterium, Pseudomonas aeruginosa, Penicillium oxalicum, Sphingomonas sp. GY2B, Miscanthus sinesis, Rhizobiales, Burkholderiales, Actinomycetales, Pseudomonas pulida, Pseudomonas putida KT2440, Rhodococcus aetherivorans BCP 1, Rhodococcus opacus R7, and Pseudomonas stutzeri 5190. Aspects of the invention also encompass methods and uses of the compositions and systems described herein in chromosome imaging, e.g. for visualizing specific sequences within live cells. In some examples, chromosome imaging is performed by fluorescently-tagging the compositions described herein.
The compositions described herein may be used to create genetically modified animal models or to create functional genomic screens. In some embodiments, the genetically modified animal models can be used for disease research. In some embodiments, the functional genomic screens can be used to identify genes involved in specific biological processes. In some embodiments, the functional genomic screens can be used to identify polynucleotide sequences related to disease pathogens. In some embodiments, the polynucleotide sequences are DNA. In some embodiments, the polynucleotide sequences are RNA. Any disease or disorder that may be detected using any of the composition or methods described herein (e.g., Fanzor systems) are contemplated for detection herein.
In some aspects, the invention provides methods comprising delivering one or more polynucleotides, such as or one or more vectors as described herein, one or more transcripts thereof, and/or one or proteins transcribed therefrom, to a host cell. In some aspects, the invention further provides cells produced by such methods, and organisms (such as animals, plants, seeds, or fungi) comprising or produced from such cells. In some embodiments, a base editor as described herein in combination with (and optionally complexed with) a guide sequence is delivered to a cell.
Exemplary delivery strategies are known in the art, and described herein, which include vector-based strategies. In some embodiments, the method of delivery provided comprises nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid.nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Exemplary methods of delivery of nucleic acids include lipofection, nucleofection, electoporation, stable genome integration (e.g., piggybac), microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™, Lipofectin™ and SF Cell Line 4D-Nucleofector X Kit™ (Lonza)). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424; WO 91/16024. Other methods of delivery known in the art are contemplated for use with Fanzor system described herein.
Delivery may be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration). Delivery methods known in the art are contemplated for use herein. As a non-limiting example, the compositions and methods of the present invention may be delivered via ex vivo administration to non-limiting cell types such as B cells, T cells, tumor infiltrating lymphocytes (TIL), CARTs, and/or stem cells (e.g., bone marrow stem cells) for the treatment of various diseases. Other cell types compatible with ex vivo administration known in the art are also contemplated for use with the compositions and methods disclosed herein. The compositions and methods of the present invention may be delivered via in vivo administration to target tissues and/or cells of target tissues using, as non-limiting examples, AAV or other programmable tissue-specific lipid nanoparticles (LNPs). Other methods of in vivo administration known in the art are also contemplated for use with the compositions and methods disclosed herein.
Delivery may be achieved through the use of RNP complexes. Examples of target polynucleotides include a sequence associated with a signaling biochemical pathway, e.g., a signaling biochemical pathway-associated gene or polynucleotide. Examples of target polynucleotides include a disease associated gene or polynucleotide. A “disease-associated” gene or polynucleotide refers to any gene or polynucleotide which is yielding transcription or translation products at an abnormal level or in an abnormal form in cells derived from a disease-affected tissues compared with tissues or cells of a non-disease control. It may be a gene that becomes expressed at an abnormally high level, it may be a gene that becomes expressed at an abnormally low level, where the altered expression correlates with the occurrence and/or progression of the disease. A disease-associated gene also refers to a gene possessing mutation(s) or genetic variation that is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease. The transcribed or translated products may be known or unknown, and may be at a normal or abnormal level. Examples of target polynucleotides include a viral associated gene or polynucleotide. A “viral-associated” gene or polynucleotide refers to any gene or polynucleotide of viral origin integrated within a host genome. It may be a gene that is involved in the replication, transcription, translation, or assembly of a virus. It may be a gene that is highly conserved among viruses. For example, in some embodiments, a method is provided that comprises administering to a subject having a viral disease an effective amount of the Fanzor editing system described herein that introduces a deactivating mutation into a viral-associated gene.
The “disease-associated” gene or polynucleotide can be associated with a monogenetic disorder selected from the group consisting of: Adenosine Deaminase (ADA) Deficiency; Alpha-1 Antitrypsin Deficiency; Cystic Fibrosis; Duchenne Muscular Dystrophy; Galactosemia; Hemochromatosis; Huntington's Disease; Maple Syrup Urine Disease; Marfan Syndrome; Neurofibromatosis Type 1; Pachyonychia Congenita; Phenylkeotnuria; Severe Combined Immunodeficiency; Sickle Cell Disease; Smith-Lemli-Opitz Syndrome; and Tay-Sachs Disease. In other embodiments, the disease-associated gene can be associated with a polygenic disorder selected from the group consisting of: heart disease; high blood pressure; Alzheimer's disease; arthritis; diabetes; cancer; and obesity. The compositions described herein may be administered to a subject in need thereof in a therapeutically effective amount to treat and/or prevent a disease or disorder the subject is suffering from. Any disease or disorder that may be treated and/or prevented using any of the composition or methods described herein (e.g., Fanzor systems) are contemplated for treatment herein. Any disease is conceivably treatable by such methods so long as delivery to the appropriate cells is feasible. The person having ordinary skill in the art will be able to choose and/or select a Fanzor delivery methodology to suit the intended purpose and the intended target cells.
For example, in some embodiments, a method is provided that comprises administering to a subject having such a disease, e.g., a cancer associated with a point mutation as described above, an effective amount of the Fanzor editing system described herein that corrects the point mutation or introduces a deactivating mutation into a disease-associated gene as mediated by homology-directed repair in the presence of a donor DNA molecule comprising desired genetic change. In some embodiments, a method is provided that comprises administering to a subject having such a disease, e.g., a cancer associated with a point mutation as described above, an effective amount of the Fanzor editing system described herein that corrects the point mutation or introduces a deactivating mutation into a disease-associated gene. In some embodiments, the disease is a proliferative disease. In some embodiments, the disease is a genetic disease. In some embodiments, the disease is a neoplastic disease. In some embodiments, the disease is a metabolic disease. In some embodiments, the disease is a lysosomal storage disease. Other diseases that can be treated by correcting a point mutation or introducing a deactivating mutation into a disease-associated gene will be known to those of skill in the art, and the disclosure is not limited in this respect.
The instant disclosure provides methods for the treatment of additional diseases or disorders, e.g., diseases or disorders that are associated or caused by a point mutation that can be corrected by Fanzor-mediated gene editing. Some such diseases are described herein, and additional suitable diseases that can be treated with the strategies and fusion proteins provided herein will be apparent to those of skill in the art based on the instant disclosure. Exemplary suitable diseases and disorders are listed below. It will be understood that the numbering of the specific positions or residues in the respective sequences depends on the particular protein and numbering scheme used. Numbering might be different, e.g., in precursors of a mature protein and the mature protein itself, and differences in sequences from species to species may affect numbering. One of skill in the art will be able to identify the respective residue in any homologous protein and in the respective encoding nucleic acid by methods well known in the art, e.g., by sequence alignment and determination of homologous residues. Exemplary suitable diseases and disorders include, without limitation: 2-methyl-3-hydroxybutyric aciduria; 3 beta-Hydroxysteroid dehydrogenase deficiency; 3-Methylglutaconic aciduria; 3-Oxo-5 alpha-steroid delta 4-dehydrogenase deficiency; 46,XY sex reversal, type 1, 3, and 5; 5-Oxoprolinase deficiency; 6-pyruvoyl-tetrahydropterin synthase deficiency; Aarskog syndrome; Aase syndrome; Achondrogenesis type 2; Achromatopsia 2 and 7; Acquired long QT syndrome; Acrocallosal syndrome, Schinzel type; Acrocapitofemoral dysplasia; Acrodysostosis 2, with or without hormone resistance; Acroerythrokeratoderma; Acromicric dysplasia; Acth-independent macronodular adrenal hyperplasia 2; Activated PI3K-delta syndrome; Acute intermittent porphyria; deficiency of Acyl-CoA dehydrogenase family, member 9; Adams-Oliver syndrome 5 and 6, Adenine phosphoribosyltransferase deficiency; Adenylate kinase deficiency; hemolytic anemia due to Adenylosuccinate lyase deficiency; Adolescent nephronophthisis; Renal-hepatic-pancreatic dysplasia; Meckel syndrome type 7; Adrenoleukodystrophy; Adult junctional epidermolysis bullosa; Epidermolysis bullosa, junctional, localisata variant; Adult neuronal ceroid lipofuscinosis; Adult neuronal ceroid lipofuscinosis; Adult onset ataxia with oculomotor apraxia; ADULT syndrome; Afibrinogenemia and congenital Afibrinogenemia; autosomal recessive Agammaglobulinemia 2; Age-related macular degeneration 3, 6, 11, and 12; Aicardi Goutieres syndromes 1, 4, and 5; Chilbain lupus 1; Alagille syndromes 1 and 2; Alexander disease; Alkaptonuria; Allan-Herndon-Dudley syndrome; Alopecia universalis congenital; Alpers encephalopathy; Alpha-1-antitrypsin deficiency; autosomal dominant, autosomal recessive, and X-linked recessive Alport syndromes; Alzheimer disease, familial, 3, with spastic paraparesis and apraxia; Alzheimer disease, types, 1, 3, and 4; hypocalcification type and hypomaturation type, IIA1 Amelogenesis imperfecta; Aminoacylase 1 deficiency; Amish infantile epilepsy syndrome; Amyloidogenic transthyretin amyloidosis; Amyloid Cardiomyopathy, Transthyretin-related; Cardiomyopathy; Amyotrophic lateral sclerosis types 1, 6, 15 (with or without frontotemporal dementia), 22 (with or without frontotemporal dementia), and 10; Frontotemporal dementia with TDP43 inclusions, TARDBP-related; Andermann syndrome; Andersen Tawil syndrome; Congenital long QT syndrome; Anemia, nonspherocytic hemolytic, due to G6PD deficiency; Angelman syndrome; Severe neonatal-onset encephalopathy with microcephaly; susceptibility to Autism, X-linked 3; Angiopathy, hereditary, with nephropathy, aneurysms, and muscle cramps; Angiotensin i-converting enzyme, benign serum increase; Aniridia, cerebellar ataxia, and mental retardation; Anonychia; Antithrombin III deficiency; Antley-Bixler syndrome with genital anomalies and disordered steroidogenesis; Aortic aneurysm, familial thoracic 4, 6, and 9; Thoracic aortic aneurysms and aortic dissections; Multisystemic smooth muscle dysfunction syndrome; Moyamoya disease 5; Aplastic anemia; Apparent mineralocorticoid excess; Arginase deficiency; Argininosuccinate lyase deficiency; Aromatase deficiency; Arrhythmogenic right ventricular cardiomyopathy types 5, 8, and 10; Primary familial hypertrophic cardiomyopathy; Arthrogryposis multiplex congenita, distal, X-linked; Arthrogryposis renal dysfunction cholestasis syndrome; Arthrogryposis, renal dysfunction, and cholestasis 2; Asparagine synthetase deficiency; Abnormality of neuronal migration; Ataxia with vitamin E deficiency; Ataxia, sensory, autosomal dominant: Ataxia-telangiectasia syndrome; Hereditary cancer-predisposing syndrome; Atransferrinemia; Atrial fibrillation, familial, 11, 12, 13, and 16; Atrial septal defects 2, 4, and 7 (with or without atrioventricular conduction defects); Atrial standstill 2; Atrioventricular septal defect 4; Atrophia bulborum hereditaria; ATR-X syndrome; Auriculocondylar syndrome 2; Autoimmune disease, multisystem, infantile-onset; Autoimmune lymphoproliferative syndrome, type 1a; Autosomal dominant hypohidrotic ectodermal dysplasia; Autosomal dominant progressive external ophthalmoplegia with mitochondrial DNA deletions 1 and 3; Autosomal dominant torsion dystonia 4; Autosomal recessive centronuclear myopathy; Autosomal recessive congenital ichthyosis 1, 2, 3, 4A, and 4B; Autosomal recessive cutis laxa type IA and 1B; Autosomal recessive hypohidrotic ectodermal dysplasia syndrome; Ectodermal dysplasia 11b; hypohidrotic/hair/tooth type, autosomal recessive; Autosomal recessive hypophosphatemic bone disease; Axenfeld-Rieger syndrome type 3; Bainbridge-Ropers syndrome; Bannayan-Riley-Ruvalcaba syndrome; PTEN hamartoma tumor syndrome; Baraitser-Winter syndromes 1 and 2; Barakat syndrome; Bardet-Biedl syndromes 1, 11, 16, and 19. Bare lymphocyte syndrome type 2, complementation group E; Bartter syndrome antenatal type 2; Bartter syndrome types 3, 3 with hypocalciuria, and 4; Basal ganglia calcification, idiopathic, 4; Beaded hair; Benign familial hematuria; Benign familial neonatal seizures 1 and 2; Seizures, benign familial neonatal, 1, and/or myokymia; Seizures, Early infantile epileptic encephalopathy 7; Benign familial neonatal-infantile seizures; Benign hereditary chorea; Benign scapuloperoneal muscular dystrophy with cardiomyopathy; Bernard-Soulier syndrome, types A1 and A2 (autosomal dominant); Bestrophinopathy, autosomal recessive; beta Thalassemia; Bethlem myopathy and Bethlem myopathy 2; Bietti crystalline corneoretinal dystrophy; Bile acid synthesis defect, congenital, 2; Biotinidase deficiency; Birk Barel mental retardation dysmorphism syndrome; Blepharophimosis, ptosis, and epicanthus inversus; Bloom syndrome; Borjeson-Forssman-Lehmann syndrome; Boucher Neuhauser syndrome; Brachydactyly types A1 and A2; Brachydactyly with hypertension; Brain small vessel disease with hemorrhage; Branched-chain ketoacid dehydrogenase kinase deficiency; Branchiootic syndromes 2 and 3; Breast cancer, early-onset; Breast-ovarian cancer, familial 1, 2, and 4; Brittle cornea syndrome 2; Brody myopathy; Bronchiectasis with or without elevated sweat chloride 3; Brown-Vialetto-Van laere syndrome and Brown-Vialetto-Van Laere syndrome 2; Brugada syndrome; Brugada syndrome 1; Ventricular fibrillation; Paroxysmal familial ventricular fibrillation; Brugada syndrome and Brugada syndrome 4; Long QT syndrome; Sudden cardiac death; Bull eye macular dystrophy; Stargardt disease 4; Cone-rod dystrophy 12; Bullous ichthyosiform erythroderma; Burn-Mckeown syndrome; Candidiasis, familial, 2, 5, 6, and 8; Carbohydrate-deficient glycoprotein syndrome type I and II; Carbonic anhydrase VA deficiency, hyperammonemia due to; Carcinoma of colon; Cardiac arrhythmia; Long QT syndrome, LQT1 subtype; Cardioencephalomyopathy, fatal infantile, due to cytochrome c oxidase deficiency; Cardiofaciocutaneous syndrome; Cardiomyopathy; Danon disease; Hypertrophic cardiomyopathy; Left ventricular noncompaction cardiomyopathy; Carnevale syndrome; Carney complex, type 1; Carnitine acylcarnitine translocase deficiency; Carnitine palmitoyltransferase I, II, II (late onset), and II (infantile) deficiency; Cataract 1, 4, autosomal dominant, autosomal dominant, multiple types, with microcornea, coppock-like, juvenile, with microcornea and glucosuria, and nuclear diffuse nonprogressive; Catecholaminergic polymorphic ventricular tachycardia; Caudal regression syndrome; Cd8 deficiency, familial; Central core disease; Centromeric instability of chromosomes 1, 9 and 16 and immunodeficiency; Cerebellar ataxia infantile with progressive external ophthalmoplegi and Cerebellar ataxia, mental retardation, and dysequilibrium syndrome 2; Cerebral amyloid angiopathy, APP-related; Cerebral autosomal dominant and recessive arteriopathy with subcortical infarcts and leukoencephalopathy; Cerebral cavernous malformations 2; Cerebrooculofacioskeletal syndrome 2; Cerebro-oculo-facio-skeletal syndrome; Cerebroretinal microangiopathy with calcifications and cysts; Ceroid lipofuscinosis neuronal 2, 6, 7, and 10; Ch\xc3\xa9diak-Higashi syndrome, Chediak-Higashi syndrome, adult type; Charcot-Marie-Tooth disease types 1B, 2B2, 2C, 2F, 2I, 2U (axonal), 1C (demyelinating), dominant intermediate C, recessive intermediate A, 2A2, 4C, 4D, 4H, IF, IVF, and X; Scapuloperoneal spinal muscular atrophy; Distal spinal muscular atrophy, congenital nonprogressive; Spinal muscular atrophy, distal, autosomal recessive, 5: CHARGE association; Childhood hypophosphatasia; Adult hypophosphatasia; Cholecystitis; Progressive familial intrahepatic cholestasis 3; Cholestasis, intrahepatic, of pregnancy 3; Cholestanol storage disease; Cholesterol monooxygenase (side-chain cleaving) deficiency; Chondrodysplasia Blomstrand type; Chondrodysplasia punctata 1, X-linked recessive and 2 X-linked dominant; CHOPS syndrome; Chronic granulomatous disease, autosomal recessive cytochrome b-positive, types 1 and 2; Chudley-McCullough syndrome; Ciliary dyskinesia, primary, 7, 11, 15, 20 and 22; Citrullinemia type I; Citrullinemia type I and II; Cleidocranial dysostosis; C-like syndrome; Cockayne syndrome type A; Coenzyme Q10 deficiency, primary 1, 4, and 7; Coffin Siris/Intellectual Disability; Coffin-Lowry syndrome; Cohen syndrome; Cold-induced sweating syndrome 1; COLE-CARPENTER SYNDROME 2; Combined cellular and humoral immune defects with granulomas; Combined d-2- and 1-2-hydroxyglutaric aciduria; Combined malonic and methylmalonic aciduria; Combined oxidative phosphorylation deficiencies 1, 3, 4, 12, 15, and 25; Combined partial and complete 17-alpha-hydroxylase/17,20-lyase deficiency; Common variable immunodeficiency 9; Complement component 4, partial deficiency of, due to dysfunctional c1 inhibitor; Complement factor B deficiency; Cone monochromatism; Cone-rod dystrophy 2 and 6; Cone-rod dystrophy amelogenesis imperfecta; Congenital adrenal hyperplasia and Congenital adrenal hypoplasia, X-linked; Congenital amegakaryocytic thrombocytopenia; Congenital aniridia; Congenital central hypoventilation; Hirschsprung disease 3; Congenital contractural arachnodactyly; Congenital contractures of the limbs and face, hypotonia, and developmental delay; Congenital disorder of glycosylation types 1B, 1D, 1G, 1H, 1J, 1K, 1N, 1P, 2C, 2J, 2K, IIm; Congenital dyserythropoietic anemia, type I and II; Congenital ectodermal dysplasia of face; Congenital erythropoietic porphyria; Congenital generalized lipodystrophy type 2; Congenital heart disease, multiple types, 2; Congenital heart disease; Interrupted aortic arch; Congenital lipomatous overgrowth, vascular malformations, and epidermal nevi; Non-small cell lung cancer; Neoplasm of ovary; Cardiac conduction defect, nonspecific; Congenital microvillous atrophy; Congenital muscular dystrophy; Congenital muscular dystrophy due to partial LAMA2 deficiency; Congenital muscular dystrophy-dystroglycanopathy with brain and eye anomalies, types A2, A7, A8, All, and A14; Congenital muscular dystrophy-dystroglycanopathy with mental retardation, types B2, B3, B5, and B15; Congenital muscular dystrophy-dystroglycanopathy without mental retardation, type B5; Congenital muscular hypertrophy-cerebral syndrome; Congenital myasthenic syndrome, acetazolamide-responsive; Congenital myopathy with fiber type disproportion; Congenital ocular coloboma; Congenital stationary night blindness, type 1A, 1B, 1C, 1E, IF, and 2A; Coproporphyria; Cornea plana 2; Corneal dystrophy, Fuchs endothelial, 4; Corneal endothelial dystrophy type 2; Corneal fragility keratoglobus, blue sclerae and joint hypermobility; Cornelia de Lange syndromes 1 and 5; Coronary artery disease, autosomal dominant 2; Coronary heart disease; Hyperalphalipoproteinemia 2; Cortical dysplasia, complex, with other brain malformations 5 and 6; Cortical malformations, occipital; Corticosteroid-binding globulin deficiency; Corticosterone methyloxidase type 2 deficiency; Costello syndrome; Cowden syndrome 1; Coxa plana; Craniodiaphyseal dysplasia, autosomal dominant; Craniosynostosis 1 and 4; Craniosynostosis and dental anomalies; Creatine deficiency, X-linked; Crouzon syndrome; Cryptophthalmos syndrome; Cryptorchidism, unilateral or bilateral; Cushing symphalangism; Cutaneous malignant melanoma 1; Cutis laxa with osteodystrophy and with severe pulmonary, gastrointestinal, and urinary abnormalities; Cyanosis, transient neonatal and atypical nephropathic; Cystic fibrosis; Cystinuria; Cytochrome c oxidase i deficiency; Cytochrome-c oxidase deficiency; D-2-hydroxyglutaric aciduria 2; Darier disease, segmental; Deafness with labyrinthine aplasia microtia and microdontia (LAMM); Deafness, autosomal dominant 3a, 4, 12, 13, 15, autosomal dominant nonsyndromic sensorineural 17, 20, and 65; Deafness, autosomal recessive 1A, 2, 3, 6, 8, 9, 12, 15, 16, 18b, 22, 28, 31, 44, 49, 63, 77, 86, and 89; Deafness, cochlear, with myopia and intellectual impairment, without vestibular involvement, autosomal dominant, X-linked 2; Deficiency of 2-methylbutyryl-CoA dehydrogenase; Deficiency of 3-hydroxyacyl-CoA dehydrogenase; Deficiency of alpha-mannosidase; Deficiency of aromatic-L-amino-acid decarboxylase; Deficiency of bisphosphoglycerate mutase; Deficiency of butyryl-CoA dehydrogenase; Deficiency of ferroxidase; Deficiency of galactokinase; Deficiency of guanidinoacetate methyltransferase; Deficiency of hyaluronoglucosaminidase; Deficiency of ribose-5-phosphate isomerase; Deficiency of steroid 11-beta-monooxygenase; Deficiency of UDPglucose-hexose-1-phosphate uridylyltransferase; Deficiency of xanthine oxidase; Dejerine-Sottas disease; Charcot-Marie-Tooth disease, types ID and IVF: Dejerine-Sottas syndrome, autosomal dominant; Dendritic cell, monocyte, B lymphocyte, and natural killer lymphocyte deficiency; Desbuquois dysplasia 2; Desbuquois syndrome; DFNA 2 Nonsyndromic Hearing Loss; Diabetes mellitus and insipidus with optic atrophy and deafness; Diabetes mellitus, type 2, and insulin-dependent, 20; Diamond-Blackfan anemia 1, 5, 8, and 10; Diarrhea 3 (secretory sodium, congenital, syndromic) and 5 (with tufting enteropathy, congenital); Dicarboxylic aminoaciduria; Diffuse palmoplantar keratoderma, Bothnian type; Digitorenocerebral syndrome; Dihydropteridine reductase deficiency; Dilated cardiomyopathy 1A, 1AA, 1C, 1G, lBB, 1DD, 1FF, 1HH, 1I, 1KK, 1N, 1S, 1Y, and 3B; Left ventricular noncompaction 3; Disordered steroidogenesis due to cytochrome p450 oxidoreductase deficiency; Distal arthrogryposis type 2B; Distal hereditary motor neuronopathy type 2B; Distal myopathy Markesbery-Griggs type; Distal spinal muscular atrophy, X-linked 3; Distichiasis-lymphedema syndrome; Dominant dystrophic epidermolysis bullosa with absence of skin; Dominant hereditary optic atrophy; Donnai Barrow syndrome; Dopamine beta hydroxylase deficiency; Dopamine receptor d2, reduced brain density of; Dowling-degos disease 4; Doyne honeycomb retinal dystrophy; Malattia leventinese; Duane syndrome type 2; Dubin-Johnson syndrome; Duchenne muscular dystrophy; Becker muscular dystrophy; Dysfibrinogenemia; Dyskeratosis congenita autosomal dominant and autosomal dominant, 3; Dyskeratosis congenita, autosomal recessive, 1, 3, 4, and 5; Dyskeratosis congenita X-linked; Dyskinesia, familial, with facial myokymia; Dysplasminogenemia; Dystonia 2 (torsion, autosomal recessive), 3 (torsion, X-linked), 5 (Dopa-responsive type), 10, 12, 16, 25, 26 (Myoclonic); Seizures, benign familial infantile, 2, Early infantile epileptic encephalopathy 2, 4, 7, 9, 10, 11, 13, and 14. Atypical Rett syndrome; Early T cell progenitor acute lymphoblastic leukemia; Ectodermal dysplasia skin fragility syndrome; Ectodermal dysplasia-syndactyly syndrome 1; Ectopia lentis, isolated autosomal recessive and dominant; Ectrodactyly, ectodermal dysplasia, and cleft lip/palate syndrome 3; Ehlers-Danlos syndrome type 7 (autosomal recessive), classic type, type 2 (progeroid), hydroxylysine-deficient, type 4, type 4 variant, and due to tenascin-X deficiency; Eichsfeld type congenital muscular dystrophy; Endocrine-cerebroosteodysplasia; Enhanced s-cone syndrome; Enlarged vestibular aqueduct syndrome; Enterokinase deficiency; Epidermodysplasia verruciformis; Epidermolysa bullosa simplex and limb girdle muscular dystrophy, simplex with mottled pigmentation, simplex with pyloric atresia, simplex, autosomal recessive, and with pyloric atresia; Epidermolytic palmoplantar keratoderma; Familial febrile seizures 8; Epilepsy, childhood absence 2, 12 (idiopathic generalized, susceptibility to) 5 (nocturnal frontal lobe), nocturnal frontal lobe type 1, partial, with variable foci, progressive myoclonic 3, and X-linked, with variable learning disabilities and behavior disorders; Epileptic encephalopathy, childhood-onset, early infantile, 1, 19, 23, 25, 30, and 32; Epiphyseal dysplasia, multiple, with myopia and conductive deafness; Episodic ataxia type 2; Episodic pain syndrome, familial, 3; Epstein syndrome; Fechtner syndrome; Erythropoietic protoporphyria; Estrogen resistance; Exudative vitreoretinopathy 6; Fabry disease and Fabry disease, cardiac variant; Factor H, VII, X, v and factor viii, combined deficiency of 2, xiii, a subunit, deficiency; Familial adenomatous polyposis 1 and 3; Familial amyloid nephropathy with urticaria and deafness; Familial cold urticarial; Familial aplasia of the vermis; Familial benign pemphigus; Familial cancer of breast; Breast cancer, susceptibility to; Osteosarcoma; Pancreatic cancer 3; Familial cardiomyopathy; Familial cold autoinflammatory syndrome 2; Familial colorectal cancer; Familial exudative vitreoretinopathy, X-linked; Familial hemiplegic migraine types 1 and 2; Familial hypercholesterolemia; Familial hypertrophic cardiomyopathy 1, 2, 3, 4, 7, 10, 23 and 24; Familial hypokalemia-hypomagnesemia; Familial hypoplastic, glomerulocystic kidney; Familial infantile myasthenia; Familial juvenile gout; Familial Mediterranean fever and Familial mediterranean fever, autosomal dominant; Familial porencephaly; Familial porphyria cutanea tarda; Familial pulmonary capillary hemangiomatosis; Familial renal glucosuria; Familial renal hypouricemia; Familial restrictive cardiomyopathy 1; Familial type 1 and 3 hyperlipoproteinemia; Fanconi anemia, complementation group E, I, N, and O; Fanconi-Bickel syndrome; Favism, susceptibility to; Febrile seizures, familial, 11; Feingold syndrome 1; Fetal hemoglobin quantitative trait locus 1; FG syndrome and FG syndrome 4; Fibrosis of extraocular muscles, congenital, 1, 2, 3a (with or without extraocular involvement), 3b; Fish-eye disease; Fleck corneal dystrophy; Floating-Harbor syndrome; Focal epilepsy with speech disorder with or without mental retardation; Focal segmental glomerulosclerosis 5; Forebrain defects; Frank Ter Haar syndrome; Borrone Di Rocco Crovato syndrome; Frasier syndrome; Wilms tumor 1; Freeman-Sheldon syndrome; Frontometaphyseal dysplasia land 3; Frontotemporal dementia; Frontotemporal dementia and/or amyotrophic lateral sclerosis 3 and 4; Frontotemporal Dementia Chromosome 3-Linked and Frontotemporal dementia ubiquitin-positive; Fructose-biphosphatase deficiency; Fuhrmann syndrome; Gamma-aminobutyric acid transaminase deficiency; Gamstorp-Wohlfart syndrome; Gaucher disease type 1 and Subacute neuronopathic; Gaze palsy, familial horizontal, with progressive scoliosis; Generalized dominant dystrophic epidermolysis bullosa; Generalized epilepsy with febrile seizures plus 3, type 1, type 2; Epileptic encephalopathy Lennox-Gastaut type; Giant axonal neuropathy; Glanzmann thrombasthenia; Glaucoma 1, open angle, e, F, and G; Glaucoma 3, primary congenital, d; Glaucoma, congenital and Glaucoma, congenital, Coloboma; Glaucoma, primary open angle, juvenile-onset; Glioma susceptibility 1; Glucose transporter type 1 deficiency syndrome; Glucose-6-phosphate transport defect; GLUT1 deficiency syndrome 2; Epilepsy, idiopathic generalized, susceptibility to, 12; Glutamate formiminotransferase deficiency; Glutaric acidemia IIA and IIB; Glutaric aciduria, type 1; Gluthathione synthetase deficiency; Glycogen storage disease 0 (muscle), II (adult form), IXa2, IXc, type 1A; type II, type IV, IV (combined hepatic and myopathic), type V, and type VI; Goldmann-Favre syndrome; Gordon syndrome; Gorlin syndrome; Holoprosencephaly sequence; Holoprosencephaly 7; Granulomatous disease, chronic, X-linked, variant; Granulosa cell tumor of the ovary; Gray platelet syndrome; Griscelli syndrome type 3; Groenouw corneal dystrophy type I; Growth and mental retardation, mandibulofacial dysostosis, microcephaly, and cleft palate; Growth hormone deficiency with pituitary anomalies; Growth hormone insensitivity with immunodeficiency; GTP cyclohydrolase I deficiency; Hajdu-Cheney syndrome; Hand foot uterus syndrome; Hearing impairment; Hemangioma, capillary infantile; Hematologic neoplasm; Hemochromatosis type 1, 2B, and 3; Microvascular complications of diabetes 7; Transferrin serum level quantitative trait locus 2; Hemoglobin H disease, nondeletional; Hemolytic anemia, nonspherocytic, due to glucose phosphate isomerase deficiency; Hemophagocytic lymphohistiocytosis, familial, 2; Hemophagocytic lymphohistiocytosis, familial, 3; Heparin cofactor II deficiency; Hereditary acrodermatitis enteropathica; Hereditary breast and ovarian cancer syndrome; Ataxia-telangiectasia-like disorder; Hereditary diffuse gastric cancer; Hereditary diffuse leukoencephalopathy with spheroids; Hereditary factors II, IX, VIII deficiency disease; Hereditary hemorrhagic telangiectasia type 2; Hereditary insensitivity to pain with anhidrosis; Hereditary lymphedema type I; Hereditary motor and sensory neuropathy with optic atrophy; Hereditary myopathy with early respiratory failure; Hereditary neuralgic amyotrophy; Hereditary Nonpolyposis Colorectal Neoplasms; Lynch syndrome I and II; Hereditary pancreatitis; Pancreatitis, chronic, susceptibility to; Hereditary sensory and autonomic neuropathy type IIB amd IIA; Hereditary sideroblastic anemia; Hermansky-Pudlak syndrome 1, 3, 4, and 6; Heterotaxy, visceral, 2, 4, and 6, autosomal; Heterotaxy, visceral, X-linked; Heterotopia; Histiocytic medullary reticulosis; Histiocytosis-lymphadenopathy plus syndrome; Holocarboxylase synthetase deficiency; Holoprosencephaly 2, 3, 7, and 9; Holt-Oram syndrome; Homocysteinemia due to MTHFR deficiency, CBS deficiency, and Homocystinuria, pyridoxine-responsive; Homocystinuria-Megaloblastic anemia due to defect in cobalamin metabolism, cblE complementation type; Howel-Evans syndrome; Hurler syndrome; Hutchinson-Gilford syndrome; Hydrocephalus; Hyperammonemia, type III; Hypercholesterolaemia and Hypercholesterolemia, autosomal recessive; Hyperekplexia 2 and Hyperekplexia hereditary; Hyperferritinemia cataract syndrome; Hyperglycinuria; Hyperimmunoglobulin D with periodic fever; Mevalonic aciduria; Hyperimmunoglobulin E syndrome; Hyperinsulinemic hypoglycemia familial 3, 4, and 5; Hyperinsulinism-hyperammonemia syndrome; Hyperlysinemia; Hypermanganesemia with dystonia, polycythemia and cirrhosis; Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome; Hyperparathyroidism 1 and 2; Hyperparathyroidism, neonatal severe; Hyperphenylalaninemia, bh4-deficient, a, due to partial pts deficiency, BH4-deficient, D, and non-pku; Hyperphosphatasia with mental retardation syndrome 2, 3, and 4; Hypertrichotic osteochondrodysplasia; Hypobetalipoproteinemia, familial, associated with apob32; Hypocalcemia, autosomal dominant 1; Hypocalciuric hypercalcemia, familial, types 1 and 3; Hypochondrogenesis; Hypochromic microcytic anemia with iron overload; Hypoglycemia with deficiency of glycogen synthetase in the liver; Hypogonadotropic hypogonadism 11 with or without anosmia; Hypohidrotic ectodermal dysplasia with immune deficiency; Hypohidrotic X-linked ectodermal dysplasia; Hypokalemic periodic paralysis 1 and 2; Hypomagnesemia 1, intestinal; Hypomagnesemia, seizures, and mental retardation; Hypomyelinating leukodystrophy 7; Hypoplastic left heart syndrome; Atrioventricular septal defect and common atrioventricular junction; Hypospadias 1 and 2, X-linked; Hypothyroidism, congenital, nongoitrous, 1; Hypotrichosis 8 and 12; Hypotrichosis-lymphedema-telangiectasia syndrome; I blood group system; Ichthyosis bullosa of Siemens; Ichthyosis exfoliativa; Ichthyosis prematurity syndrome; Idiopathic basal ganglia calcification 5; Idiopathic fibrosing alveolitis, chronic form; Dyskeratosis congenita, autosomal dominant, 2 and 5; Idiopathic hypercalcemia of infancy; Immune dysfunction with T-cell inactivation due to calcium entry defect 2; Immunodeficiency 15, 16, 19, 30, 31C, 38, 40, 8, due to defect in cd3-zeta, with hyper IgM type I and 2, and X-Linked, with magnesium defect, Epstein-Barr virus infection, and neoplasia; Immunodeficiency-centromeric instability-facial anomalies syndrome 2; Inclusion body myopathy 2 and 3; Nonaka myopathy; Infantile convulsions and paroxysmal choreoathetosis, familial; Infantile cortical hyperostosis; Infantile GM1 gangliosidosis; Infantile hypophosphatasia; Infantile nephronophthisis; Infantile nystagmus, X-linked; Infantile Parkinsonism-dystonia; Infertility associated with multi-tailed spermatozoa and excessive DNA; Insulin resistance; Insulin-resistant diabetes mellitus and acanthosis nigricans; Insulin-dependent diabetes mellitus secretory diarrhea syndrome; Interstitial nephritis, karyomegalic; Intrauterine growth retardation, metaphyseal dysplasia, adrenal hypoplasia congenita, and genital anomalies; Iodotyrosyl coupling defect; IRAK4 deficiency; Iridogoniodysgenesis dominant type and type 1; Iron accumulation in brain; Ischiopatellar dysplasia; Islet cell hyperplasia; Isolated 17,20-lyase deficiency; Isolated lutropin deficiency; Isovaleryl-CoA dehydrogenase deficiency; Jankovic Rivera syndrome; Jervell and Lange-Nielsen syndrome 2; Joubert syndrome 1, 6, 7, 9/15 (digenic), 14, 16, and 17, and Orofaciodigital syndrome xiv; Junctional epidermolysis bullosa gravis of Herlitz; Juvenile GM>1<gangliosidosis; Juvenile polyposis syndrome; Juvenile polyposis/hereditary hemorrhagic telangiectasia syndrome; Juvenile retinoschisis; Kabuki make-up syndrome; Kallmann syndrome 1, 2, and 6; Delayed puberty; Kanzaki disease; Karak syndrome; Kartagener syndrome; Kenny-Caffey syndrome type 2; Keppen-Lubinsky syndrome; Keratoconus 1; Keratosis follicularis; Keratosis palmoplantaris striata 1; Kindler syndrome; L-2-hydroxyglutaric aciduria; Larsen syndrome, dominant type; Lattice corneal dystrophy Type III; Leber amaurosis; Zellweger syndrome; Peroxisome biogenesis disorders; Zellweger syndrome spectrum; Leber congenital amaurosis 11, 12, 13, 16, 4, 7, and 9; Leber optic atrophy; Aminoglycoside-induced deafness; Deafness, nonsyndromic sensorineural, mitochondrial; Left ventricular noncompaction 5; Left-right axis malformations; Leigh disease; Mitochondrial short-chain Enoyl-CoA Hydratase 1 deficiency; Leigh syndrome due to mitochondrial complex I deficiency; Leiner disease; Leri Weill dyschondrosteosis; Lethal congenital contracture syndrome 6; Leukocyte adhesion deficiency type I and III; Leukodystrophy, Hypomyelinating, 11 and 6; Leukoencephalopathy with ataxia, with Brainstem and Spinal Cord Involvement and Lactate Elevation, with vanishing white matter, and progressive, with ovarian failure; Leukonychia totalis; Lewy body dementia; Lichtenstein-Knon Syndrome; Li-Fraumeni syndrome 1; Lig4 syndrome; Limb-girdle muscular dystrophy, type 1B, 2A, 2B, 2D, Cl, C5, C9, C14; Congenital muscular dystrophy-dystroglycanopathy with brain and eye anomalies, type A14 and B14; Lipase deficiency combined; Lipid proteinosis; Lipodystrophy, familial partial, type 2 and 3; Lissencephaly 1, 2 (X-linked), 3, 6 (with microcephaly), X-linked; Subcortical laminar heterotopia, X-linked; Liver failure acute infantile; Loeys-Dietz syndrome 1, 2, 3; Long QT syndrome 1, 2, 2/9, 2/5, (digenic), 3, 5 and 5, acquired, susceptibility to; Lung cancer; Lymphedema, hereditary, id; Lymphedema, primary, with myelodysplasia; Lymphoproliferative syndrome 1, 1 (X-linked), and 2; Lysosomal acid lipase deficiency; Macrocephaly, macrosomia, facial dysmorphism syndrome; Macular dystrophy, vitelliform, adult-onset; Malignant hyperthermia susceptibility type 1; Malignant lymphoma, non-Hodgkin; Malignant melanoma; Malignant tumor of prostate; Mandibuloacral dysostosis; Mandibuloacral dysplasia with type A or B lipodystrophy, atypical; Mandibulofacial dysostosis, Treacher Collins type, autosomal recessive; Mannose-binding protein deficiency; Maple syrup urine disease type 1A and type 3; Marden Walker like syndrome; Marfan syndrome; Marinesco-Sj\xc3∴xb6gren syndrome; Martsolf syndrome; Maturity-onset diabetes of the young, type 1, type 2, type 11, type 3, and type 9; May-Hegglin anomaly; MYH9 related disorders; Sebastian syndrome; McCune-Albright syndrome; Somatotroph adenoma; Sex cord-stromal tumor; Cushing syndrome; McKusick Kaufman syndrome; McLeod neuroacanthocytosis syndrome; Meckel-Gruber syndrome; Medium-chain acyl-coenzyme A dehydrogenase deficiency; Medulloblastoma; Megalencephalic leukoencephalopathy with subcortical cysts 1 and 2a; Megalencephaly Cutis marmorata telangiectatica congenital; PIK3CA Related Overgrowth Spectrum; Megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome 2; Megaloblastic anemia, thiamine-responsive, with diabetes mellitus and sensorineural deafness; Meier-Gorlin syndromes land 4; Melnick-Needles syndrome; Meningioma; Mental retardation, X-linked, 3, 21, 30, and 72; Mental retardation and microcephaly with pontine and cerebellar hypoplasia; Mental retardation X-linked syndromic 5; Mental retardation, anterior maxillary protrusion, and strabismus; Mental retardation, autosomal dominant 12, 13, 15, 24, 3, 30, 4, 5, 6, and 9; Mental retardation, autosomal recessive 15, 44, 46, and 5; Mental retardation, stereotypic movements, epilepsy, and/or cerebral malformations; Mental retardation, syndromic, Claes-Jensen type, X-linked; Mental retardation, X-linked, nonspecific, syndromic, Hedera type, and syndromic, wu type; Merosin deficient congenital muscular dystrophy; Metachromatic leukodystrophy juvenile, late infantile, and adult types; Metachromatic leukodystrophy; Metatrophic dysplasia; Methemoglobinemia types I and 2; Methionine adenosyltransferase deficiency, autosomal dominant; Methylmalonic acidemia with homocystinuria; Methylmalonic aciduria cblB type; Methylmalonic aciduria due to methylmalonyl-CoA mutase deficiency; METHYLMALONIC ACIDURIA, mut(0) TYPE; Microcephalic osteodysplastic primordial dwarfism type 2; Microcephaly with or without chorioretinopathy, lymphedema, or mental retardation; Microcephaly, hiatal hernia and nephrotic syndrome; Microcephaly; Hypoplasia of the corpus callosum; Spastic paraplegia 50, autosomal recessive; Global developmental delay; CNS hypomyelination; Brain atrophy; Microcephaly, normal intelligence and immunodeficiency; Microcephaly-capillary malformation syndrome; Microcytic anemia; Microphthalmia syndromic 5, 7, and 9; Microphthalmia, isolated 3, 5, 6, 8, and with coloboma 6; Microspherophakia; Migraine, familial basilar; Miller syndrome; Minicore myopathy with external ophthalmoplegia; Myopathy, congenital with cores; Mitchell-Riley syndrome; mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency; Mitochondrial complex I, II, III, III (nuclear type 2, 4, or 8) deficiency; Mitochondrial DNA depletion syndrome 11, 12 (cardiomyopathic type), 2, 4B (MNGIE type), 8B (MNGIE type); Mitochondrial DNA-depletion syndrome 3 and 7, hepatocerebral types, and 13 (encephalomyopathic type); Mitochondrial phosphate carrier and pyruvate carrier deficiency; Mitochondrial trifunctional protein deficiency; Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency; Miyoshi muscular dystrophy 1; Myopathy, distal, with anterior tibial onset; Mohr-Tranebjaerg syndrome; Molybdenum cofactor deficiency, complementation group A; Mowat-Wilson syndrome; Mucolipidosis III Gamma; Mucopolysaccharidosis type VI, type VI (severe), and type VII; Mucopolysaccharidosis, MPS-I-H/S, MPS-II, MPS-III-A, MPS-III-B, MPS-III-C, MPS-IV-A, MPS-IV-B; Retinitis Pigmentosa 73; Gangliosidosis GM1 type1 (with cardiac involvement) 3; Multicentric osteolysis nephropathy; Multicentric osteolysis, nodulosis and arthropathy; Multiple congenital anomalies; Atrial septal defect 2; Multiple congenital anomalies-hypotonia-seizures syndrome 3; Multiple Cutaneous and Mucosal Venous Malformations; Multiple endocrine neoplasia, types land 4; Multiple epiphyseal dysplasia 5 or Dominant; Multiple gastrointestinal atresias; Multiple pterygium syndrome Escobar type; Multiple sulfatase deficiency; Multiple synostoses syndrome 3; Muscle AMP guanine oxidase deficiency; Muscle eye brain disease; Muscular dystrophy, congenital, megaconial type; Myasthenia, familial infantile, 1; Myasthenic Syndrome, Congenital, 11, associated with acetylcholine receptor deficiency; Myasthenic Syndrome, Congenital, 17, 2A (slow-channel), 4B (fast-channel), and without tubular aggregates; Myeloperoxidase deficiency; MYH-associated polyposis; Endometrial carcinoma; Myocardial infarction 1; Myoclonic dystonia; Myoclonic-Atonic Epilepsy; Myoclonus with epilepsy with ragged red fibers; Myofibrillar myopathy 1 and ZASP-related; Myoglobinuria, acute recurrent, autosomal recessive; Myoneural gastrointestinal encephalopathy syndrome; Cerebellar ataxia infantile with progressive external ophthalmoplegia; Mitochondrial DNA depletion syndrome 4B, MNGIE type; Myopathy, centronuclear, 1, congenital, with excess of muscle spindles, distal, 1, lactic acidosis, and sideroblastic anemia 1, mitochondrial progressive with congenital cataract, hearing loss, and developmental delay, and tubular aggregate, 2; Myopia 6; Myosclerosis, autosomal recessive; Myotonia congenital; Congenital myotonia, autosomal dominant and recessive forms; Nail-patella syndrome; Nance-Horan syndrome; Nanophthalmos 2; Navajo neurohepatopathy; Nemaline myopathy 3 and 9; Neonatal hypotonia; Intellectual disability; Seizures; Delayed speech and language development; Mental retardation, autosomal dominant 31; Neonatal intrahepatic cholestasis caused by citrin deficiency; Nephrogenic diabetes insipidus, Nephrogenic diabetes insipidus, X-linked; Nephrolithiasis/osteoporosis, hypophosphatemic, 2; Nephronophthisis 13, 15 and 4; Infertility; Cerebello-oculo-renal syndrome (nephronophthisis, oculomotor apraxia and cerebellar abnormalities); Nephrotic syndrome, type 3, type 5, with or without ocular abnormalities, type 7, and type 9; Nestor-Guillermo progeria syndrome; Neu-Laxova syndrome 1; Neurodegeneration with brain iron accumulation 4 and 6; Neuroferritinopathy; Neurofibromatosis, type 1 and type 2; Neurofibrosarcoma; Neurohypophyseal diabetes insipidus; Neuropathy, Hereditary Sensory, Type IC; Neutral 1 amino acid transport defect; Neutral lipid storage disease with myopathy; Neutrophil immunodeficiency syndrome; Nicolaides-Baraitser syndrome; Niemann-Pick disease type C1, C2, type A, and type Cl, adult form; Non-ketotic hyperglycinemia; Noonan syndrome 1 and 4, LEOPARD syndrome 1; Noonan syndrome-like disorder with or without juvenile myelomonocytic leukemia; Normokalemic periodic paralysis, potassium-sensitive; Norum disease; Epilepsy, Hearing Loss, And Mental Retardation Syndrome; Mental Retardation, X-Linked 102 and syndromic 13; Obesity; Ocular albinism, type I; Oculocutaneous albinism type 1B, type 3, and type 4; Oculodentodigital dysplasia; Odontohypophosphatasia; Odontotrichomelic syndrome; Oguchi disease; Oligodontia-colorectal cancer syndrome; Opitz G/BBB syndrome; Optic atrophy 9; Oral-facial-digital syndrome; Ornithine aminotransferase deficiency; Orofacial cleft 11 and 7, Cleft lip/palate-ectodermal dysplasia syndrome; Orstavik Lindemann Solberg syndrome; Osteoarthritis with mild chondrodysplasia; Osteochondritis dissecans; Osteogenesis imperfecta type 12, type 5, type 7, type 8, type I, type III, with normal sclerae, dominant form, recessive perinatal lethal; Osteopathia striata with cranial sclerosis, Osteopetrosis autosomal dominant type 1 and 2, recessive 4, recessive 1, recessive 6; Osteoporosis with pseudoglioma; Oto-palato-digital syndrome, types I and H; Ovarian dysgenesis 1; Ovarioleukodystrophy; Pachyonychia congenita 4 and type 2; Paget disease of bone, familial; Pallister-Hall syndrome; Palmoplantar keratoderma, nonepidermolytic, focal or diffuse; Pancreatic agenesis and congenital heart disease; Papillon-Lef\xc3\xa8vre syndrome; Paragangliomas 3; Paramyotonia congenita of von Eulenburg; Parathyroid carcinoma; Parkinson disease 14, 15, 19 (juvenile-onset), 2, 20 (early-onset), 6, (autosomal recessive early-onset, and 9; Partial albinism; Partial hypoxanthine-guanine phosphoribosyltransferase deficiency; Patterned dystrophy of retinal pigment epithelium; PC-K6a; Pelizaeus-Merzbacher disease; Pendred syndrome; Peripheral demyelinating neuropathy, central dysmyelination; Hirschsprung disease; Permanent neonatal diabetes mellitus; Diabetes mellitus, permanent neonatal, with neurologic features; Neonatal insulin-dependent diabetes mellitus; Maturity-onset diabetes of the young, type 2; Peroxisome biogenesis disorder 14B, 2A, 4A, 5B, 6A, 7A, and 7B; Perrault syndrome 4; Perry syndrome; Persistent hyperinsulinemic hypoglycemia of infancy; familial hyperinsulinism; Phenotypes; Phenylketonuria; Pheochromocytoma; Hereditary Paraganglioma-Pheochromocytoma Syndromes; Paragangliomas 1; Carcinoid tumor of intestine; Cowden syndrome 3; Phosphoglycerate dehydrogenase deficiency; Phosphoglycerate kinase 1 deficiency; Photosensitive trichothiodystrophy; Phytanic acid storage disease; Pick disease; Pierson syndrome; Pigmentary retinal dystrophy; Pigmented nodular adrenocortical disease, primary, 1; Pilomatrixoma; Pitt-Hopkins syndrome; Pituitary dependent hypercortisolism; Pituitary hormone deficiency, combined 1, 2, 3, and 4; Plasminogen activator inhibitor type 1 deficiency; Plasminogen deficiency, type I; Platelet-type bleeding disorder 15 and 8; Poikiloderma, hereditary fibrosing, with tendon contractures, myopathy, and pulmonary fibrosis; Polycystic kidney disease 2, adult type, and infantile type; Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy; Polyglucosan body myopathy 1 with or without immunodeficiency; Polymicrogyria, asymmetric, bilateral frontoparietal; Polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract; Pontocerebellar hypoplasia type 4; Popliteal pterygium syndrome; Porencephaly 2; Porokeratosis 8, disseminated superficial actinic type; Porphobilinogen synthase deficiency; Porphyria cutanea tarda; Posterior column ataxia with retinitis pigmentosa; Posterior polar cataract type 2; Prader-Willi-like syndrome; Premature ovarian failure 4, 5, 7, and 9; Primary autosomal recessive microcephaly 10, 2, 3, and 5; Primary ciliary dyskinesia 24; Primary dilated cardiomyopathy; Left ventricular noncompaction 6; 4, Left ventricular noncompaction 10; Paroxysmal atrial fibrillation; Primary hyperoxaluria, type I, type, and type III; Primary hypertrophic osteoarthropathy, autosomal recessive 2; Primary hypomagnesemia; Primary open angle glaucoma juvenile onset 1; Primary pulmonary hypertension; Primrose syndrome; Progressive familial heart block type 1B; Progressive familial intrahepatic cholestasis 2 and 3; Progressive intrahepatic cholestasis; Progressive myoclonus epilepsy with ataxia; Progressive pseudorheumatoid dysplasia; Progressive sclerosing poliodystrophy; Prolidase deficiency; Proline dehydrogenase deficiency; Schizophrenia 4; Properdin deficiency, X-linked; Propionic academia; Proprotein convertase 1/3 deficiency; Prostate cancer, hereditary, 2; Protan defect; Proteinuria; Finnish congenital nephrotic syndrome; Proteus syndrome; Breast adenocarcinoma; Pseudoachondroplastic spondyloepiphyseal dysplasia syndrome; Pseudohypoaldosteronism type 1 autosomal dominant and recessive and type 2; Pseudohypoparathyroidism type 1A, Pseudopseudohypoparathyroidism; Pseudoneonatal adrenoleukodystrophy; Pseudoprimary hyperaldosteronism; Pseudoxanthoma elasticum; Generalized arterial calcification of infancy 2; Pseudoxanthoma elasticum-like disorder with multiple coagulation factor deficiency; Psoriasis susceptibility 2; PTEN hamartoma tumor syndrome; Pulmonary arterial hypertension related to hereditary hemorrhagic telangiectasia; Pulmonary Fibrosis And/Or Bone Marrow Failure, Telomere-Related, 1 and 3; Pulmonary hypertension, primary, 1, with hereditary hemorrhagic telangiectasia; Purine-nucleoside phosphorylase deficiency; Pyruvate carboxylase deficiency; Pyruvate dehydrogenase E1-alpha deficiency; Pyruvate kinase deficiency of red cells; Raine syndrome; Rasopathy; Recessive dystrophic epidermolysis bullosa; Nail disorder, nonsyndromic congenital, 8; Reifenstein syndrome; Renal adysplasia; Renal carnitine transport defect; Renal coloboma syndrome; Renal dysplasia; Renal dysplasia, retinal pigmentary dystrophy, cerebellar ataxia and skeletal dysplasia; Renal tubular acidosis, distal, autosomal recessive, with late-onset sensorineural hearing loss, or with hemolytic anemia; Renal tubular acidosis, proximal, with ocular abnormalities and mental retardation; Retinal cone dystrophy 3B; Retinitis pigmentosa; Retinitis pigmentosa 10, 11, 12, 14, 15, 17, and 19; Retinitis pigmentosa 2, 20, 25, 35, 36, 38, 39, 4, 40, 43, 45, 48, 66, 7, 70, 72; Retinoblastoma; Rett disorder; Rhabdoid tumor predisposition syndrome 2; Rhegmatogenous retinal detachment, autosomal dominant; Rhizomelic chondrodysplasia punctata type 2 and type 3; Roberts-SC phocomelia syndrome; Robinow Sorauf syndrome; Robinow syndrome, autosomal recessive, autosomal recessive, with brachy-syn-polydactyly; Rothmund-Thomson syndrome; Rapadilino syndrome; RRM2B-related mitochondrial disease; Rubinstein-Taybi syndrome; Salla disease; Sandhoff disease, adult and infantil types; Sarcoidosis, early-onset; Blau syndrome; Schindler disease, type 1; Schizencephaly; Schizophrenia 15; Schneckenbecken dysplasia; Schwannomatosis 2; Schwartz Jampel syndrome type 1; Sclerocornea, autosomal recessive; Sclerosteosis; Secondary hypothyroidism; Segawa syndrome, autosomal recessive; Senior-Loken syndrome 4 and 5; Sensory ataxic neuropathy, dysarthria, and ophthalmoparesis; Sepiapterin reductase deficiency; SeSAME syndrome; Severe combined immunodeficiency due to ADA deficiency, with microcephaly, growth retardation, and sensitivity to ionizing radiation, atypical, autosomal recessive, T cell-negative, B cell-positive, NK cell-negative of NK-positive; Severe congenital neutropenia; Severe congenital neutropenia 3, autosomal recessive or dominant; Severe congenital neutropenia and 6, autosomal recessive; Severe myoclonic epilepsy in infancy; Generalized epilepsy with febrile seizures plus, types 1 and 2; Severe X-linked myotubular myopathy; Short QT syndrome 3; Short stature with nonspecific skeletal abnormalities; Short stature, auditory canal atresia, mandibular hypoplasia, skeletal abnormalities; Short stature, onychodysplasia, facial dysmorphism, and hypotrichosis; Primordial dwarfism; Short-rib thoracic dysplasia 11 or 3 with or without polydactyly; Sialidosis type I and II; Silver spastic paraplegia syndrome; Slowed nerve conduction velocity, autosomal dominant; Smith-Lemli-Opitz syndrome; Snyder Robinson syndrome; Somatotroph adenoma; Prolactinoma; familial, Pituitary adenoma predisposition; Sotos syndrome 1 or 2; Spastic ataxia 5, autosomal recessive, Charlevoix-Saguenay type, 1, 10, or 11, autosomal recessive; Amyotrophic lateral sclerosis type 5; Spastic paraplegia 15, 2, 3, 35, 39, 4, autosomal dominant, 55, autosomal recessive, and 5A; Bile acid synthesis defect, congenital, 3; Spermatogenic failure 11, 3, and 8; Spherocytosis types 4 and 5; Spheroid body myopathy; Spinal muscular atrophy, lower extremity predominant 2, autosomal dominant; Spinal muscular atrophy, type II; Spinocerebellar ataxia 14, 21, 35, 40, and 6; Spinocerebellar ataxia autosomal recessive 1 and 16; Splenic hypoplasia; Spondylocarpotarsal synostosis syndrome; Spondylocheirodysplasia, Ehlers-Danlos syndrome-like, with immune dysregulation, Aggrecan type, with congenital joint dislocations, short limb-hand type, Sedaghatian type, with cone-rod dystrophy, and Kozlowski type; Parastremmatic dwarfism; Stargardt disease 1; Cone-rod dystrophy 3; Stickler syndrome type 1; Kniest dysplasia; Stickler syndrome, types 1 (nonsyndromic ocular) and 4; Sting-associated vasculopathy, infantile-onset; Stormorken syndrome; Sturge-Weber syndrome, Capillary malformations, congenital, 1; Succinyl-CoA acetoacetate transferase deficiency; Sucrase-isomaltase deficiency; Sudden infant death syndrome; Sulfite oxidase deficiency, isolated; Supravalvar aortic stenosis; Surfactant metabolism dysfunction, pulmonary, 2 and 3; Symphalangism, proximal, 1b; Syndactyly Cenani Lenz type; Syndactyly type 3; Syndromic X-linked mental retardation 16; Talipes equinovarus; Tangier disease; TARP syndrome; Tay-Sachs disease, B1 variant, Gm2-gangliosidosis (adult), Gm2-gangliosidosis (adult-onset); Temtamy syndrome; Tenorio Syndrome; Terminal osseous dysplasia; Testosterone 17-beta-dehydrogenase deficiency; Tetraamelia, autosomal recessive; Tetralogy of Fallot; Hypoplastic left heart syndrome 2; Truncus arteriosus; Malformation of the heart and great vessels; Ventricular septal defect 1; Thiel-Behnke corneal dystrophy; Thoracic aortic aneurysms and aortic dissections; Marfanoid habitus; Three M syndrome 2; Thrombocytopenia, platelet dysfunction, hemolysis, and imbalanced globin synthesis; Thrombocytopenia, X-linked; Thrombophilia, hereditary, due to protein C deficiency, autosomal dominant and recessive; Thyroid agenesis; Thyroid cancer, follicular; Thyroid hormone metabolism, abnormal; Thyroid hormone resistance, generalized, autosomal dominant; Thyrotoxic periodic paralysis and Thyrotoxic periodic paralysis 2; Thyrotropin-releasing hormone resistance, generalized; Timothy syndrome; TNF receptor-associated periodic fever syndrome (TRAPS); Tooth agenesis, selective, 3 and 4; Torsades de pointes; Townes-Brocks-branchiootorenal-like syndrome; Transient bullous dermolysis of the newborn; Treacher collins syndrome 1; Trichomegaly with mental retardation, dwarfism and pigmentary degeneration of retina; Trichorhinophalangeal dysplasia type I; Trichorhinophalangeal syndrome type 3; Trimethylaminuria; Tuberous sclerosis syndrome; Lymphangiomyomatosis; Tuberous sclerosis 1 and 2; Tyrosinase-negative oculocutaneous albinism; Tyrosinase-positive oculocutaneous albinism; Tyrosinemia type I; UDPglucose-4-epimerase deficiency; Ullrich congenital muscular dystrophy; Ulna and fibula absence of with severe limb deficiency; Upshaw-Schulman syndrome; Urocanate hydratase deficiency; Usher syndrome, types 1, 1B, 1D, 1G, 2A, 2C, and 2D; Retinitis pigmentosa 39; UV-sensitive syndrome; Van der Woude syndrome; Van Maldergem syndrome 2; Hennekam lymphangiectasia-lymphedema syndrome 2; Variegate porphyria; Ventriculomegaly with cystic kidney disease; Verheij syndrome; Very long chain acyl-CoA dehydrogenase deficiency; Vesicoureteral reflux 8; Visceral heterotaxy 5, autosomal; Visceral myopathy; Vitamin D-dependent rickets, types land 2; Vitelliform dystrophy; von Willebrand disease type 2M and type 3; Waardenburg syndrome type 1, 4C, and 2E (with neurologic involvement); Klein-Waardenberg syndrome; Walker-Warburg congenital muscular dystrophy; Warburg micro syndrome 2 and 4; Warts, hypogammaglobulinemia, infections, and myelokathexis; Weaver syndrome; Weill-Marchesani syndrome 1 and 3; Weill-Marchesani-like syndrome; Weissenbacher-Zweymuller syndrome; Werdnig-Hoffmann disease; Charcot-Marie-Tooth disease; Werner syndrome; WFS1-Related Disorders; Wiedemann-Steiner syndrome; Wilson disease, Wolfram-like syndrome, autosomal dominant; Worth disease; Van Buchem disease type 2; Xeroderma pigmentosum, complementation group b, group D, group E, and group G; X-linked agammaglobulinemia; X-linked hereditary motor and sensory neuropathy; X-linked ichthyosis with steryl-sulfatase deficiency; X-linked periventricular heterotopia; Oto-palato-digital syndrome, type I; X-linked severe combined immunodeficiency; Zimmermann-Laband syndrome and Zimmermann-Laband syndrome 2; and Zonular pulverulent cataract 3. Reference is made to PCT Publication No. WO2020/191249A1, the entirety of which is incorporated by reference herein.
Globisporangium
ultimum DAOM
Globisporangium
ultimum DAOM
Globisporangium
ultimum DAOM
Globisporangium
ultimum DAOM
Globisporangium
ultimum DAOM
Globisporangium
ultimum DAOM
Globisporangium
ultimum DAOM
Globisporangium
ultimum DAOM
Globisporangium
ultimum DAOM
Globisporangium
ultimum DAOM
Mayetiola destructor
Mayetiola destructor
Mayetiola destructor
Mayetiola destructor
Mayetiola destructor
Mayetiola destructor
Mayetiola destructor
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Allomyces
macrogynus ATCC
Allomyces
macrogynus ATCC
Allomyces
macrogynus ATCC
Ectocarpus siliculosus
Ectocarpus siliculosus
Phytophthora lateralis
Phytophthora lateralis
Phytophthora cryptogea
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Rhizomucor miehei
Rhizomucor miehei
Phytophthora rubi
Klebsormidium nitens
Podila verticillata
Phytophthora pisi
Schizochytrium sp.
Acanthamoeba
astronyxis
Acanthamoeba
astronyxis
Acanthamoeba
royreba
Acanthamoeba
royreba
Acanthamoeba
divionensis
Acanthamoeba
divionensis
Acanthamoeba quina
Picochlorum sp.
Picochlorum sp.
Picochlorum sp.
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Tilleta horrida
Sporisorium
scitamineum
Balamuthia
mandrillaris
Balamuthia
mandrillaris
Balamuthia
mandrillaris
Balamuthia
mandrillaris
Balamuthia
mandrillaris
Balamuthia
mandrillaris
Balamuthia
mandrillaris
Balamuthia
mandrillaris
Balamuthia
mandrillaris
Balamuthia
mandrillaris
Balamuthia
mandrillaris
Balamuthia
mandrillaris
Balamuthia
mandrillaris
Balamuthia
mandrillaris
Balamuthia
mandrillaris
Balamuthia
mandrillaris
Balamuthia
mandrillaris
Balamuthia
mandrillaris
Balamuthia
mandrillaris
Balamuthia
mandrillaris
Phytophthora
multivora
Phytophthora
multivora
Phytophthora
multivora
Phytophthora
multivora
Phylophthora taxon
totara
Phytophthora
agathidicida
Phytophthora
agathidicida
Phytophthora
agathidicida
Aurantiochytrium sp.
Aurantiochytrium sp.
Aurantiochytrium sp.
Aurantiochytrium sp.
Aurantiochytrium sp.
Gonapodya prolifera
Gonapodya prolifera
Gonapodya prolifera
Gonapodya prolifera
Gonapodya prolifera
Gonapodya prolifera
Gonapodya prolifera
Gonapodya prolifera
Parhyale hawaiensis
Parhyale hawaiensis
Parhyale hawaiensis
Parhyale hawaiensis
Parhyale hawaiensis
Cystobasidium
pallidum
Meira nashicola
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Pilasporangium
apinafurcum
Chlamydomonas
asymmetrica
Chlamydomonas
asymmetrica
Chlamydomonas
asymmetrica
Chlamydomonas
asymmetrica
Chlamydomonas
sphaeroides
Chlamydomonas
sphaeroides
Tilletia indica
Tilletia indica
Tilletia indica
Tilletia indica
Tilletia indica
Tilletia indica
Nothophytophthora
Nothophytophthora
Nothophytophthora
Nothophytophthora
Nothophytophthora
Nothophytophthora
Cladosiphon
okamuranus
Cladosiphon
okamuranus
Cladosiphon
okamuranus
Cladosiphon
okamuranus
Cladosiphon
okamuranus
Byssochlamys sp. IMV
Byssochlamys sp. IMV
Erythrobasidium
hasegawianum
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Trebouxia sp.
Trebouxia sp.
Trebouxia sp.
Trebouxia sp.
Rhizophlyctis rosea
Phytophthora plurivora
Phytophthora plurivora
Bifiguratus adelaidae
Bifiguratus adelaidae
Dunaliella salina
Dunaliella salina
Dunaliella salina
Notospermus
geniculatus
Notospermus
geniculatus
Coemansia reversa
Coemansia reversa
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Prototheca stagnorum
Prototheca stagnorum
Prototheca stagnorum
Tetrabaena socialis
Prototheca cutis
Prototheca cutis
Prototheca cutis
Prototheca cutis
Aurantiochytrium sp.
Aurantiochytrium sp.
Aurantiochytrium sp.
Aurantiochytrium sp.
Aurantiochytrium sp.
Aurantiochytrium sp.
Aurantiochytrium sp.
Aurantiochytrium sp.
Eudorina sp. 2006-
Raphidocelis
subcapitala
Raphidocelis
subcapitala
Raphidocelis
subcapitala
Raphidocelis
subcapitala
Raphidocelis
subcapitala
Phytophthora
nicotianae
Phytophthora
nicotianae
Phytophthora
nicotianae
Phytophthora
nicotianae
Phytophthora
nicotianae
Diversispora epigaea
Gigaspora rosea
Caulochytrium
protostelioides
Caulochytrium
protostelioides
Caulochytrium
protostelioides
Caulochytrium
protostelioides
Caulochytrium
protostelioides
Caulochytrium
protostelioides
Caulochytrium
protostelioides
Caulochytrium
protostelioides
Caulochytrium
protostelioides
Caulochytrium
protostelioides
Caulochytrium
protostelioides
Caulochytrium
protostelioides
Caulochytrium
protostelioides
Caulochytrium
protostelioides
Caulochytrium
protostelioides
Zygotorulaspora
florentina
Torulaspora
franciscae
Torulaspora
franciscae
Torulaspora
franciscae
Lipomyces
mesembrius
Lipomyces
mesembrius
Lipomyces
mesembrius
Lipomyces
mesembrius
Lipomyces
mesembrius
Lipomyces
mesembrius
Lipomyces sp. NRRL
Lipomyces sp NRRL
Lipomyces sp. NRRL
Lipomyces sp. NRRL
Lipomyces arxii
Lipomyces arxii
Lipomyces arxii
Brevipaipus yothers
Pseudoperonospora
humuli
Acaulopage tetraceros
Nannochloris sp RS
Haematococcus sp.
Chloroidium sp. JM
Chloroidium sp. JM
Chloroidium sp. CF
Chloroidium sp. CF
Chloromonas sp.
Chloromonas sp
Chloromonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Phocoena phocoena
Nannochloropsis
oceanica
Nannochloropsis
oceanica
Nannochloropsis
oceanica
Nannochloropsis
oceanica
Nannochloropsis
oceanica
Ectocarpus sp. Ec32
Ectocarpus sp. Ec32
Schizochytrium sp.
Schizochytrium sp.
Schizochytrium sp.
Schizochytrium sp.
Schizochytrium sp.
Schizochytrium sp.
Schizochytrium sp.
Schizochytrium sp.
Schizochytrium sp.
Schizochytrium sp.
Schizochytrium sp.
Schizochytrium sp.
Schizochytrium sp.
Schizochytrium sp.
Schizochytrium sp.
Schizochytrium sp.
Schizochytrium sp.
Schizochytrium sp.
Schizochytrium sp.
Schizochytrium sp.
Schizochytrium sp.
Globisporangium
splendens
Globisporangium
splendens
Stentor roeselii
Pseudokeronopsis
carnea
Pseudokeronopsis
carnea
Synchytrium
endobioticum
Synchytrium
endobioticum
Chytnomyces
confervae
Powellomyces hirtus
Powellomyces hirtus
Powellomyces hirtus
Dreissena rostriformis
Dreissena rostriformis
Neoporphyra
haitanensis
Neoporphyra
haitanensis
Neoporphyra
haitanensis
Neoporphyra
haitanensis
Neoporphyra
haitanensis
Neoporphyra
haitanensis
Neoporphyra
haitanensis
Neoporphyra
haitanensis
Neoporphyra
haitanensis
Neoporphyra
haitanensis
Neoporphyra
haitanensis
Neoporphyra
haitanensis
Neoporphyra
haitanensis
Neoporphyra
haitanensis
Neoporphyra
haitanensis
Neoporphyra
haitanensis
Neoporphyra
haitanensis
Neoporphyra
haitanensis
Neoporphyra
haitanensis
Neoporphyra
haitanensis
Neoporphyra
haitanensis
Neoporphyra
haitanensis
Neoporphyra
haitanensis
Saccharina japonica
Saccharina japonica
Saccharina japonica
Nymphicus
hollandicus
Nymphicus
hollandicus
Nymphicus
hollandicus
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Neopyropia yezoensis
Andalucia godoyi
Andalucia godoyi
Andalucia godoyi
Andalucia godoyi
Taenaris catops
Actias luna
Actias luna
Actias luna
Psitteuteles goldiei
Psitteuteles goldiei
Psitteuteles goldiei
Psitteuteles goldiei
Psitteuteles goldiei
Carybdea marsupialis
Picochlorum sp.
Picochlorum sp.
Picochlorum sp.
Picochlorum sp.
Picochlorum sp.
Picochlorum sp.
Picochlorum sp
Picochlorum sp.
Picochlorum sp.
Picochlorum sp.
Picochlorum sp.
Picochlorum sp.
Picochlorum sp.
Picochlorum sp.
Picochlorum sp
Picochlorum sp.
Picochlorum sp.
Picochlorum sp.
Picochlorum sp.
Picochlorumsp.
Picochlorumsp.
Picochlorum
costavermella
Picochlorum
costavermella
Picochlorum
costavermella
Androctonus
mauritanicus
Androctonus
mauritanicus
Androctonus
mauritanicus
Androctonus
mauritanicus
Androctonus
mauritanicus
Androctonus
mauritanicus
Androctonus
mauritanicus
Androctonus
mauritanicus
Androctonus
mauritanicus
Androctonus
mauritanicus
Androctonus
mauritanicus
Babylonia areolata
Catotricha
subobsoleta
Catotricha
subobsoleta
Catotricha
subobsoleta
Callirhytis sp.
Heteractis magnifica
Isoetes engelmannii
Ostreococcus
mediterraneus
Brettanomyces
custersianus
Phytophthora
chlamydospora
Phytophthora
chlamydospora
Phytophthora
chlamydospora
Triparma laevis f.
inornata
Phytophthora syringae
Phytophthora syringae
Phytophthora syringae
Phytophthora syringae
Phytophthora syringae
Phytophthora syringae
Phytophthora syringae
Undaria pinnatifida
Undaria pinnatifida
Undaria pinnatifida
Undaria pinnatifida
Undaria pinnatifida
Undaria pinnatifida
Undaria pinnatifida
Undaria pinnatifida
Thraustochytrium
aureum
Parietichytrium sp.
Parietichytrium sp.
Parietichytrium sp.
Daphnia sinensis
Daphnia sinensis
Cyclotella cryptica
Cyclotella cryptica
Paralithodes platypus
Paralithodes platypus
Paralithodes platypus
Paralithodes platypus
Paralithodes platypus
Paralithodes platypus
Paralithodes platypus
Paralithodes platypus
Paralithodes platypus
Paralithodes platypus
Paralithodes platypus
Paralithodes platypus
Paralithodes platypus
Paralithodes platypus
Thraustochytrium sp.
Thraustochytrium sp.
Thraustochytrium sp
Hypothenemus
hampei
Hypothenemus
hampei
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Anthracocystis panici-
leucophaei
Anthracocystis panici-
leucophaei
Apophysomyces
ossiformis
Fucus vesiculosus
Scenedesmus sp.
Scenedesmus sp.
Macrobrachium
nipponense
Mortierella sp. GBA35
Mortierella sp. AD010
Mortierella sp. AD010
Haplosporangium sp.
Haplosporangium sp.
Haplosporangium sp.
Mortierella sp. NVP85
Mortierella sp. AD011
Mortierella hygrophila
Aphidius ervi
Aphidius ervi
Aphidius ervi
Aphidius ervi
Aphidius ervi
Aphidius ervi
Aphidius ervi
Aphidius ervi
Aphidius ervi
Aphidius ervi
Aphidius ervi
Aphidius ervi
Aphidius ervi
Linnemannia zychae
Mortierella antarctica
Gryganskiella
cystojenkinii
Gryganskiella
cystojenkinii
Apophysomyces sp.
Dissophora globulifera
Dissophora globulifera
Mortierella sp. GBA43
Mortierella sp. GBA43
Mortierella sp GBA43
Mortierella sp NVP41
Actinomortierella
ambigua
Actinomortierella
ambigua
Mortierella
polycephala
Daphnia obtusa
Daphnia obtusa
Daphnia obtusa
Chioncecetes opilio
Chionoecetes opilio
Neovahikampfia
damariscottae
Neovahikampfia
damariscottae
Neovahikampfia
damariscottae
Neovahikampfia
damariscottae
Neovahikampfia
damariscottae
Neovahikampfia
damariscottae
Thamnidium elegans
Thamnidium elegans
Thamnidium elegans
Thamnidium elegans
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Bradysia odoriphaga
Bradysia odoriphaga
Bradysia odoriphaga
Bradysia odoriphaga
Bradysia odoriphaga
Bradysia odoriphaga
Bradysia odoriphaga
Isochrysis galbana
Isochrysis galbana
Isochrysis galbana
Isochrysis galbana
Isochrysis galbana
Isochrysis galbana
Isochrysis galbana
Isochrysis galbana
Isochrysis galbana
Isochrysis galbana
Isochrysis galbana
Isochrysis galbana
Isochrysis galbana
Isochrysis galbana
Isochrysis galbana
Isochrysis galbana
Euura lappo
Euura lappo
Phytophthora capsici
Phytophthora capsici
Phytophthora capsici
Phytophthora capsici
Phytophthora capsici
Phytophthora capsici
Phytophthora capsici
Phytophthora capsici
Phytophthora capsici
Paralithodes
camtschaticus
Propsilocerus akamusi
Propsilocerus akamusi
Propsilocerus akamusi
Propsilocerus akamusi
Phytophthora
pseudosyringae
Phytophthora
pseudosyringae
Apostasia ramifera
Listronotus
oregonensis
Lisironotus
oregonensis
Leptopilina boulardi
Leptopilina boulardi
Leptopilina boulardi
Leptopilina boulardi
Leptopilina boulardi
Leptopilina boulardi
Leptopilina boulardi
Leptopilina boulardi
Leptopilina boulardi
Leptopilina boulardi
Leptopilina boulardi
Leptopilina boulardi
Leptopilina boulardi
Cystobasidium
slooffiae
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Chlorops oryzae
Chlorops oryzae
Chlorops oryzae
Chlorops oryzae
Chlorops oryzae
Chlorops oryzae
Dreissens polymorpha
Dreissens polymorpha
Dreissens polymorpha
Dreissens polymorpha
Dreissens polymorpha
Dreissens polymorpha
Dreissens polymorpha
Dreissens polymorpha
Dreissens polymorpha
Dreissens polymorpha
Dreissens polymorpha
Dreissens polymorpha
Dreissens polymorpha
Dreissens polymorpha
Dreissens polymorpha
Dreissens polymorpha
Vermamoeba
vermiformis
Vermamoeba
vermiformis
Vermamoeba
vermiformis
Vermamoeba
vermiformis
Vermamoeba
vermiformis
Vermamoeba
vermiformis
Vermamoeba
vermiformis
Vermamoeba
vermiformis
Vermamoeba
vermiformis
Vermamoeba
vermiformis
Vermamoeba
vermiformis
Vermamoeba
vermiformis
Vermamoeba
vermiformis
Vermamoeba
vermiformis
Vermamoeba
vermiformis
Vermamoeba
vermiformis
Vermamoeba
vermiformis
Vermamoeba
vermiformis
Vermamoeba
vermiformis
Vermamoeba
vermiformis
Vermamoeba
vermiformis
Vermamoeba
vermiformis
Vermamoeba
vermiformis
Phytophthora
ramorum
Mythimna separata
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Anadara
kagoshimensis
Anadara
kagoshimensis
Sphagnum fallax
Sphagnum fallax
Rhodotorula sp. CC01
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Microglena sp. YARC
Begonia
darthvaderiana
Begonia
darthvaderiana
Triops longicaudatus
Chloropicon primus
Idolea baltica
Amoeboaphelidium
protococcarum
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Ustilago bromivora
Ustilago bromivora
Ustilago bromivora
Synstelium
polycarpum
Tetradesmus obliquus
Rhizomucor pusillus
Rhizomucor pusillus
Rhizomucor pusillus
Rhizomucor pusillus
Ectocarpus sp. CCAP
Ectocarpus sp. CCAP
Tetradesmus
acuminalus
Tetradesmus
acuminalus
Tetradesmus
acuminalus
Tetradesmus
acuminalus
Tetradesmus
acuminalus
Tetradesmus
acuminalus
Tetradesmus
acuminalus
Tetradesmus
acuminalus
Tilletia controversa
Tilletia controversa
Tilletia controversa
Tilletia controversa
Tilletia controversa
Tilletia controversa
Tilletia controversa
Tilletia controversa
Tilletia controversa
Tilletia controversa
Tilletia controversa
Tilletia controversa
Tilletia controversa
Tilletia controversa
Tilletia controversa
Hypena proboscidalis
Hypena proboscidalis
Apotomis turbidana
Xestia xanthographa
Mamestra brassicae
Craniophora ligustri
Lysandra bellargus
Atethmia centrago
Atethmia centrago
Autographa pulchrina
Hemaris fuciformis
Tenebrio molitor
Tenebrio molitor
Tenebrio molitor
Cydia splendana
Peribalodes
rhomboidaria
Pammene fasciana
Dunaliella primolecta
Dunaliella primolecta
Dunaliella primolecta
Dunaliella primolecta
Dunaliella primolecta
Dunaliella primolecta
Dunaliella primolecta
Dunaliella primolecta
Dunaliella primolecta
Dunaliella primolecta
Bellardia pandia
Bellardia pandia
Bellardia pandia
Bellardia pandia
Bellardia pandia
Bellardia pandia
Platycheirus
albimanus
Platycheirus
albimanus
Platycheirus
albimanus
Platycheirus
albimanus
Platycheirus
albimanus
Platycheirus
albimanus
Platycheirus
albimanus
Platycheirus
albimanus
Platycheirus
albimanus
Platycheirus
albimanus
Platycheirus
albimanus
Platycheirus
albimanus
Platycheirus
albimanus
Platycheirus
albimanus
Platycheirus
albimanus
Platycheirus
albimanus
Platycheirus
albimanus
Platycheirus
albimanus
Platycheirus
albimanus
Platycheirus
albimanus
Platycheirus
albimanus
Steromphala cineraria
Dryobotodes eremita
Philereme vetulata
Pollenia angustigena
Pollenia angustigena
Pollenia angustigena
Pollenia angustigena
Pollenia angustigena
Pollenia angustigena
Pollenia angustigena
Pollenia angustigena
Pollenia angustigena
Pollenia angustigena
Pollenia angustigena
Pollenia angustigena
Pollenia angustigena
Pollenia angustigena
Pollenia angustigena
Pollenia angustigena
Pollenia angustigena
Pollenia angustigena
Pollenia angustigena
Pollenia angustigena
Catocala fraxini
Apotomis betuletana
Apotomis betuletana
Bombylius major
Bombylius major
Bombylius major
Bombylius major
Bombylius major
Bombylius major
Bombylius major
Bombylius major
Bombylius major
Bombylius major
Bombylius major
Bombylius major
Bombylius major
Bombylius major
Bombylius major
Bombylius major
Nephrotoma
flavescens
Nephrotoma
flavescens
Nephrotoma
flavescens
Nephrotoma
flavescens
Nephrotoma
flavescens
Nephrotoma
flavescens
Nephrotoma
flavescens
Nephrotoma
flavescens
Nephrotoma
flavescens
Nephrotoma
flavescens
Nephrotoma
flavescens
Nephrotoma
flavescens
Nephrotoma
flavescens
Nephrotoma
flavescens
Nephrotoma
flavescens
Aplysia californica
Aplysia californica
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Volvox carteri f.
nagariensis
Phytophthora sojae
Phytophthora sojae
Phytophthora sojae
Phytophthora sojae
Phytophthora sojae
Phytophthora sojae
Phytophthora sojae
Phytophthora sojae
Phytophthora sojae
Phytophthora sojae
Phytophthora sojae
Phytophthora sojae
Phytophthora sojae
Phytophthora sojae
Spizellomyces
punctatus DAOM
Spizellomyces
punctatus DAOM
Spizellomyces
punctatus DAOM
Spizellomyces
punctatus DAOM
Spizellomyces
punctatus DAOM
Spizellomyces
punctatus DAOM
Spizellomyces
punctatus DAOM
Spizellomyces
punctatus DAOM
Ostreococcus tauri
Phytophthora
parasitica INRA-310
Phytophthora
parasitica INRA-310
Phytophthora
parasitica INRA-310
Guillardia theta
Guillardia theta
Guillardia theta
Guillardia theta
Guillardia theta
Guillardia theta
Guillardia theta
Guillardia theta
Guillardia theta
Guillardia theta
Guillardia theta
Guillardia theta
Guillardia theta
Guillardia theta
Guillardia theta
Guillardia theta
Guillardia theta
Guillardia theta
Guillardia theta
Guillardia theta
Guillardia theta
Guillardia theta
Guillardia theta
Copidosoma
floridanum
Copidosoma
floridanum
Copidosoma
floridanum
Copidosoma
floridanum
Auxenochlorella
protothecoides
Auxenochlorella
protothecoides
Auxenochlorella
protothecoides
Auxenochlorella
protothecoides
Auxenochlorella
protothecoides
Auxenochlorella
protothecoides
Auxenochlorella
protothecoides
Auxenochlorella
protothecoides
Auxenochlorella
protothecoides
Auxenochlorella
protothecoides
Auxenochlorella
protothecoides
Auxenochlorella
protothecoides
Sphaeroforma arctica
Sphaeroforma arctica
Branchiostoma
belcher
Phycomyces
blakesleeanus NRRL
Rhagoletis zephyria
Rhagoletis zephyria
Rhagoletis zephyria
Rhagoletis zephyria
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Rhagoletis pomonella
Rhagoletis pomonella
Rhagoletis pomonella
Rhagoletis pomonella
Rhagoletis pomonelia
Bradysia coprophila
Bradysia coprophila
Bradysia coprophila
Mercenaria
mercenaria
Aphidius gifuensis
Aphidius gifuensis
Aphidius gifuensis
Aphidius gifuensis
Aphidius gifuensis
Aphidius gifuensis
Aphidius gifuensis
Aphidius gifuensis
Aphidius gifuensis
Aphidius gifuensis
Aphidius gifuensis
Aphidius gifuensis
Aphidius gifuensis
Aphidius gifuensis
Aphidius gifuensis
Aphidius gifuensis
Aphidius gifuensis
Aphidius gifuensis
Aphidius gifuensis
Aphidius gifuensis
Aphidius gifuensis
Aphidius gifuensis
Aphidius gifuensis
Hydra vulgaris
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Allomyces macrogynus
Ectocarpus siliculosus
Ectocarpus siliculosus
Moesziomyces aphidis
Klebsormidium nitens
Klebsormidium nitens
Klebsormidium nitens
Klebsormidium nitens
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Parasitella parasitica
Mucor ambiguus
Mucor ambiguus
Gonapodya prolifera
Gonapodya prolifera
Gonapodya prolifera
Gonapodya prolifera
Nothophytophthora sp.
Nothophytophthora sp.
Nothophytophthora sp.
Nothophytophthora sp.
Nothophytophthora sp.
Phytophthora
megakarya
Bifiguratus adelaidae
Dunaliella salina
Dunaliella salina
Chlamydomonas
eustigma
Coemansia reversa
Coemansia reversa
Coemansia reversa
Coemansia reversa
Tetrabaena socialis
Tetrabaena socialis
Tetrabaena socialis
Tetrabaena socialis
Tetrabaena socialis
Tetrabaena socialis
Tetrabaena socialis
Tetrabaena socialis
Tetrabaena socialis
Tetrabaena socialis
Tetrabaena socialis
Tetrabaena socialis
Rhodotorula taiwanensis
Raphidocelis
subcapitata
Raphidocelis
subcapitata
Raphidocelis
subcapitata
Raphidocelis
subcapitata
Raphidocelis
subcapitata
Diversispora epigaea
Diversispora epigaea
Diversispora epigaea
Diversispora epigaea
Diversispora epigaea
Diversispora epigaea
Diversispora epigaea
Diversispora epigaea
Diversispora epigaea
Diversispora epigaea
Diversispora epigaea
Diversispora epigaea
Diversispora epigaea
Diversispora epigaea
Diversispora epigaea
Diversispora epigaea
Diversispora epigaea
Diversispora epigaea
Diversispora epigaea
Diversispora epigaea
Gigaspora rosea
Caulochytrium
protostelioides
Caulochytrium
protostelioides
Elysia chlorotica
Elysia chlorotica
Rhodotorula sp. CCFEE
Globisporangium
splendens
Globisporangium
splendens
Globisporangium
splendens
Globisporangium
splendens
Globisporangium
splendens
Globisporangium
splendens
Synchytrium
endobioticum
Synchytrium
endobioticum
Synchytrium
endobioticum
Synchytrium
endobioticum
Synchytrium
endobioticum
Synchytrium
endobioticum
Synchytrium
endobioticum
Synchytrium
endobioticum
Synchytrium
endobioticum
Synchytrium
endobioticum
Synchytrium
endobioticum
Synchytrium
endobioticum
Spizellomyces sp.
Powellomyces hirtus
Trebouxia sp. A1-2
Trebouxia sp. A1-2
Trebouxia sp. A1-2
Trebouxia sp A1-2
Trebouxia sp. A1-2
Trebouxia sp. A1-2
Trebouxia sp. A1-2
Trebouxia sp. A1-2
Trebouxia sp. A1-2
Trebouxia sp. A1-2
Trebouxia sp. A1-2
Trebouxia sp. A1-2
Trichomonascus ciferrii
Tilletia walkeri
Tilletia walkeri
Apophysomyces
ossiformis
Apophysomyces
ossiformis
Apophysomyces
ossiformis
Apophysomyces
ossiformis
Apophysomyces
ossiformis
Scenedesmus sp.
Scenedesmus sp.
Dissophora ornata
Dissophora ornata
Haplosporangium
gracile
Haplosporangium
gracile
Mortierella sp. GBA35
Mortierella sp. GBA35
Mortierella sp. AD031
Mortierella sp. GBA39
Haplosporangium sp. Z 11
Haplosporangium sp. Z 11
Haplosporangium sp. Z 11
Haplosporangium sp. Z 767
Haplosporangium sp. Z 767
Mortierella sp. NVP85
Podila horticola
Podila horticola
Podila horticola
Mortierella sp. AD094
Mortierella hygrophila
Mortierella hygrophila
Linnemannia zychae
Linnemannia zychae
Linnemannia zychae
Mortierella antarctica
Entomortierella
chiamydospora
Podila clonocystis
Gryganskiella
cystojenkinii
Gryganskiella
cystojenkinii
Gryganskiella
cystojenkinii
Podila epicladia
Apophysomyces sp.
Apophysomyces sp.
Apophysomyces sp.
Apophysomyces sp.
Apophysomyces sp.
Apophysomyces sp.
Apophysomyces sp.
Apophysomyces sp.
Apophysomyces sp.
Apophysomyces sp.
Apophysomyces sp.
Apophysomyces sp.
Linnemannia gamsil
Linnemannia gamsil
Linnemannia gamsil
Linnemannia exigua
Dissophora globulifera
Podila minutissima
Podila minutissima
Gamsiella
multidivaricata
Mortierella sp. AD032
Mortierella sp. GBA30
Mortierella sp. GBA30
Mortierella sp. GBA30
Mortierella sp. GBA43
Mortierella sp. GBA43
Mortierella sp. GBA43
Mortierella sp. GBA43
Mortierella sp. GBA43
Mortierella sp. GBA43
Mortierella sp. NVP41
Mortierella sp. NVP41
Mortierella sp. NVP41
Actinomortierella
ambigua
Actinomortierella
ambigua
Actinomortierella
ambigua
Mortierella polycephala
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Geosiphon pyriformis
Umbelopsis vinacea
Umbelopsis isabellina
Umbelopsis isabellina
Thamnidium elegans
Thamnidium elegans
Thamnidium elegans
Thamnidium elegans
Thamnidium elegans
Thamnidium elegans
Thamnidium elegans
Thamnidium elegans
Thamnidium elegans
Thamnidium elegans
Thamnidium elegans
Thamnidium elegans
Thamnidium elegans
Thamnidium elegans
Thamnidium elegans
Thamnidium elegans
Thamnidium elegans
Thamnidium elegans
Thamnidium elegans
Mucor plumbeus
Mucor plumbeus
Mucor plumbeus
Mucor plumbeus
Mucor plumbeus
Mucor plumbeus
Mucor plumbeus
Mucor plumbeus
Mucor plumbeus
Mucor plumbeus
Mucor plumbeus
Mucor plumbeus
Mucor plumbeus
Mucor plumbeus
Mucor plumbeus
Mucor plumbeus
Mucor plumbeus
Mucor plumbeus
Mucor plumbeus
Mucor plumbeus
Mucor circinatus
Mucor circinatus
Mucor circinatus
Mucor circinatus
Mucor circinatus
Mucor circinatus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Mucor saturninus
Phytophthora idaei
Phytophthora idaei
Phytophthora idaei
Phytophthora idaei
Bradysia odonphaga
Phytophthora aleatoria
Phytophthora aleatoria
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Dreissena polymorpha
Dreissena polymorpha
Dreissena polymorpha
Dreissena polymorpha
Dreissena polymorpha
Dreissena polymorpha
Dreissena polymorpha
Dreissena polymorpha
Dreissena polymorpha
Dreissena polymorpha
Dreissena polymorpha
Dreissena polymorpha
Dreissena polymorpha
Dreissena polymorpha
Dreissena polymorpha
Dreissena polymorpha
Dreissena polymorpha
Dreissena polymorpha
Dreissena polymorpha
Dreissena polymorpha
Dreissena polymorpha
Dreissena polymorpha
Dreissena polymorpha
Dreissena polymorpha
Phytophthora ramorum
Phytophthora ramorum
Coccomyxa sp. Obi
Coccomyxa sp. Obi
Sphagnum fallax
Mortierella claussenii
Mortierella claussenii
Mortierella ciaussenii
Mortierella claussenii
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chloropicon primus
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Chlorella vulgaris
Cichorium endivia
Cichorium endivia
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
protococcarum
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Amoeboaphelidium
occidentale
Absidia glauca
Absidia glauca
Absidia glauca
Absidia glauca
Absidia glauca
Absidia glauca
Absidia glauca
Absidia glauca
Absidia glauca
Absidia glauca
Ustilago bromivora
Sporisorium reilianum f.
Ectocarpus sp. CCAP
Ectocarpus sp. CCAP
Tilletia controversa
Tilletia controversa
Tilletia controversa
Tilletia controversa
Tilletia controversa
Tilletia controversa
Tilletia controversa
Tilletia controversa
Tilletia controversa
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Heterostelium album
Heterostelium album
Heterostelium album
Heterostelium album
Heterostelium album
Eremothecium gossypii
Cyanidioschyzon
merolae strain 10D
Phytophthora sojae
Phytophthora sojae
Phytophthora sojae
Phytophthora sojae
Phytophthora sojae
Phytophthora sojae
Phytophthora sojae
Phytophthora sojae
Phytophthora sojae
Phytophthora sojae
Spizellomyces punctatus
Spizellomyces punctatus
Spizellomyces punctatus
Spizellomyces punctatus
Spizellomyces punctatus
Spizellomyces punctatus
Spizellomyces punctatus
Spizellomyces punctatus
Spizellomyces punctatus
Spizellomyces punctatus
Spizellomyces punctatus
Spizellomyces punctatus
Spizellomyces punctatus
Spizellomyces punctatus
Spizellomyces punctatus
Spizellomyces punctatus
Spizellomyces punctatus
Spizellomyces punctatus
Spizellomyces punctatus
Spizellomyces punctatus
Spizellomyces punctatus
Spizellomyces punctatus
Spizellomyces punctatus
Salpingoeca rosetta
Salpingoeca rosetta
Salpingoeca rosetta
Salpingoeca rosetta
Salpingoeca rosetta
Cavenderia fasciculata
Cavenderia fasciculata
Cavenderia fasciculata
Cavenderia fasciculata
Cavenderia fasciculata
Cavenderia fasciculata
Cavenderia fasciculata
Cavenderia fasciculata
Cavenderia fasciculata
Cavenderia fasciculata
Cavenderia fasciculata
Cavenderia fasciculata
Cavenderia fasciculata
Torulaspora delbrueckii
Torulaspora delbrueckii
Torulaspora delbrueckii
Torulaspora delbrueckii
Torulaspora delbrueckii
Torulaspora delbrueckii
Torulaspora delbrueckii
Phytophthora parasitica
Phytophthora parasitica
Phytophthora parasitica
Phytophthora parasitica
Phytophthora parasitica
Phytophthora parasitica
Phytophthora parasitica
Phytophthora parasitica
Phytophthora parasitica
Phytophthora parasitica
Phytophthora parasitica
Phytophthora parasitica
Phytophthora parasitica
Phytophthora parasitica
Phytophthora parasitica
Phytophthora parasitica
Acanthamoeba
castellanii str. Neff
Acanthamoeba
castellanii str. Neff
Acanthamoeba
castellanii str. Neff
Guillardia theta
Rhodotorula toruloides
Rhodotorula toruloides
Rhodotorula toruloides
Rhodotorula toruloides
Pseudozyma hubeiensis
Pseudozyma hubeiensis
Pseudozyma flocculosa
Pseudozyma flocculosa
Kalmanozyma
brasiliensis GHG001
Auxenochlorella
protothecoides
Auxenochlorella
protothecoides
Auxenochlorella
protothecoides
Auxenochlorella
protothecoides
Auxenochlorella
protothecoides
Auxenochlorella
protothecoides
Auxenochlorella
protothecoides
Auxenochlorella
protothecoides
Auxenochlorella
protothecoides
Auxenochlorella
protothecoides
Auxenochlorella
protothecoides
Phycomyces
blakesleeanus NRRL
Phycomyces
blakesleeanus NRRL
Phycomyces
blakesleeanus NRRL
Phycomyces
blakesleeanus NRRL
Phycomyces
blakesleeanus NRRL
Phycomyces
blakesleeanus NRRL
Phycomyces
blakesleeanus NRRL
Phycomyces
blakesleeanus NRRL
Phycomyces
blakesleeanus NRRL
Phycomyces
blakesleeanus NRRL
Phycomyces
blakesleeanus NRRL
Phycomyces
blakesleeanus NRRL
Phycomyces
blakesleeanus NRRL
Phycomyces
blakesleeanus NRRL
Phycomyces
blakesleeanus NRRL
Phycomyces
blakesleeanus NRRL
Phycomyces
blakesleeanus NRRL
Phycomyces
blakesleeanus NRRL
Phycomyces
blakesleeanus NRRL
Phycomyces
blakesleeanus NRRL
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Tilletiopsis
washingtonensis
Tilletiopsis
washingtonensis
Tilletiopsis
washingtonensis
Tilletiopsis
washingtonensis
Naegleria lovaniensis
Naegleria lovaniensis
Naegleria lovaniensis
Naegleria lovaniensis
Naegleria lovaniensis
Naegleria lovaniensis
Naegleria lovaniensis
Naegleria lovaniensis
Naegleria lovaniensis
Naegleria lovaniensis
Naegleria lovaniensis
Naegleria lovaniensis
Naegleria lovaniensis
Naegleria lovaniensis
Naegleria lovaniensis
Naegleria lovaniensis
Naegleria lovaniensis
Naegleria lovaniensis
Morchella importuna
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Bradysia coprophila
Bradysia coprophila
Bradysia coprophila
Bradysia coprophila
Bradysia coprophila
Bradysia coprophila
Bradysia coprophila
Bradysia coprophila
Bradysia coprophila
Bradysia coprophila
Bradysia coprophila
Bradysia coprophila
Bradysia coprophila
Bradysia coprophila
Mercenaria mercenaria
Mercenaria mercenaria
Mercenaria mercenaria
Mercenaria mercenaria
Mercenaria mercenaria
Aphidius gifuensis
Mayetiola destructor
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Rhizopus delemar RA
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Mucor irregularis B50
Rhizomucor miehei
Rhizomucor miehei
Rhizomucor miehei
Rhizomucor miehei
Rhizomucor miehei
Rhizomucor miehei
Rhizomucor miehei
Rhizomucor miehei
Rhizomucor miehei
Rhizomucor miehei
Rhizomucor miehei
Rhizomucor miehei
Rhizomucor miehei
Rhizomucor miehei
Rhizomucor miehei
Rhizomucor miehei
Rhizomucor miehei
Rhizomucor miehei
Rhizomucor miehei
Rhizomucor miehei
Rhizomucor miehei
Rhizomucor miehei
Eremothecium coryli
Belgica antarctica
Belgica antarctica
Coccomyxa sp.
Coccomyxa sp.
Coccomyxa sp.
Coccomyxa sp.
Coccomyxa sp.
Coccomyxa sp.
Coccomyxa sp.
Parasitella parasitica
Parasitella parasitica
Sporisorium
scitamineum
Arabis nordmanniana
Heliconius ismenius
Heliconius ismenius
Gonapodya prolifera
Mucor circinelloides
Mucor circinelloides
Mucor circinelloides
Mucor circinelloides
Mucor circinelloides
Mucor circinelloides
Mucor circinelloides
Mucor circinelloides
Mucor circinelloides
Mucor circinelloides
Mucor circinelloides
Mucor circinelloides
Mucor circinelloides
Mucor circinelloides
Mucor circinelloides
Mucor circinelloides
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Gongronella sp. w5
Chlamydomonas
asymmetrica
Chlamydomonas
asymmetrica
Chlamydomonas
asymmetrica
Chlamydomonas
asymmetrica
Chlamydomonas
asymmetrica
Chlamydomonas
asymmetrica
Leptopilina clavipes
Candida sp. JCM 15000
Candida sp. JCM 15000
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Porphyra umbilicalis
Mamestra configurata
Ammotragus lervia
Ammotragus lervia
Chlamydomonas
eustigma
Chlamydomonas
eustigma
Notospermus
geniculatus
Notospermus
geniculatus
Notospermus
geniculatus
Notospermus
geniculatus
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Clitarchus hooker
Tetrabaena socialis
Tetrabaena socialis
Tetrabaena socialis
Tetrabaena socialis
Prototheca cufis
Prototheca cufis
Prototheca cufis
Prototheca cufis
Prototheca cufis
Prototheca cufis
Prototheca cufis
Prototheca cufis
Prototheca cufis
Prototheca cufis
Prototheca cufis
Prototheca cufis
Prototheca cufis
Prototheca cufis
Prototheca cufis
Prototheca cufis
Prototheca cufis
Prototheca cufis
Prototheca cufis
Prototheca cufis
Prototheca cufis
Prototheca cufis
Prototheca cufis
Prototheca cufis
Prototheca cufis
Prototheca cufis
Prototheca cufis
Prototheca cufis
Prototheca cufis
Periplaneta americana
Eudorina sp. 2006-703-
Rhizopus azygosporus
Diversispora epigaea
Diversispora epigaea
Diversispora epigaea
Diversispora epigaea
Diversispora epigaea
Diversispora epigaea
Glomus cerebriforme
Glomus cerebriforme
Glomus cerebriforme
Glomus cerebriforme
Glomus cerebriforme
Glomus cerebriforme
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Gigaspora rosea
Pogostemon cablin
Pogostemon cablin
Pogostemon cablin
Torulaspora franciscae
Torulaspora franciscae
Torulaspora franciscae
Torulaspora franciscae
Torulaspora franciscae
Torulaspora franciscae
Torulaspora franciscae
Lipomyces sp. NRRL Y-
Brevipalpus yothersi
Procavia capensis
Characiochloris sp.
Characiochloris sp.
Tuta absoluta
Drosophila neonasuta
Drosophila neonasuta
Lichtheimia ramosa
Lichtheimia ramosa
Lichtheimia ramosa
Lichtheimia ramosa
Lichtheimia ramosa
Lichtheimia ramosa
Lichtheimia ramosa
Lichtheimia ramosa
Ursus thibetanus
thibetanus
Saccharomyces
cerevisiae ×
Saccharomyces
eubayanus ×
Saccharomyces
kudriavzevii ×
Saccharomyces uvarum
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Gigaspora margarita
Zostera nigricaulis
Nymphicus hollandicus
Nymphicus hollandicus
Neopyropia yezcensis
Neopyropia yezcensis
Neopyropia yezcensis
Neopyropia yezcensis
Neopyropia yezcensis
Neopyropia yezcensis
Neopyropia yezcensis
Neopyropia yezcensis
Neopyropia yezcensis
Neopyropia yezcensis
Neopyropia yezcensis
Neopyropia yezcensis
Neopyropia yezcensis
Neopyropia yezcensis
Neopyropia yezcensis
Neopyropia yezcensis
Neopyropia yezcensis
Neopyropia yezcensis
Neopyropia yezoensis
Taenaris catops
Actias luna
Actias luna
Actias luna
Actias luna
Actias luna
Actias luna
Actias luna
Actias luna
Actias luna
Actias luna
Ara chloropterus
Ara chloropterus
Stiretrus anchorago
Danaus melanipous
Danaus melanippus
Psitleuteles goldiei
Psitteuteles goldiei
Lorius garrulus
Ara militaris
Picochlorum sp. ‘celeri’
Picochlorum sp. ‘celeri’
Picochlorum sp. ‘celeri’
Picochlorum sp. ‘celeri’
Androctonus
mauritanicus
Androctonus
mauritanicus
Androctonus
mauritanicus
Androctonus
mauritanicus
Androctonus
mauritanicus
Androctonus
mauritanicus
Androctonus
mauritanicus
Androctonus
mauritanicus
Androctonus
mauritanicus
Androctonus
mauritanicus
Androctonus
mauritanicus
Androctonus
mauritanicus
Androctonus
mauritanicus
Androctonus
mauritanicus
Androctonus
mauritanicus
Androctonus
mauritanicus
Androctonus
mauritanicus
Androctonus
mauritanicus
Androctonus
mauritanicus
Androctonus
mauritanicus
Pionus senilis
Pionus senilis
Rhinocypha anisoptera
Chrysaora chesapeakei
Papilio bianor
Papilio bianor
Magicicada
septendecula
Magicicada
septendecula
Ostreococcus
mediterraneus
Ostreococcus
mediterraneus
Cyclina sinensis
Cyclina sinensis
Cyclina sinensis
Cyclotella cryptica
Cyclotella cryptica
Cyclotella cryptica
Cyclotella cryptica
Cyclotella cryptica
Hypothenemus hampei
Chlorella sp. BAC 9706
Graphium doson
Graphium doson
Graphium doson
Anthracocystis panici-
leucophaei
Apophysomyces
ossiformis
Apophysomyces
ossiformis
Apophysomyces
ossiformis
Apophysomyces
ossiformis
Apophysomyces
ossiformis
Apophysomyces
ossiformis
Apophysomyces
ossiformis
Apophysomyces
ossiformis
Apophysomyces
ossiformis
Apophysomyces
ossiformis
Apophysomyces
ossiformis
Apophysomyces
ossiformis
Apophysomyces
ossiformis
Scenedesmus sp.
Scenedesmus sp.
Scenedesmus sp.
Scenedesmus sp.
Scenedesmus sp.
Scenedesmus sp.
Scenedesmus sp.
Scenedesmus sp.
Scenedesmus sp.
Scenedesmus sp.
Scenedesmus sp.
Chrysaora achlyos
Apophysomyces sp.
Apophysomyces sp.
Apophysomyces sp.
Apophysomyces sp.
Apophysomyces sp
Apophysomyces sp.
Apophysomyces sp.
Apophysomyces sp.
Apophysomyces sp.
Apophysomyces sp.
Apophysomyces sp.
Apophysomyces sp.
Apophysomyces sp.
Apophysomyces sp.
Apophysomyces sp.
Apophysomyces sp.
Apophysomyces sp.
Apophysomyces sp.
Apophysomyces sp.
Apophysomyces sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Chlamydomonas sp.
Thamnidium elegans
Mucor plumbeus
Mucor saturninus
Bradysia odoriphaga
Bradysia odoriphaga
Bradysia odoriphaga
Bradysia odoriphaga
Bradysia odoriphaga
Bradysia odoriphaga
Bradysia odoriphaga
Bradysia odoriphaga
Bradysia odoriphaga
Bradysia odoriphaga
Bradysia odoriphaga
Bradysia odoriphaga
Bradysia odoriphaga
Bradysia odoriphaga
Euura lappo
Euura lappo
Euura lappo
Euura lappo
Euura lappo
Euura lappo
Euura lappo
Euura lappo
Euura lappo
Euura lappo
Euura lappo
Euura lappo
Euura lappo
Euura lappo
Euura lappo
Euura lappo
Skeletonema costatum
Skeletonema costatum
Cotesia chilonis
Cotesia chilonis
Listronotus oregonensis
Listronotus oregonensis
Listronotus oregonensis
Listronotus oregonensis
Listronotus oregonensis
Listronotus oregonensis
Listronotus oregonensis
Leptopilina boulardi
Leptopilina boulardi
Leptopilina boulardi
Leptopilina boulardi
Leptopilina boulardi
Melopolophium
dirhodum
Dreissena polymorpha
Dreissena polymorpha
Dreissena polymorpha
Mythimna separata
Mythimna separata
Drosophila nannoptera
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Sitodiplosis mosellana
Anadara kagoshimensis
Anadara kagoshimensis
Anadara kagoshimensis
Anadara kagoshimensis
Anadara kagoshimensis
Anadara kagoshimensis
Anadara kagoshimensis
Anadara kagoshimensis
Spodoptera exigua
Spodoptera exigua
Spodoptera exigua
Begonia darthvaderiana
Begonia darthvaderiana
Begonia darthvaderiana
Begonia darthvaderiana
Begonia darthvaderiana
Micropterna sequax
Zelostemma sp. ZL-
Saxidomus purpurata
Saxidomus purpurata
Saxidomus purpurata
Saxidomus purpurata
Saxidomus purpurata
Saxidomus purpurata
Saxidomus purpurata
Saxidomus purpurata
Saxidomus purpurata
Saxidomus purpurata
Saxidomus purpurata
Saxidomus purpurata
Saxidomus purpurata
Chloropicon primus
Pseudozyma pruni
Pseudozyma pruni
Pseudozyma pruni
Pseudozyma pruni
Pseudozyma pruni
Sporisorium sorghi
Sporisorium sorghi
Sporisorium sorghi
Sporisorium sorghi
Coffea canephora
Absidia giauca
Tetradesmus obliquus
Rhizomucor pusillus
Rhizomucor pusillus
Rhizomucor pusillus
Rhizomucor pusillus
Rhizomucor pusillus
Rhizomucor pusillus
Rhizomucor pusillus
Rhizomucor pusillus
Rhizomucor pusillus
Rhizomucor pusillus
Rhizomucor pusillus
Rhizomucor pusillus
Rhizomucor pusillus
Rhizomucor pusillus
Rhizomucor pusillus
Rhizomucor pusillus
Rhizomucor pusillus
Rhizomucor pusillus
Rhizomucor pusillus
Rhizomucor pusillus
Pharmaceum exiguum
Odontarrhena argentea
Raparia bulbosa
Ormyrus nitidulus
Arapaima gigas
Arapaima gigas
Hippocampus kuda
Ectocarpus sp. CCAP
Ectocarpus sp. CCAP
Tetradesmus
acuminatus
Autographa gamma
Autographa gamma
Autographa gamma
Autographa gamma
Hypena proboscidalis
Hypena proboscidalis
Hypena proboscidalis
Hypena proboscidalis
Hypena proboscidalis
Hypena proboscidalis
Hypena proboscidalis
Hypena proboscidalis
Hypena proboscidalis
Apotomis turbidana
Apotomis turbidana
Apotomis turbidana
Apotomis turbidana
Apotomis turbidana
Apotomis turbidana
Apotomis turbidana
Apotomis turbidana
Xestia xanthographa
Xestia xanthographa
Xestia xanthographa
Xestia xanthographa
Xestia xanthographa
Xestia xanthographa
Xestia xanthographa
Xestia xanthographa
Xestia xanthographa
Noctua fimbriata
Mamestra brassicae
Mamestra brassicae
Cosmia trapezina
Cosmia trapezina
Cosmia trapezina
Cosmia trapezina
Celastrina argiolus
Celastrina argiolus
Cyaniris semiargus
Cyaniris semiargus
Cyaniris semiargus
Cyaniris semiargus
Cyaniris semiargus
Cyaniris semiargus
Amphipyra tragopoginis
Amphipyra tragopoginis
Amphipyra tragopoginis
Lysandra coridon
Lysandra coridon
Lysandra coridon
Lycaena phlaeas
Lysandra bellargus
Lysandra bellargus
Lysandra bellargus
Lysandra bellargus
Lysandra bellargus
Lysandra bellargus
Lysandra bellargus
Atethmia centrago
Atethmia centrago
Abrostola tripartita
Abrostola tripartita
Abrostola tripartita
Abrostola tripartita
Abrostola tripartita
Abrostola tripartita
Abrostola tripartita
Glaucopsyche alexis
Glaucopsyche alexis
Glaucopsyche alexis
Plebejus argus
Plebejus argus
Plebejus argus
Autographa pulchrina
Autographa pulchrina
Autographa pulchrina
Ochropleura plecta
Hemaris fuciformis
Hemaris fuciformis
Hemaris fuciformis
Hemaris fuciformis
Hemaris fuciformis
Hemaris fuciformis
Hemaris fuciformis
Hemaris fuciformis
Hemaris fuciformis
Hemaris fuciformis
Hemaris fuciformis
Hemaris fuciformis
Hemaris fuciformis
Hemaris fuciformis
Idaea aversata
Mythimna ferrago
Chrysoteuchia culmella
Chrysoteuchia culmella
Cydia splendana
Cydia splendana
Cydia splendana
Cydia splendana
Cydia splendana
Cydia splendana
Cydia splendana
Cydia splendana
Cydia splendana
Apamea monoglypha
Apamea monoglypha
Pammene fasciana
Pammene fasciana
Pammene fasciana
Pammene fasciana
Pammene fasciana
Pammene fasciana
Pammene fasciana
Dunaliella primolecta
Dunaliella primolecta
Dunaliella primolecta
Dunaliella primolecta
Dunaliella primolecta
Dunaliella primolecta
Hydraecia micacea
Agrochola circellaris
Agrochola circellaris
Agrochola circellaris
Agrochola circellaris
Agrochola circellaris
Agrochola circellaris
Agrochola circellaris
Griposia aprilina
Griposia aprilina
Agrochola macilenta
Erebia ligea
Erebia ligea
Erebia ligea
Dryobotodes eremita
Dryobotodes eremita
Dryobotodes eremita
Philereme vetulata
Philereme vetulata
Philereme vetulata
Philereme vetulata
Philereme vetulata
Philereme vetulata
Philereme vetulata
Philereme vetulata
Philereme vetulata
Euplexia lucipara
Euplexia lucipara
Euplexia lucipara
Euplexia lucipara
Euplexia lucipara
Euplexia lucipara
Euplexia lucipara
Euplexia lucipara
Euplexia lucipara
Macaria notata
Catocala fraxini
Catocala fraxini
Catocala fraxini
Catocala fraxini
Andricus quercusramuli
Andricus quercusramuli
Andricus curvator
Apotomis betuletana
Apotomis betuletana
Apotomis betuletana
Apotomis betuletana
Apotomis betuletana
Diarsia rubi
Diarsia rubi
Diarsia rubi
Diarsia rubi
Diarsia rubi
Epinotia nisella
Epinotia nisella
Epinotia nisella
Epinotia nisella
Epinotia nisella
Epinotia nisella
Epinotia nisella
Epinotia nisella
Diachrysia chrysitis
Diachrysia chrysitis
Diachrysia chrysitis
Diachrysia chrysitis
Diachrysia chrysitis
Diachrysia chrysitis
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Volvox carteri f.
nagariensis
Eremothecium
cymbalariae
Torulaspora delbrueckii
Copidosoma floridanum
Auxenochlorella
protothecoides
Auxenochlorella
protothecoides
Auxenochlorella
protothecoides
Auxenochlorella
protothecoides
Auxenochlorella
protothecoides
Ziziphus jujuba
Phycomyces
blakesleeanus NRRL
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Sipha flava
Sipha flava
Naegleria lovaniensis
Naegleria lovaniensis
Naegleria lovaniensis
Ostrinia furnacalis
Ostrinia furnacalis
Ostrinia furnacalis
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Spodoptera frugiperda
Spodoptera frugiperda
Spodoptera frugiperda
Bradysis coprophila
Bradysis coprophila
Bradysis coprophila
Bradysis coprophila
Bradysis coprophila
Bradysis coprophila
Bradysis coprophila
Bradysis coprophila
Bradysia coprophila
Mercenaria mercenaria
Mercenaria mercenaria
Mercenaria mercenaria
Mercenaria mercenaria
Mercenaria mercenaria
Mercenaria mercenaria
Mercenaria mercenaria
Mercenaria mercenaria
Mercenaria mercenaria
Mercenaria mercenaria
Globisporangium
ultimum DAOM BR144
Globisporangium
ultimum DAOM BR144
Globisporangium
ultimum DAOM BR144
Globisporangium
ultimum DAOM BR144
Globisporangium
ultimum DAOM BR144
Globisporangium
ultimum DAOM BR144
Globisporangium
ultimum DAOM BR144
Globisporangium
ultimum DAOM BR144
Globisporangium
ultimum DAOM BR144
Globisporangium
ultimum DAOM BR144
Globisporangium
ultimum DAOM BR144
Globisporangium
ultimum DAOM BR144
Globisporangium
ultimum DAOM BR144
Globisporangium
ultimum DAOM BR144
Allomyces macrogynus
Coccomyxa sp.
Arabis nordmanniana
Tilletia indica
Phytophthora plurivora
Dunaliella salina
Dunaliella salina
Tetrabaena socialis
Rhodotorula
mucilaginosa
Phytophthora nicotianae
Phytophthora nicotianae
Phytophthora nicotianae
Phytophthora nicotianae
Phytophthora nicotianae
Phytophthora nicotianae
Phytophthora nicotianae
Phytophthora nicotianae
Elysia chlorotica
Ectocarpus sp. Ec32
Ectocarpus sp. Ec32
Drosophila neonasuta
Drosophila neonasuta
Globisporangium
splendens
Synchytrium
endobioticum
Synchytrium
endobioticum
Trebouxia sp A1-2
Gigaspora margarita
Androctonus
mauritanicus
Androctonus
mauritanicus
Phytophthora
chlamydospora
Phytophthora syringae
Hydra viridissima
Cotesia chilonis
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Carpediemonas
membranifera
Pythium oligandrum
Dreissena polymorpha
Sitodiplosis mosellana
Begonia darthvaderiana
Begonia darthvaderiana
Begonia darthvaderiana
Begonia darthvaderiana
Begonia darthvaderiana
Begonia darthvaderiana
Begonia darthvaderiana
Clogmia albipunctata
Papaver nudicaule
Odontarrhena argentea
Erysimum pusillum
Noccaea caerulescens
Raparia bulbosa
Raparia bulbosa
Tilletia controversa
Tilletia controversa
Tilletia controversa
Craniophora ligustri
Autographa puichrina
Autographa pulchrina
Griposia aprilina
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Cyanidioschyzon
merolae strain 10D
Cyanidioschyzon
merolae strain 10D
Spizellomyces punctatus
Spizellomyces punctatus
Phytophthora parasitica
Phytophthora parasitica
Phytophthora parasitica
Phytophthora parasitica
Phytophthora parasitica
Phytophthora parasitica
Phycomyces
blakesleeanus NRRL
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Rhizopus microsporus
Naegleria lovaniensis
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Contarinia nasturtii
Acanthamoeba
polyphaga lentillevirus
Acanthamoeba
polyphaga lentillevirus
Acanthamoeba
polyphaga lentillevirus
Megavirus Iba
Megavirus courdo11
Bandra megavirus
Indivirus ILV1
Indivirus ILV1
Indivirus ILV1
Indivirus ILV1
Catovirus CTV1
Catovirus CTV1
Catovirus CTV1
Catovirus CTV1
Catovirus CTV1
Hokovirus HKV1
Hokovirus HKV1
Hokovirus HKV1
Klosneuvirus KNV1
Klosneuvirus KNV1
Klosneuvirus KNV1
Klosneuvirus KNV1
Klosneuvirus KNV1
Acanthamoeba
castellanii mamavirus
Acanthamoeba
castellanii mamavirus
Acanthamoeba
castellanii mamavirus
Acanthamoeba
castellanii mamavirus
Acanthamoeba
castellanii mamavirus
Acanthamoeba
castellanii mamavirus
Acanthamoeba
castellanii mamavirus
Acanthamoeba
castellanii mamavirus
Acanthamoeba
castellanii mamavirus
Acanthamoeba
castellanii mamavirus
Acanthamoeba
castellanii mamavirus
Acanthamoeba
castellanii mamavirus
Acanthamoeba
polyphaga mimivirus
Acanthamoeba
polyphaga mimivirus
Acanthamoeba
polyphaga mimivirus
Acanthamoeba
polyphaga mimivirus
Acanthamoeba
polyphaga mimivirus
Acanthamoeba
polyphaga mimivirus
Acanthamoeba
polyphaga mimivirus
Acanthamoeba
polyphaga mimivirus
Acanthamoeba
polyphaga mimivirus
Mimivirus Bombay
Mimivirus Bombay
Tupanvirus soda lake
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Dasosvirus sp.
Edafosvirus sp.
Edafosvirus sp.
Edafosvirus sp.
Edafosvirus sp.
Edafosvirus sp.
Gaeavirus sp.
Gaeavirus sp
Harvfovirus sp.
Harvfovirus sp.
Hyperionvirus sp.
Hyperionvirus sp.
Hyperionvirus sp.
Hyperionvirus sp.
Hyperionvirus sp.
Satyrvirus sp.
Satyrvirus sp.
Sylvanvirus sp.
Sylvanvirus sp.
Sylvanvirus sp.
Sylvanvirus sp.
Terrestrivirus sp.
Megavirus baoshan
Megavirus baoshan
Moumouvirus
australiensis
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Faustovirus
Haliotid herpesvirus 1
Mamestra configurata
nucleopolyhedrovirus B
Spodoptera frugiperda
ascovirus 1a
Spodoptera frugiperda
ascovirus 1a
Heliothis virescens
ascovirus 3e
Hellothis virescens
ascovirus 3e
Heliothis virescens
ascovirus 3e
Hellothis virescens
ascovirus 3e
Hellothis virescens
ascovirus 3e
Helicoverpa armigera
granulovirus
Helicoverpa armigera
granulovirus
Pseudalatia unipuncta
granulovirus
Pseudalatia unipuncta
granulovirus
Acanthamoeba
polyphaga mimivirus
Acanthamoeba
polyphaga mimivirus
Acanthamoeba
polyphaga mimivirus
Acanthamoeba
polyphaga mimivirus
Acanthamoeba
polyphaga mimivirus
Megavirus chiliensis
Megavirus chiliensis
Abalone herpesvirus
Acanthamoeba
polyphaga moumouvirus
Acanthamoeba
polyphaga moumouvirus
Pandoravirus dulcis
Pandoravirus dulcis
Mimivirus terra2
Mimivirus terra2
Mimivirus terra2
Mimivirus terra2
Pandoravirus
inopinatum
Tipula oleracea
nudivirus
Lambdina fiscellaria
nucleopolyhedrovirus
Kaumoebavirus
Kaumcebavirus
Heliothis virescens
ascovirus 3f
Heliothis virescens
ascovirus 3f
Heliothis virescens
ascovirus 3g
Heliothis virescens
ascovirus 3g
Heliothis virescens
ascovirus 3g
Heliothis virescens
ascovirus 3g
Trichoplusia ni
granulovirus LBIV-12
Pandoravirus
neocaledonia
The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.
Both prokaryotic and eukaryotic genomes are replete with diverse transposons, a broad class of mobile genetic elements (MGE), that widely differ in abundance. Transposons of the highly abundant IS200/605 family encode the TnpA protein, which is a DDE class transposase responsible for the single-strand ‘peel and paste’ transposition mechanism of these MGEs, and TnpB protein the role of which in transposition remains unclear. Numerous non-autonomous transposons encode TnpB alone, requiring a transposase to be supplied in trans. RNA-programmable DNA nucleases serve multiple roles in prokaryotes, including in mobile element defense and spread. These nucleases include argonaut, CRISPR, and the obligate mobile element-guided activity (OMEGA) systems, the latter of which include the TnpB, IscB, IsrB, and IshB nucleases. TnpB contains a RuvC-like nuclease domain (RNase H fold) that is specifically related to the homologous nuclease domain of CasI2, the effector nuclease of type V CRISPR-Cas systems, specifically, CAS12F, suggesting that TnpB is the evolutionary ancestor of Cas12. Phylogenetic analysis of the RuvC-like domains, indeed, supports independent origins of Cas12s of different type V subtypes from distinct groups of TnpBs. Recently, it has been demonstrated experimentally (through biochemical and cellular experiments) that these TnpBs are components of OMEGA (obligate mobile element-guided activity) systems that encode the ωRNA next to the nuclease gene (often overlapping with the 3′-end or the coding region of the latter). The ωRNA-TnpB complex is a RNA-guided DNA endonuclease. The ωRNA resembles a crRNA structurally but is larger and contains a spacer-like, target recognition sequence that lies immediately outside the transposon end suggesting that these nuclease are involved in RNA-guided transposition although other roles in the transposon life cycle cannot be ruled out. The OMEGA nucleases are programmable, that is, cleavage can be directed to any genomic region by replacing the spacer-like region by an arbitrary sequence. Hence these OMEGA nucleases have considerable potential as genome editing tools, and first attempts in this direction have been reported.
While TnpBs are highly abundant in bacteria and archaea, TnpB homologs (denoted Fanzors) have also been identified in diverse eukaryotes, including metazoans, fungi and many unicellular organisms, and some double-stranded (ds)DNA viruses with large genomes infecting unicellular eukaryotes. Two major groups of Fanzors have been identified: 1) Fanzor1 that are associated with eukaryotic transposons, including Mariners, IS4-like elements, Sola, Helitron, and MuDr, and 2) Fanzor2 systems that are found in IS607-like transposons and are present in dsDNA viral genomes. Despite the similarities between TnpB and Fanzors, Fanzors have not been surveyed comprehensively throughout eukaryotic diversity and, unlike the OMEGA nucleases, neither the biochemical activity of Fanzors nor their role in transposons have been studied experimentally.
The Examples herein report a comprehensive census of Fanzors in eukaryotic and viral genomes, phylogenetic analysis clarifying their prokaryotic origins and tracing their evolution, and RNA sequencing (RNA-seq) and biochemical experiments demonstrating the programmable RNA-guided endonuclease activity of the Fanzors, showcasing their utility as new genome editing tools.
To identify putative RuvC nucleases in eukaryotic and viral genomes, a comprehensive search was performed across eukaryotic and viral genomes using a profile derived from the multiple alignment of the RuvC domains from bacterial TnpB, IscB and IsrB, and the previously identified Fanzor1 and Fanzor2 proteins. This search yielded Fanzor proteins occurring across metazoans, fungi, plants, and diverse unicellular eukaryotesas well as giant viruses of the family Mimiviridae (FIGS. 1A-1B). Clustering these putative nucleases with selected representatives of TnpB, IscB, and IsrB revealed several distinct families of eukaryotic RuvC containing nucleases. One Family, which contains the previously discovered Fanzor1 proteins, occurred in diverse eukaryotes, including fungi, plants, various protists, and animals (
To characterize the Fanzor2 family, the Fanzor2 from Acanthamoeba polyphaga mimivirus (1svMimi Fanzor2) was selected. Leveraging the fact that IsvMimi, is present multiple times in the mimivirus genome, all copies of this Fanzor2 were aligned to find conserved elements both in the ORF and in the surrounding neighborhood. Similar to bacterial TnpB and IscB systems, a strong conservation both within protein-coding regions and in the non-coding region at the 3′ end of the IS607 MGE was found. This non-coding sequence conservation extended 200 base pairs past the end of IsvMimi ORF before reaching the right inverted repeat element IRR, in contrast to the more ORF-proximal IRR found in TnpB MGE. (
To evaluate whether the strongly conserved fRNA was associating with the Fanzor2 protein, the Isvmimi locus containing the non-coding RNA region and E. coli codon-optimized Isvmimi Fanzor2 protein in E. coli were co-expressed (
It was hypothesized herein that Isvmimi Fanzor2 is guided by the proximal fRNA to target and cleave DNA sequences. To reprogram this activity, an fRNA with the last 21 nucleotides targeting a novel 21 bp sequence was designed. Rosetta cells were co-transformed with both the fRNA with reprogrammed 3′ guide sequence and a Strep tagged Isvmimi to directly obtain the RNP in E. coli. (
Having determined the constraints on Isvmimi Fanzor2 cleavage, the location of this cleavage within the target was then mapped. The products from Isvmimi Fanzor2 cleavage were isolated and the locations of the ends were mapped using Sanger sequencing, finding that cleavage occurred in the TAM, with multiple nicks within the non-target strand (NTS) and a single nick in the targeted strand (TS). (
To understand if the glutamate rearrangement drives the unique cleavage properties, including cutting inside of the TAM and lack of collateral, the TnpB (Istvo5 TnpB) was purified, which also processes this glutamate rearrangement.
Having demonstrated that the Fanzor2 family had RNA programmable cleavage, the characterization shown herein was expanded to the additional families spanning viruses, plants, metazoans, fungi, and protists. Unlike the Fanzor2 systems, many of these broader family members are associated with diverse transposable element associations and sometimes lack readily identifiable MGE scars, complicating fRNA determination. To characterize an additional family member from plants, the Fanzor1 systems from the green algae Chlamydomonas reinhardtii (Cre Fanzor1) were selected, which contains multiple Fanzor1 copies. Cre Fanzor1 is associated with the eukaryotic Helitron 2 transposons, which do have identifiable short asymmetrical terminal inverted repeats (ATIRs) flanking the MGE insertion ends. The homologous Cre Fanzor1 was aligned to determine the putative conserved fRNA, and, similar to the Fanzor2 families, a strong conservation of fRNA regions was found.
To determine the relevant fRNA species, the region containing the putative Cre-1 Fanzor1 fRNA and a codon optimized Cre-1 Fanzor1 protein in E. coli were co-expressed. Similar to the Fanzor2 family, the Fanzor1 protein required fRNA co-expression for production of stable protein and RNA sequencing on purified RNP revealed a precise fRNA species processed near the 3′ end of the Fanzor1 protein, overlapping the 3′ ATIR of the MGE. This fRNA had strong predicted secondary structure, but was distinct from the Fanzor2 clade. The conservation of this non-coding RNA was further studied with the closest systems to the Cre systems in terms of protein sequence similarity and found that the non-coding RNA was conserved in both sequence and structure.
To reprogram Cre Fanzor1 protein cleavage using the putative fRNA, a Cre Fanzor1 RNP containing a guide against the previously used TAM library was purified. As with Isvmimi Fanzor2, Cre Fanzor1 stability was fRNA dependent. Co-incubation of this complex with the TAM library generated two significant bands in a guide and magnesium dependent fashion. Sequencing the uncleaved TAM targets determined a specific TAM preference that validated upon testing individual TAM targets enriched in the screen. The in vitro activity of Cre Fanzor1 showcases that active Fanzor proteins are evolutionarily widespread across diverse lineages.
To test whether programmable Fanzor nuclease could be applied for genome editing given their mesophilic operating temperature, the fRNA guide was engineered for expression in mammalian cells. Because there are two poly U stretches (>5 U) in the putative guide scaffolds for Isvmimi that can block U6 promoter expression, the fifth U inside the guide stem-loop region to interrupt the poly U stretch was mutated. 21 nt guides were designed using this redesigned scaffold against several positions inside the human EMX1 gene and tested for its indel activity in HEK293FT cells.
As Fanzors extend programmable nucleases into eukaryotes, the emergence of introns across Fanzor diversity was explored. Among the Fanzor families, a wide range of intron numbers was found. Using RNA sequencing data, the presence of three to four introns within the Cre Fanzor1 genes that are removed from the mature mRNA transcript was confirmed. Analyzing the conservation of the locus, it was surprisingly found that the introns are substantially less conserved than exonic sequences, implying that ancestral Fanzors inserted into host genomes via horizontal transfer and acquired introns overtime. It is unclear how splicing plays into the regulation of Fanzor expression and transposition activity.
Notably, Fanzor2 proteins occur within the IS607 transposon, which is similar to the TnpA family of proteins, suggesting Fanzor2 might serve as the eukaryotic TnpB counterpart for the known bacterial IS200/605 superfamily. Because of these associations, the full extent of Fanzor2 association with transposase domains was analyzed first, finding primarily an association with IS607 element transposases. These proteins are closely associated and can be found within readily identifiable inverted repeat element ends. By analyzing the host genome junctions with the IRL and IRR, it was found that the Fanzor2 transposons primarily insert in A/T rich target sequences. Many of these target motifs appear similar to the Isvmimi TAM preference, suggesting that Fanzor2 cleavage may be directly related to the insertion site preference for the transposon.
Unlike Fanzor2 systems, many previously found Fanzor1 proteins are associated with eukaryotic transposons, including DNA transposons from different superfamilies including Helitron, Mariner, IS4-like, Sola and MuDr, however, the full extent of transposons acquiring Fanzor1 into their MGE by analyzing nearby ORFs with transposon domains has not been previously characterized. While helitron and MuDr transposase ORFs do not directly associate with Fanzor1 inside the transposon, the other transposases do strictly associate within the transposon, motivating our guilt by association approach for finding additional transposase associations.
RNA-guided nucleases serve vital roles in horizontal gene transfer in prokaryotic hosts and mobile elements, allowing for both adaptive immunity a programmable gene flow. RNA programmable DNA nucleases shown herein are similarly abundant in eukaryotic nuclear genomes and viruses, including plant, fungal, and metazoan groups. These Fanzor nucleases, which contain the previously discovered Fanzor1 and Fanzor2 systems (Bao and Jurka 2013), are evolutionarily similar to the TnpB nucleases associated with 1S200/IS605 family transposons. This transfer of these nucleases from a prokaryotic to eukaryotic context may have occurred through large DNA viruses acquiring TnpBs via horizontal gene transfer from bacteria and phages in amoebae, serving as “melting pots” of HGT between prokaryotes and eukaryotes (Boyer et al. 2009). As Fanzor systems spread throughout eukaryotic diversity, introns were acquired within the Fanzor nucleases, likely driven by the improved fitness of spliced genes from enhanced nucleocytoplasmic transport (Dimaano and Ullman 2004). The co-evolution of Fanzors with the nuclear genomes of their eukaryotic hosts is supported by the intron density of Fanzor genes matching the intron density of their host genomes (Basu et al. 2008, Csuros et al. 2011). The co-evolution of Fanzor systems with their hosts nuclear genomes reported herein suggests preferential movement within hosts compared to HGT. The Fanzor family persistence and spread within eukaryotic genomes implies Fanzor systems spread within host genomes with minimal fitness cost or potential fitness gain to the host. Without wishing to be bound by theory, one possible mechanism of positive fitness of Fanzors could be maintenance of genome stability, as is the case with non-LTR retrotransposons that insert in repetitive regions and help maintain repetitive genes (Nelson et al. 2021).
Fanzor families are associated with diverse transposases, strongly suggesting multiple events capturing Fanzor proteins by these transposons during evolution and a putative role of RNA guided nuclease activity of Fanzors in transposition. This role could be through a variety of mechanisms, including: 1) precise excision of the transposon from the genome via self-homing, 2) passive homing of the transposon to new alleles via leveraging nuclease-induced DSBs and DNA repair mechanisms, such as homologous recombination, and 3) active homing of the transposon using RNA guided DNA binding or cleavage for direct targeting of transposase activity. The latter mechanism would be analogous to the CRISPR-associated Tn7-like transposons that possess RNA-guided transposition via acquisition of RNA-guided DNA binding CRISPR effectors in conjunction with transposase components (Strecker et al. 2019; Klompe et al. 2019). Moreover, as Fanzor-containing transposons harbor associated genes of diverse putative functions and multiple Fanzor families possess N-terminal domains of varying predicted functions, Fanzor families may have additional undetermined roles.
The biochemical characterization of Fanzor nucleases shown herein revealed both similarities with the related TnpB and CRISPR-Cas12f nuclease, as well as several important distinctions. Similar to the Cas12 and TnpB nucleases, Fanzors generate double stranded breaks through a single RuvC domain; however, unliked the Cas12 and TnpBs, which cut DNA targets distal from the 5′ PAM/TAM on the 3′ end of the guide, Fanzor proteins unexepectedly cut within the 5′ TAM region. Potentially related to the unique cleavage position is the surprising apparent loss of collateral activity from the Fanzor family. Without wishing to be bound by theory, it is hypothesized that because the TAM is more internal to the RNP:DNA complex, it is possible that the activated RuvC domain is not solvent exposed, preventing trans DNA cleavage upon target recognition. As opposed to the more T rich sequence constraints of Cas12 and TnpB families, the Fanzor TAM preference is surprisingly diverse, with AT rich preference for the Fanzor2 family and a GC-rich preference for Fanzor1 proteins. Lastly, while the non-coding RNA of Fanzor2 overlaps with the transposon IRR, much like TnpB's ωRNA, it is further downstream of the Fanzor ORF, whereas the muRNAs are contained within the 3′ of the TnpB ORF. Therefore, the Fanzors are a unique family of eukaryotic programmable nucleases distantly related to TnpBs and Cas12f systems.
It is surprisingly shown herein that Fanzors can be applied for genome editing with detectable cleavage and indel generation activity in human cells. The Fanzor enzymes provide multiple advantages including precise nuclease activity, a small size, and eukaryotic origins, which may reduce the immunogenicity of these nucleases in humans. The broad distribution of Fanzor proteins across the multiple eukaryotic kingdoms and associated viruses suggests a further, as yet-discovered abundance of RNA-guided systems. The evolution of these nucleases expands the field's understanding of horizontal gene transfer, transposition systems in eukaryotes, the evolution of programmable nucleases, and the spread of mobile genetic elements from prokaryotes to eukaryotes. Future studies utilizing improved abilities to infer spliced genes from eukaryotic diversity will likely uncover more RNA-guided enzymatic systems that might have broad biotechnological promise. Taken together, the Fanzor diversity leaves many systems and associated proteins to be explored and will expand the nuclease toolbox for new human therapeutics.
Fanzors are predicted to be programmable nucleases. Fanzors (Fanzor1 and Fanzor2) are proteins that were found to contain RuvC nuclease domains in eukaryotic genomes. They are predicted to be programmable nucleases based on RuvC domain and similarity to bacterial TnpBs. Computational analyses conducted herein show how the presence of a conserved non-coding region near the Fanzor genes that is likely the guide RNA for the protein. In this example, a number of these proteins were tested and verified that they are programmable nucleases. The impact of these are that they can be new enzymes for genome editing and they come from eukaryotic systems making them safer and potentially better for human therapeutics.
Putative RNA-guided nucleases were identified throughout eukaryotic genomes and their viral genomes by comprehensively mining 22,497 eukaryotic and viral assemblies from NCBI GenBank. This present search, seeded with a multiple alignment of RuvC domains from the previously identified Fanzor1 and Fanzor2 proteins (Bao et al. 2013), yielded 3,655 putative nucleases occurring across metazoans, fungi, algae, choanoflagellida, rhodophyta, unicellular eukaryotes, and multiple viral families (
Given the association of Fanzors with different transposons (Bao et al. 2013), a comprehensive eukaryotic transposon search was performed (Riehl et al, 2022) within 10 kb of all Fanzor MGE sequences (
Analyzing associations of Fanzor nucleases with surrounding proteins revealed numerous instances of transposase domains, including the serine resolvase found in IS607 elements, further demonstrating the inclusion of Fanzor in transposons (
Since TnpB and IscB systems are known to process either the 3′ end or 5′ end of the MGE RNA into ωRNA and subsequently bind to ωRNA for guided dsDNA cleavage activity (Karvelis et al., 2021; Altae-Train et al. 2021; Nety et al. 2023) a comprehensive noncoding RNA alignment search was performed on all Fanzor loci. The search revealed significantly longer Fanzor noncoding conservation on both the 3′ and 5′ ends of the MGEs compared to TnpB and IscB systems (
The conservation of the fRNA and similarity of Fanzor nucleases to prokaryotic RNA-guided nucleases suggested that the fRNA could associate with ApmHNuc and program DNA cleavage through ApmHNuc's conserved RuvC domains. To investigate potential fRNA-ApmHNuc binding, the A. polyphaga mimivirus Fanzor locus, containing the non-coding RNA region, and E. coli codon-optimized ApmHNuc, were co-expressed in E. coli (
It was hypothesized that ApmHNuc is guided by its associated fRNA to target and cleave DNA sequences. Testing this activity required both the engineering of a reprogrammed fRNA and the determination of sequence preferences, akin to a target adjacent motif (TAM) (Karvelis et al. 2021; Altae-Tran et al. 2021). A synthetic fRNA was generated by combining a 3′-terminal 21-nt targeting sequence with the fRNA scaffold (ending at the IRR) determined through RNA profiling. Rosetta cells were co-transformed with plasmids coding for both the synthetic fRNA and ApmHNuc, and isolated the RNP complex from E. coli. To determine potential sequence preferences of ApmHNuc, cleavage on a DNA target containing a randomized 7 nucleotide TAM 5′ of a 21 bp target region complementary to the fRNA targeting sequence was tested. The TAM library was co-incubated with purified ApmHNuc RNPs containing either targeting or scrambled synthetic fRNA guide sequences, and the relative depletion of sequences was profiled with next-generation sequencing (NGS). TAM depletion analysis revealed a strong 5′ GGG motif adjacent to the target site (
Similar to TnpB (Nakagawa et al. 2023; Sasnauskas et al. 2021), cleavage by ApmHNuc is likely mediated by conserved acidic residues in the RuvC domain (
Cleavage locations of RNA-guided nucleases vary substantially, with cleavage sites both up and downstream from the target location. To profile ApmHNuc cleavage patterns, ApmHNuc reaction products were purified and the locations of the cleavage ends were mapped using Sanger sequencing. Cleavage occurred in the 3′ regions of the target sequence, with multiple nicks in both the target strand (TS) and the non-target strand (NTS) (
Compared to a majority of TnpB families, Fanzor nucleases contain a substitution in the canonical catalytic RuvC-II site from a glutamate residue to a catalytically inert residue (proline, glycine) (
To compare the structural conformations of the canonical and alternative catalytic sites, a TnpB from Thermoplasma volcanium GSS1 (TvoTnpB) harboring a rearranged site was selected, and compared experimentally determined or computationally predicted structures between ApmHNuc, TvoTnpB (re-arranged RuvC-II), TnpB from Deinococcus radiodurans R1 (Isdra2; canonical RuvC domain), and Cas12f from uncultured archaeon (UnCas12f) and compared the spatial configurations of the canonical and alternative catalytic glutamic acids (
To generalize the activity of the rearranged RuvC domain beyond ApmHNuc, the nuclease activity of TvoTnpB was evaluated, which contains the alternative glutamic acid catalytic residue. TvoTnpB RNPs were generated by co-expressing the TvoTnpB protein with its native locus in E coli, and these RNP were isolated to profile the associated noncoding RNA by NGS. A significant enrichment of noncoding RNA expression was found near the right end (RE) element, similar to other TnpB systems (
Lastly, it was hypothesized that the rearranged RuvC catalytic site of the Fanzor might be less solvent exposed, as suggested by the structural analysis (
Whereas the Family 5 Fanzor systems are closely related to TnpB systems, it was found that most Fanzor orthologs, including Fanzor1 nucleases, are distantly related and have radiated throughout all eukaryotic branches of life, including amoeba, fungi, plates, and animals (
To demonstrate that these expanded Fanzor family members actively process and associate with their cognate fRNAs, family 1 Fanzor from the unicellular green alga Chlamydomonas reinhardtii (CreHNuc) was focused on (
The CreHNuc systems are associated with Helitron 2 transposons, which contain identifiable short target site duplications (TSDs) and asymmetrical terminal inverted repeats (ATIRs). In the CreHNuc-1 system, we found defined TSD and ATIR sequences flanking 5′ and 3′ of the CreHNuc MGE. The CreHNuc-1 system lacks the RepHel domain, indicating that it is an non-autonomous Helitron. It was hypothesized that either the 3′ TSD or the 3′ ATIR sequence indicates the end of the fRNA of CreHNuc-1 and performed small RNA sequencing directly from the native green algae organism, finding significant enrichment of small non-coding RNAs aligning to the 3′ UTR of the CreHNuc-1 mRNA (
All 6 full-length copies of CreHNuc systems inside the genome were aligned and strong alignment was found near the C-terminal coding region of the Fanzor nuclease, which contains the RuvC domain, and variable N-terminal compositions (
To evaluate the functional role of the CreHNuc-1 fRNA, the CreHNuc-1 protein was co-expressed either with its native fRNA on the 3′ end of the MGE or a scramble RNA sequences. It was found that CreHNuc is only stable when coexpressed with its fRNA, suggesting that CreHNuc actively associates with its fRNA for stability (
To evaluate the functional activity of the identified ApmHNuc NLS, the N-terminus NLS tag of ApmHNuc was fused to either the N-terminus or C-terminus of super-folded GFP (sfGFP). The sfGFP was also attached onto the N-terminus of wild-type ApmHNuc and visualized its location via fluorescent microscopy. It was found that compared to a wild-type sfGFP, the N-terminus NLS tag of ApmHNuc fused to either terminus of sfGFP resulted in a strong nuclear localization of sfGFP (
Next, to test whether Fanzor systems could be applied for mammalian genome editing given their mesophilic operating temperature and eukaryotic nature, ApmHNuc was codon-optimized for mammalian expression and engineered its fRNA guide for expression in mammalian cells. Since the fRNA is longer in length than typical ωRNAs (>350 nt), HEK293T cells were co-transfected with a T7 promoter-driven guide expression plasmid along with human codon-optimized T7 polymerase and wild-type ApmHNuc protein. A reporter plasmid that carries the 21 nt target matching the T7-driven guide was designed in front of a Gaussia luciferase (Gluc) out of frame from the start codon along with a cypridina luciferase (Cluc) driven by a constitutive promoter on the same plasmid to normalize for transfection efficiency. Indel activity would knock the Gluc into frame, allowing for detectable Gluc luciferase activity. Using this reporter system, a significant increase in normalized luciferase was found in the targeting guide condition compared to a non-targeting guide control, suggesting that indel were generated by the ApmHNuc protein (
Putative RNA-guided nucleases were identified across 22,497 eukaryotic and viral assemblies from NCBI GenBank by searching for similarity to a multiple alignment of RuvC domains from known Fanzor1 and Fanzor2 proteins (Bao et al. 2013). There were 3,655 putative nucleases with unique sequences (using a 70/o similarity clustering threshold) that occurred across metazoans, fungi, choanoflagellates, algae, rhodophyta, diverse unicellular eukaryotes, and multiple viral families (
Phylogenetic analysis of the expanded set of Fanzor nucleases and a selection of closely related TnpBs revealed 5 distinct Fanzor clades supported by bootstrap analysis, with four Fanzor1 families (Fanzor1a-1d) and a single Fanzor2 clade (
Projecting Fanzor hosts onto the eukaryotic tree of life shows broad spread into amoebozoa, several other groups of unicellular eukaryotes, plants, fungi, and animals, including Chordata and Arthopoda (
Fanzors commonly associate with different transposons (Bao et al. 2013). A comprehensive transposon search was performed (Chen et al. 2018) within 10 kb of Fanzors, analyzing the identity of the associated ORFs by domain search (
Table 3. Fanzor families in eukaryotics genomes and their identified transposon associations.
Fanzor elements are named after the host species. Fanzor2 elements are indicated by *. The left and right termini are indicated by L. and R. respectively, in the orientation of the encoded Fanzor protein. N: none; n.a.: not available; i.e.: incomplete. #: The encoded Tpase (or coding sequences). If a given Fanzor element does not encode Tpase, but the superfamily it belongs can be determined, the superfamily name is parenthesized. Rows highlighted in white correspond to Fanzor-Transposon associations previously identified (Bao et al. 2013). Bold rows correspond to new transposon associations identified in this study.
3 (TAN)
4? (TTAA)
4-bp (ATAN)
TnpB and IscB nucleases process the ends of the transposon-encoded RNA transcript into ωRNA, which complex with the respective nucleases to form a RNA-guided dsDNA endonuclease ribonucleoprotein (RNP) (Karvel et al. 2021; Altae-Tran et al. 202; Nety et al. 2023). Fanzor loci were searched for putative regions encoding OMEGA-like RNAs, based on conservation of non-coding sequence. There was conservation extending beyond the detectable Fanzor ORF on both 5′ and 3′ ends of the ORF, with the conserved regions significantly longer for some Fanzor families than those in TnpB and IscB loci, although some families like the viral-enriched Fanzor2 have non-coding lengths similar to those of TnpB systems (
The Fanzor2 from the Acanthamoeba polyphaga mimivirus (ApmFNuc) that is encoded within a IS607 transposon and contains a TnpA transposase and defined inverted terminal repeats was further investigated to explore the potential activity and expression of these conserved regions (
It was hypothesized that the fRNA forms a complex with ApmFNuc and directs binding and DNA cleavage to a specific sequence in the target. To investigate potential fRNA-ApmFNuc binding, the A. polyphaga mimivirus Fanzor locus, containing the non-coding RNA region, and an E. coli codon-optimized ApmFNuc was co-expressed in E coli (
Testing RNP cleavage activity required both the engineering of a reprogrammed fRNA and the determination of any sequence preferences, akin to the target adjacent motif (TAM) in the case of TnpB and IscB (Karvelis et al. 2021, Altae-Train et al. 2021). A 3′-terminal 21-nt targeting sequence was combined with the fRNA scaffold determined through RNA profiling to engineer a synthetic fRNA, co-expressed the synthetic fRNA and ApmFNuc in E co/i, and isolated the reprogrammed RNP complex. Cleavage on a DNA target containing a randomized 7 nucleotide TAM 5′ of a 21 nt target region complementary to the fRNA targeting sequence was tested to determine potential sequence preferences of ApmFNuc. This TAM library was co-incubated with purified ApmFNuc RNPs containing either targeting or scrambled synthetic fRNA guide sequences. The relative depletion of sequences was profiled with next-generation sequencing (NGS). TAM depletion analysis revealed a strong 5′ GGG motif adjacent to the target site (
Cleavage locations of RNA-guided nucleases vary substantially, with cleavage sites located either upstream or downstream of the target sequence. To profile ApmFNuc cleavage patterns, ApmFNuc reaction products were purified and mapped the locations of the cleavage ends using Sanger sequencing. Cleavage occurred in the 3′ regions of the target sequence, with multiple nicks in both the target strand (TS) and the non-target strand (NTS) (
This study sought to explore whether Fanzor2 proteins from diverse eukaryotes also are active RNA-guided nucleases. Three Fanzor2 representatives from three animals and a Fanzor1 representative from a plant were chosen for this study: 1) Fanzor2 from Aercenaria mercenaria (Venus clam, MmFNuc), 2) Fanzor2 from Dreissena polymorpha (Zebra mussel, DpFNuc), 3) Fanzor2 from Batillaria attramentaria (Japanese mud snail; BaFNuc), and 4) Fanzor1 from Klebsormidium nitens (freshwater green algae; KnFNuc) (
Next, a 7N TAM library was challenged with MmFNuc, DpFNuc, BaFNuc, and KnFNuc RNPs with fRNA guide sequences complementary to the library target. There was strong TAM selection corresponding to TTTA, TA, TTA, and TTA TAMs for MmFNuc, DpFNuc, BaFNuc, and KnFNuc, respectively (
Alignment of Fanzor nucleases and TnpB members shows that, compared to the majority of TnpBs, Fanzor nucleases contain a substitution in the catalytic RuvC-11 motif from a glutamate to a catalytically inert residue (proline or glycine) (
AlphaFold2-generated structural models of ApmFNuc, MmFNuc, DpFNuc, BaFNuc, KnFNuc, and a TnpB from Thermoplasma volcanium GSS1 (TvTnpB) that both contain a rearranged catalytic site with the Cryo-EM structures of TnpB from Deinococcus radiodurans R1 (Isdra2) and Cas12f from uncultured archaeon (UnCas12f) containing the canonical catalytic site were compared (
To test the predicted role of the conserved alternative glutamate in Fanzor activity, two ApmFNuc RNP with mutations at predicted catalytic sites in RuvC-I (D324A) or the alternative glutamate in RuvC-11 (E467A) were purified (
The activity of the TnpB2, TvTnpB, was then profiled to determine if these re-arranged TnpBs were similarly active. TvTnpB RNPs were isolated by co-expressing the enzyme with its native locus in E. coli and profiled associated noncoding RNA by NGS (
Although the rearranged RuvC catalytic site of the Fanzors and TnpB2 did not impact on target cleavage, it was hypothesized that it could affect the collateral cleavage activity of the enzyme (Chen et al. 2018, Abudayyeh et al. 2016). ApmFNuc, MmFNuc, DpFNuc, BaFNuc, TvTnpB, and the canonical TnpB Isdra2TnpB were profiled for either RNA or DNA collateral cleavage activity by co-incubating the RNP complexes with their cognate targets along with either RNA or DNA cleavage reporters, single-stranded nucleic acid substrates functionalized with a quencher and fluorophore that become fluorescent upon nucleolytic cleavage. While all nucleases had similar on-target cleavage efficiencies (
As eukaryotic RNA-guided endonucleases would need to enter the nucleus to access their genomic targets, it was hypothesized that Fanzor nucleases might have harbor nuclear localization signals to actively cross the nuclear membrane. In the Alphafold2 predicted structures of ApmFNuc, a disordered region of 64 amino acids was discovered at the N-terminus (
To evaluate the localization of ApmFNuc and its NLS, a super-folder GFP (sfGFP) was fused to the N-terminus of ApmFNuc and attached the N-terminal portion of ApmFNuc containing the NLS to either the N-terminus or C-terminus of sfGFP. sfGFP localization was visualized via fluorescent microscopy, finding that sfGFP with the NLS from ApmFNuc fused to either terminus had strong nuclear localization (
Next, a broad search for Fanzor-encoded NLS sequences was performed by analyzing each Fanzor ORF for a predicted NLS. Across all Fanzor families, ˜60% of ORFs had readily identifiable NLS sequences, on par with the prediction accuracy of a validated set of NLS-containing proteins (Nguyen et al. 2009) and substantially greater than the fraction of NLS sequences predicted for cytosolic human proteins (
Next, codon-optimizing ApmFNuc, DpFNuc, MmFNuc, and BaFNuc were then tested for mammalian genome editing by engineering their fRNA guide scaffolds for optimal U6-based expression in mammalian cells by removing poly-U stretches (
RNA-guided DNA endonucleases are prominent in prokaryotes including roles in innate immunity mediated by prokaryotic Argonautes (Swarts et al. 2014); adaptive immunity by CRISPR systems (Hsu et al. 2014, Hille et al. 2018, Doudna et al. 2014); RNA-guided transposition by CRISPR-associated transposases (Strecker et al. 2019, Klompe et al. 2019), and still uncharacterized functions of OMEGA nucleases in transposon life cycles (Karvelis et al. 2021, Altae-Tran et al. 2021). In eukaryotes, whereas RNA-guided cleavage of RNA is the cornerstone of the RNA-interference defense machinery and post-transcriptional regulation (Hannon et al. 2002, Hutvagner et al. 2008), RNA-guided cleavage of genomic DNA has not been demonstrated, to our knowledge. The examples show that the previously uncharacterized eukaryotic homologs (Bao et al. 2013) of the OMEGA effector nuclease TnpB are RNA-guided, programmable DNA nucleases. Extensive searching of diverse genomes of eukaryotes and their viruses enabled the discovery of thousands of RuvC-containing Fanzor nucleases. While this manuscript was in review, additional work characterized Fanzor nucleases biochemically and in mammalian cells, further confirming Fanzors as RNA-guided nucleases (Saito et al. 2023).
Phylogenetic analysis of the Fanzors together with their closest TnpB relatives revealed 5 major Fanzor families, which all contain Fanzor nucleases interspersed with prokaryotic TnpBs, suggesting that TnpBs entered the eukaryotic genomes on multiple, independent occasions. Considering the high abundance of TnpBs in bacteria and archaea, and their mobility, along with the exposure of unicellular eukaryotes to bacteria, this apparent history of multiple jumps into eukaryotic genomes does not appear surprising. Furthermore, given the wide spread of Fanzors in eukaryotes, together with the near ubiquity of TnpBs in bacteria and archaea, it appears likely that TnpBs were originally inherited from both archaeal and bacterial partners in the original endosymbiosis that triggered eukaryogenesis (Lopez-Garcia and Moreira 2023). Subsequent events of TnpB capture by eukaryotes could occur via additional endosymbioses as well as sporadic contacts with bacterial DNA. Notably, however, the high intron density in many Fanzors implies their long evolution in many groups of eukaryotes. The history of Fanzor2, however, is quite distinct from the four Fanzor1 families. This variety of Fanzors are enriched in viruses and in IS607 transposons and are far more closely similar to TnpB than members of other Fanzor families, suggesting likely origin from phagocytosis of TnpB-containing bacteria by amoeba and subsequent spread via amoeba-trophic giant viruses (Boyer et al. 2009).
Association of Fanzor nucleases with transposases suggests a role for their RNA-guided nuclease activity in transposition similarly to the case of TnpB. The exact nature of that role, however, remains unknown. TnpB has been reported to boost the persistence of the associated transposons in bacterial populations (Pastemak et al. 2013, Meers et al. 2023). TnpB and Fanzors potentially could perform different mechanistic roles in transposon maintenance. In particular, these RNA-guided nucleases could target sites from which a transposon was excised, initiating homology directed repair through a transposon-containing locus, restoring the transposon in the original site and thus serving as an alternate mechanism of transposon propagation (Meers et al. 2023). The association of TnpBs and Fanzors with diverse types of transposases suggests that the function(s) of the RNA-guided nucleases do not strictly depend on the transposition mechanism.
The biochemical characterization of both viral and eukaryotic Fanzor nucleases revealed both similarities with the homologous TnpB and Cas12 RNA-guided nucleases and several notable distinctions. Like TnpB and Cas12, Fanzor nucleases generate double-stranded breaks through a single RuvC domain and cleave the target DNA near the 3′ end of the target. However, unlike canonical TnpB and Cas12 enzymes, which possess strong collateral activity against free ssDNA, Fanzor nucleases and a subset of related TnpBs contain rearranged catalytic sites that are not conducive to collateral activity. In contrast to the T-rich TAMs of TnpB and PAMs of Cas12, the Fanzor TAM preference is diverse, with a GC preference observed for the viral ApmFNuc and A/T rich preferences for the eukaryotic MmFNuc, DpFNuc, and BaFNuc. In some cases, the TAM preference agrees with the insertion site sequence, which is compatible with the role of Fanzors in transposition. Finally, the fRNA of Fanzors overlaps with the transposon IRR and TIR, much like TnpB's ωRNA, but extends farther downstream of the Fanzor ORF, in contrast to the ωRNAs that ends near the 3′ regions of the TnpB ORF. Furthermore, although the Fanzor nucleases originated from TnpB, some features of these eukaryotic RNA-guided nucleases notably differ from those of the prokaryotic ones, reflecting their adaptation functioning in eukaryotic cells, such as the acquisition of introns and functional NLS sequences for nuclear localization.
The examples demonstrate that Fanzor nucleases can be applied for efficient genome editing with detectable cleavage and indel generation activity in human cells. While the Fanzor nucleases are compact (˜600 amino acids), which could facilitate delivery, and their eukaryotic origins might help to mitigate the immunogenicity of these nucleases in humans, additional engineering is needed to further improve the activity of these systems in human cells, as has been accomplished for other miniature RNA-guided nucleases such as Cas12f (Bigelyte et al. 2021, Wu et al. 2021, Xu et al. 2021, Kin et al. 2021). The broad distribution of Fanzor nucleases among diverse eukaryotic lineages and associated viruses suggests many more currently unknown RNA-guided systems could exist in eukaryotes, serving as a rich resource for future characterization and development of new biotechnologies.
Following protein purification and sequencing, variants of Fanzor proteins are evolved using PACE systems to form a large library of Fanzor mutants. Mutants are then subjected to selection based on the lack of DNA collateral activity using an antibiotic resistance selection system. Cells harboring Fanzor mutants that restore antibiotic resistance are isolated and subjected to additional successive rounds of mutation and selection under varying selection stringencies.
Those Fanzor mutants that conferred a survival advantage are tested for base editing activity in mammalian cells across >5 endogenous genomic loci to assess editing efficiency, product purity, the size of the editing window, and sequence context preferences. Successive rounds of directed evolution are then performed until the resulting Fanzors perform at a useful level (e.g., >20% editing, >50% product purity, <5% indels, and an editing window of 2-8 nucleotides).
For each position that is experimentally screened for single mutation effects on Fanzor activity, each residue is computationally mutated into other amino acid types. Single sequence structure prediction is performed using AlphaFold2. The model with the highest per-residue confidence score (pLDDT) is computationally evaluated for enzyme and substrate binding free energy. Candidate Fanzor proteins are physically synthesized and evaluated for their genome editing activity using methods described herein.
A profile of the Fanzor RuvC domain (Fanzor profile) was constructed by aligning the previously discovered Fanzor proteins (seed sequences) with MUSCLE v5 (-align), extracting the RuvC domain, and building a profile HMM with hmmbuild (default options) from the HMMER v3 suite of programs. An initial set of putative Fanzor proteins was gathered by searching all annotated proteins and translated ORFs (stop codon to stop codon) longer than 100 residues in NCBI eukaryotic and viral assemblies (one assembly per species) as well as all full length proteins annotated on eukaryotic and viral sequences in GenBank (hmmsearch-E 0.001-Z 61295632). To predict introns in Fanzor ORFs, AUGUSTUS v3.5.0 and Spain v2.4.13f were applied to the genomic region containing the ORF (10 kb upstream/downstream). AUGUSTUS was used for ab initio gene prediction when there was an available parameter set of the same class as the target species. Tantan was used to soft-mask the genome prior to gene prediction using an “-r” parameter of 0.01 if the genome AT fraction was less than 0.8 and 0.02 otherwise (with the suggested scoring matrix for AT-rich genomes). Spain was used to splice-align Fanzor proteins to the Fanzor ORFs (default options). The protein query set for Spain was generated by searching UniClust90 and GenBank eukaryotic proteins with the Fanzor profile. The Fanzor profile was iteratively refined by repeatedly searching the initial set of proteins (hmmsearch-E 0.0001-domE 1000-Z 69000000), extracting the RuvC domain, clustering with Mmseq2 (-min-seq-id 0.5-c 0.9), aligning the cluster representatives with the profile seed sequences, manually refining the alignment, building a new profile, and using the new profile for the next round. Three rounds of refinement were completed. The refined profile was used for a final round of searches and clusters that would have been included in the profile were kept for the subsequent filtering steps. To reduce the likelihood of including genome assembly contaminants in downstream analysis, all Fanzor proteins from NCBI assemblies marked as contig level completeness or those originating from contigs shorter than 50 kb (only from assemblies) were discarded. The remaining sequences were clustered using a combination of Diamond v2. 1.6 (-evalue 0.0001-id 70-query-cover 90-subject-cover 90-max-target-seqs 500-comp-based-stats 3) and MCL (-I 4.0). Each cluster was aligned with MUSCLE and a consensus sequence was computed using a custom python script. The RuvC domains were extracted from each consensus sequence and all aligned with MUSCLE. The alignment was manually inspected and filtered to yield a final set of Fanzor sequences.
A profile HMM was constructed from a multiple sequence alignment of each Fanzor family and used to query a custom database of prokaryotic and metagenomic assemblies using HMMER (-E 0.0001-Z 61295632). Sequences identical to another sequence were discarded and the remaining were clustered with Mmseqs2 (-min-seq-id 0.7-c 0.9-s 7). Each TnpB sequence was assigned to a Fanzor family based on the profile that matched it with the highest domain bitscore. The split-RuvC domain was extracted from each cluster representative and further clustered with Mmseqs2 (-min-seq-id 0.5-c 0.9-s 7) for a two-step clustering process. These cluster representatives were aligned with MUSCLE and sequences without alignment to the conserved DED motif were discarded.
To make a combined tree of TnpBs and Fanzor sequences, the split-RuvC domain was extracted from every Fanzor consensus sequence and clustered with Mmseqs2 (-min-seq-id 0.9-c 0.9). These cluster representatives were aligned, along with the TnpB split-RuvC domain cluster representatives, using MUSCLE. To make a tree of only Fanzor sequences, the extracted split-RuvC domains were aligned with MUSCLE without clustering. In both cases, a approximately-maximum-likelihood phylogenetic tree was constructed with FastTree2 (-lg-gamma) and visualized with R and the ggtree suite of packages.
To make a phylogenetic tree of TnpB and Fanzor sequences, the split-RuvC domain was extracted from every Fanzor consensus sequence and aligned to the split-RuvC domain of a 3 k random subset of the two-step clustered TnpB representatives using MUSCLE (-supers). Sequences appearing to be fragments were discarded from the alignment and the remaining sequences were realigned. An approximately-maximum-likelihood phylogenetic tree was constructed with FastTree2 (-lg-gamma). All branches with a local support value (as computed by FastTree) less than 0.7 were collapsed and the tree rooted at the midpoint. The subsequent tree was visualized with R and the ggtree suite of packages.
NLStradamus was used with default threshold at 0.6 and model option 2 (four-state bipartite model) to predict NLS domains. For background false positive rate determination, a comprehensive search on Uniprot is performed by looking for Homo sapiens cytosolic proteins (with reviewed status) and a total of 1126 proteins are pulled out for analysis. For on target false negative rate determination, the original set of training sequences that include known NLS containing proteins from NLStradamus is used (Nguyen Ba et al. 2009). NLS sequences cloned for experimental testing are listed in Table 5.
Prototheca cutis
Andricus curvator
Torulaspora
delbrueckii
Globisporangim
ultimum
ultimum
Scenedesmus sp.
Scenedesmus sp.
Chlamydomonas
Chlamydomonas
Chlamydomonas
Chlamydomonas
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Apophysomyces
variabilis
Cyanidischyzon
merolae
Cyanidischyzon
merolae
Chlamydomonas
reinhardtii
Contarinia
NLStradamus was used with default options to predict NLS domains.
NLStradamus was used with default threshold at 0.6 and model option 2 (four-state bipartite model) to predict NLS domains. For background false positive rate determination, a comprehensive search on Uniprot is performed by looking for Homo sapiens cytosolic proteins (with reviewed status) and a total of 1126 proteins are pulled out for analysis. For on target false negative rate determination, the original set of training sequences that include known NLS containing proteins from NLStradamus is used (Nguyen Ba et al 2009). NLS sequences cloned for experimental testing are listed in Table 5.
Prediction of Transposon Associations with Fanzor Systems
RFSB transposon classifier (Riehl et al. 2022) is used to classify Fanzor-transposon associations by inputting the surrounding 10 kb genomic sequence around the Fanzor protein. The classify mode is used with default parameters to make the prediction. Afterward, all predicted DNA transposon is mapped back to the phylogenetic tree. For all Fanzor nucleases that were classified with transposons, cd-hit is used to cluster these sets of Fanzor proteins with default parameters to find any clusters with two or more sequences for multiple sequence alignments. Then these clusters containing (>2 Fanzor systems) were blasted against all Repbase documented transposons (Bao et al. 2015). Left and right end elements, terminal inverted repeats (TIR), and their associated transposons are then determined by either protein homology to known transposons in Repbase or high similarity of TIR/LE/RE element to known transposon profiles.
Prediction of Fanzor-Associated ncRNA
Fanzor that were not simply ORF translations were clustered along their entire length at 70% sequence identity and 95% coverage with Mmseqs2 (-min-seq-id 0.7-c 0.95). Each cluster with at least two sequences was subject to ncRNA prediction. For each cluster, the 5′ region of the first exon plus 1.5 kb upstream bases and 3′ region of the last exon plus 1.5 kb downstream bases were cut from sequence. The 5′ and 3′ regions were aligned separately with MAFFT (default options). Each column of the alignment was scored for conservation and the change point in conservation scores was predicted with the R changepoint package to detect a drop in conservation. If the predicted change point was found to be at least 13 bases outside of the exon boundary of every sequence in the alignment, the conserved portion of the exon, plus 11 bases past the change point, were folded with RNAalifold from the ViennaRNA software suite.
To purify Fanzor or TnpB protein, Rosetta2 DE3 pLys cells were transformed with a twin-strep-sumo tag fused to the N-term of a Fanzor or TnpB construct along with the predicted fRNA/ωRNA driven by a separate vector. Following transformation, single colonies were picked from the agar plate containing antibiotics and picked into a starter culture of 10 mL for overnight incubation at 37 degree Celsius. The starter culture was transferred to 2 L of TB with the designated antibiotics and grown until the OD reached between 0.6-0.8. The culture was moved to 4C for 30 minutes prior to induction with 0.5 mM IPTG induction. The cultures were then grown at 16 degree Celsius overnight and harvested by centrifugation the next day. The pellet is then flash frozen at −80° C. and subsequently homogenized in lysis buffer (0.02M Tris-HCl pH8.0, 0.5M NaCl, 1 mM DTT, and 0.1M cOmplete™, EDTA-free Protease Inhibitor Cocktail (Merck Millipore) with high-pressure sonication for 15 minutes. The homogenized lysates are then centrifuged at 14,000 RPM for 30 minutes at 4° C. The clarified supernatant is isolated from the subsequent bacterial pellet and incubated with Strep-Tactin® XT 4Flow® high capacity resin (Cat. No. 2-5030-010) for 1 hour. Following incubation, the crude solution is loaded onto a Glass Econo-Column® Column for gravity flow chromatography and washed three times with the previously described lysis buffer. To elute tagged protein, 10 units of sumo protease is then added onto the column for on-column cleavage overnight at 4° C. The next day, the eluent is collected and concentrated through an Amicon® Ultra-15 Centrifugal Filter (Cat. No. UFC9030) before continuing to FPLC. To purify desired protein from added sumo protease, the concentrated eluent is loaded onto a Superdex® 200 Increase 10/300 GL gel filtration column (GE Healthcare). The column was equilibrated with running buffer (10 mM HEPES (pH 7.0 at 25° C.), 1 M NaCl, 5 mM MgCl2, 2 mM DTT). The Peak fractions containing RNP are pulled and analyzed by SDS-PAGE. Correct fractions are concentrated again with amicon filter tubes and subsequently buffer is exchanged into storage buffer (0.02M Tris HCL PH8, 0.25M NaCl, 50% glycerol, 2 mM DTT) and stored at −20 for further use. TnpB proteins follow the same purification procedure with the following modifications: T7 express (NEB) pLys strain is used for transformation and subsequent culture.
Fanzor protein sequences were E. coli codon optimized using the IDT codon optimization tool, and fRNA scaffolds were synthesized by IDT eBlock gene fragments. Cell-free transcription/translation reactions were carried out using a PURExpress In Vitro Protein Synthesis Kit (NEB) as per the manufacturer's protocol with half-volume reactions, using 75 ng of template for the protein of interest, 125 ng of template for the corresponding fRNA or ωRNA with a guide targeting the TAM library and 30 ng of TAM library plasmid. Reactions were incubated at 37° C. for 4 hours, then quenched by heating up to 95 degree Celcius for 15 minutes and cooling down to 4° C. 10 ug RNase A (Qiagen) is added followed by a 15 min incubation at 50° C. DNA was extracted by PCR purification and adaptors were ligated using an NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) using the NEBNext Adaptor for Illumina (NEB) as per the manufacturer's protocol. Following adaptor ligation, cleaved products were amplified specifically using one primer specific to the TAM library backbone and one primer specific to the NEBNext adaptor with a 10-cycle PCR using NEBNext High Fidelity 2×PCR Master Mix (NEB) with an annealing temperature of 65° C., followed by a second 12-cycle round of PCR to further add the Illumina i5 adaptor. Amplified libraries were gel extracted, quantified by qubit (Invitrogen) and subjected to paired-end sequencing on an Illumina MiSeq with Read 1 200 cycles, Index 1 8 cycles, Index 2 8 cycles and Read 2 80 cycles. TAMs were extracted and position weight matrix based on the enrichment score was generated and Weblogos were visualized based on this position weight matrix using a custom Python script. All sequencing primers used are listed in Table 6.
Fanzor protein sequences were E. coli codon optimized using the IDT codon optimization tool, and fRNA scaffolds were synthesized by IDT eBlock gene fragments. Cell-free transcription/translation reactions were carried out using a PURExpress In Vitro Protein Synthesis Kit (NEB) as per the manufacturer's protocol with half-volume reactions, using 75 ng of template for the protein of interest, 125 ng of template for the corresponding fRNA or ωRNA with a guide targeting the TAM library and 30 ng of TAM library plasmid. Reactions were incubated at 37° C. for 4 hours, then quenched by heating up to 95 degree Celcius for 15 minutes and cooling down to 4° C. 10 ug RNase A (Qiagen) is added followed by a 15 min incubation at 50° C. DNA was extracted by PCR purification and adaptors were ligated using an NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) using the NEBNext Adaptor for Illumina (NEB) as per the manufacturer's protocol. Following adaptor ligation, cleaved products were amplified specifically using one primer specific to the TAM library backbone and one primer specific to the NEBNext adaptor with a 10-cycle PCR using NEBNext High Fidelity 2×PCR Master Mix (NEB) with an annealing temperature of 65° C., followed by a second 12-cycle round of PCR to further add the Illumina i5 adaptor. Amplified libraries were gel extracted, quantified by qubit (Invitrogen) and subjected to paired-end sequencing on an Illumina MiSeq with Read 1 200 cycles, Index 1 8 cycles, Index 2 8 cycles and Read 2 80 cycles. TAMs were extracted and position weight matrix based on the enrichment score was generated and Weblogos were visualized based on this position weight matrix using a custom Python script. All sequencing primers used are listed in table S4.
1 uM of purified RNP and 100 ng of the 7N TAM library is incubated at 37 degree Celsius in NEB buffer 3 for 3 hours. Subsequently, reaction is purified and analyzed following the same procedure as cell-free transcription/translation TAM screen. TAM library sequence and guides used are listed in table S5.
Cell-free transcription/translation reactions were carried out using a PURExpress In Vitro Protein Synthesis Kit (NEB) as per the manufacturer's protocol with half-volume reactions using 75 ng of template for the protein of interest and a 100 ng of fRNA or ωRNA. Reactions were incubated at 37° C. for 4 hours to allow for RNP formation, then placed on ice to quench in vitro transcription/translation. 50-100 ng of target substrate was then added, and the reactions were incubated at the specified temperature for 1 additional hour. Reactions were then quenched by heating up to 95 degrees for 15 minutes and cooling back down to 50-degrees Celcius for addition of 10 ug RNase A (Qiagen) for 10 minutes incubation. DNA was extracted by PCR purification using minElute columns (Qiagen) and run on 6% Novex TBE gels (Thermo Fisher Scientific) as per the manufacturer's protocols, as specified in figures. Gels were stained with 1×SYBR Gold (Thermo Fisher Scientific) for 10-15 min and imaged on a ChemiDoc imager (BioRad) with optimal exposure settings. Each condition was performed twice for replicability.
Heterologous expression in E. coli Rosetta2 chemically competent E. coli were transformed with plasmids containing the locus of interest. A single colony was used to seed a 5 mL overnight culture. Following overnight growth, cultures were spun down, resuspended in 750 μL TRI reagent (Zymo) and incubated for 5 min at room temperature. 0.5 mm zirconia/silica beads (BioSpec Products) were added and the culture was vortexed for approximately 1 minute to mechanically lyse cells. 200 μL chloroform (Sigma Aldrich) was then added, culture was inverted gently to mix and incubated at room temperature for 3 min, followed by spinning at 12000×g at 4° C. for 15 min. The aqueous phase was used as input for RNA extraction using a Direct-zol RNA miniprep plus kit (Zymo). Extracted RNA was treated with 10 units of DNase I (NEB) for 30 min at 37° C. to remove residual DNA and purified again with an RNA Clean & Concentrator-25 kit (Zymo). Ribosomal RNA was removed using a RiboMinus Transcriptome Isolation Kit for bacteria (Thermo Fisher Scientific) as per the manufacturer's protocol using half-volume reactions. The purified sample was then treated with 20 units of T4 polynucleotide kinase (NEB) for 6 h at 37° C. and purified again with an RNA Clean & Concentrator-25 (Zymo) kit. The purified RNA was treated with 20 units of 5′ RNA phosphatase (Lucigen) for 30 min at 37° C. and purified again using an RNA Clean & Concentrator-5 kit (Zymo). Purified RNA was used as input to an NEBNext Small RNA Library Prep for Illumina (NEB) as per the manufacturer's protocol with an extension time of 60 s and 16 cycles in the final PCR. Amplified libraries were gel extracted, quantified by qPCR using a KAPA Library Quantification Kit for Illumina (Roche) on a StepOne Plus machine (Applied Biosystems/Thermo Fisher Scientific) and sequenced on an Illumina NextSeq with Read 1 42 cycles, Read 2 42 cycles and Index 1 6 cycles. Adapters were trimmed using CutAdapt and mapped to loci of interest using BWA-align. Reads were visualized using Genious.
Ribonucleoprotein: RNPs were purified as described. 100 μL concentrated RNP was used as input. The above protocol was followed with the following modifications: 300 μL TRI reagent (Zymo) and 60 μL chloroform (Sigma Aldrich) were used for RNA extraction.
PureExpress RNPs: 75 ng of plasmid encoding the Fanzor ORF and 125 ng of the plasmid containing the locus were incubated in 1 unit of pureexpress reactions for 4 hours at 37 degrees Celcius. Afterward, the RNP is affinity purified using the protocol described above for heterologous Rosetta cell protein production and subjected to the same pipeline for small RNA sequencing.
Chlamydomonas reinhardtii was obtained from the University of Minnesota (CRC). The algae was lysed in trizol with glass beads vigorously shaken for 2 hours at room temperature. Then the above protocol was followed with the following modifications: Ribosomal RNA was removed using a plant specific ribominus rRNA depletion kits as per the manufacturer's protocol and the rRNA-depleted sample was purified using Agencourt RNAClean XP beads (Beckman Coulter) prior to T4 PNK treatment. T4 PNK treatment was performed for 1.5 h and purified with an RNA Clean & Concentrator-5 kit (Zymo). Final PCR in the small RNA library prep contained 10 cycles.
DNase alert and Rnase alert were purchased from IDT. 1 uM of RNP or 10 uL of PureExpress generated RNP and 10 nM of DNA target containing either the target spacer or a scramble spacer are diluted in 1× DNase/Rnase alert reaction buffer into 50 uL reactions. The solution is mixed well in the reaction test tube and subsequently aliquoted into 384 well plates. The plates are loaded onto applied biosystems qPCR machines and reactions were ran at 37 degree Celsius for ApmHNuc, AmpFNuc2, DrpFNuc2, BaaFNuc2, MemFNuc2, and Isdra2 TnpB, and 60 degree Celsius for TvoTnpB. The SYBR and HEX channel fluorescence intensity is recorded every minute for a duration of 60 minutes. The intensity is normalized by subtracting the non-target DNA sequence from the target DNA sequence group. A positive control DNase (2 uL) and RNAse (2 uL) is ran along with the Fanzor/TnpB group as a positive control to monitor the assay.
Target sequences with 7N degenerate flanking sequences were synthesized by IDT and amplified by PCR with NEBNext High Fidelity 2× Master Mix (NEB). Backbone plasmid was digested with restriction enzymes (pUC19: KPNI and HindIII, Thermo Fisher Scientific) and treated with FastAP alkaline phosphatase (Thermo Fisher Scientific). The amplified library fragment was inserted into the backbone plasmid by Gibson assembly at 50° C. for 1 hour using 2× Gibson Assembly Master Mix (NEB) with an 8:1 molar ratio of insert:vector. The Gibson assembly reaction was then isopropanol precipitated by the addition of an equal volume of isopropanol (Sigma Aldrich), the final concentration of 50 mM NaCl, and 1 μL of GlycoBlue nucleic acid co-precipitant (Thermo Fisher Scientific). After a 15 min incubation at room temperature, the solution was spun down at max speed at 4° C. for 15 min, then the supernatant was pipetted off and the pelleted DNA has resuspended in 12 μL TE and incubated at 50° C. for 10 minutes to dissolve. 2 μL were then transformed by electroporation into Endura Electrocompetent F co/i (Lucigen) as per the manufacturer's instructions, recovered by shaking at 37° C. for 1 h, then plated across 5 22.7 cm×22.7 cm BioAssay plates with the appropriate antibiotic resistance. After 12-16 hours of growth at 37° C., cells were scraped from the plates and midi- or maxi-prepped using a NucleoBond Midi- or Maxi-prep kit (Machery Nagel). The sequence is provided in Table 7.
1 μM of RNP and 25 ng of TAM library plasmid were incubated at 37 degree for 2 hours in NEB Buffer 3. Reactions were quenched by placing at 4° C. or on ice and adding 10 ug Rnase A (Qiagen) and 8 units Proteinase K (NEB) each followed by a 5 min incubation at 37° C. DNA was extracted by PCR purification and adaptors were ligated using an NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) using the NEBNext Adaptor for Illumina (NEB) as per the manufacturer's protocol. Following adaptor ligation, cleaved products were amplified specifically using one primer specific to the TAM library backbone and one primer specific to the NEBNext adaptor with a 12-cycle PCR using NEBNext High Fidelity 2×PCR Master Mix (NEB) with an annealing temperature of 63° C., followed by a second 20-cycle round of PCR to further add the Illumina i5 adaptor. Amplified libraries were gel extracted, quantified by qubit dsDNA kit (Invitrogen) and subject to single-end sequencing on an Illumina MiSeq with Read 1 200 cycles, Index 1 8 cycles and Index 2 8 cycles. TAMs were extracted and visualized by Weblogo3. Alternatively, a primer set targeting the TAM library plasmid is used to amplify the uncleaved product for 12 cycle and followed by a second 20 cycle rounds of PCR to add the Illumina i5 adaptor. Amplified libraries were gel extracted and subjected to single end sequencing on an Illumina MiSeq with Read 1 200 cycles, Index 1 8 cycles and Index 2 8 cycles. Depletion of TAMs were calculated by comparing to a non-targeting RNP as control and normalized to the original plasmid library distribution. Primers used are listed in Table 8.
Double-stranded DNA (dsDNA) substrates were produced by PCR amplification of pUC19 plasmids containing the target sites and the TAM sequences. All ωRNA and fRNA used in the biochemical assays was in vitro transcribed using the HiScribe T7 Quick High Yield RNA Synthesis kit (NEB) from the DNA templates purchased from IDT. Target cleavage assays performed with ApmHNuc contained 10 nM of DNA substrate, 1 μM of protein, and 4 μM of fRNA in a final 1× reaction buffer of NEB Buffer 3. Assays were allowed to proceed at 37° C. for 2 hour, then briefly shifted to 50° C. for 5 min, and immediately placed on ice to help relax the RNA structure prior to RNA digestion. Reactions were then treated with Rnase A (Qiagen), and Proteinase K (NEB), and purified using a PCR cleanup kit (Qiagen). DNA was resolved by gel electrophoresis on Novex 6% TBE polyacrylamide gels (Thermo Fisher Scientific). 1 uM of purified RNP and 100 ng of the 7N TAM library is incubated at 37 degree Celsius in NEB buffer 3 for 3 hours. Subsequently, reaction is purified and analyzed following the same procedure as cell-free transcription/translation TAM screen. TAM library sequence and guides used are listed in Table 7.
1 μM of RNP and 100 ng of the target plasmid were incubated at 37 degree for 3 hours in NEB Buffer 3. Reactions were quenched by placing at 4° C. or on ice and adding 10 ug RNase A (Qiagen) and 8 units Proteinase K (NEB) each followed by a 5 min incubation at 37° C. DNA was extracted by PCR purification and adaptors were ligated using an NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) using the NEBNext Adaptor for Illumina (NEB) as per the manufacturer's protocol. Following adaptor ligation, cleaved products were amplified specifically using one primer specific to the target plasmid (one on 5′ site of the cleavage and one on 3′ side of the cleavage) and one primer specific to the NEBNext adaptor with a 12-cycle PCR using NEBNext High Fidelity 2×PCR Master Mix (NEB) with an annealing temperature of 63° C., followed by a second 20-cycle round of PCR to further add the Illumina i5 adaptor. Amplified libraries were gel extracted, quantified by qubit dsDNA kit (Invitrogen) and subject to single-end sequencing on an Illumina MiSeq with Read 1 100 cycles, Index 1 8 cycles and Index 2 8 cycles. All sequencing primers are listed in Table 6.
The N-terminal predicted NLS sequences of Fanzor is cloned onto N-terminal of sfGFP by Gibson assembly into a pCMV promoter backbone (NLS sequences cloned are listed in Table 5). 24 hours before transfection, 15,000 HEK293FT cells were plated onto a glass bottom 96 well plates pre-coated with poly-D lysine. 100 ng of NLS-sfGFP construct is transfected into HEK293FT cells using lipofectamine 3000 and 24 hours after transfection, cells were fixed and permeabilized using Fix and Pern kit (Thermofisher) and subsequently stained by either DAPI or SYTO-Red nuclear stain (Thermofisher). All wells were measured via confocal microscopy at room temperature. Cells were focused in the 488 nm channel on the basis of the sfGFP protein. For each well, a 2×2 field of view image at 20× magnification was collected under the following settings and stitched around the center point. Images were collected in 488 nm (32.8% power, 100 ms exposure), 359 nm (35.2% power, 100 ms exposure), and 633 nm (80% power, 100 ms exposure).
Mammalian cell culture experiments were performed in the HEK293FT line (Thermo Fisher) grown in Dulbecco's Modified Eagle Medium with high glucose, sodium pyruvate, and GlutaMAX (Thermo Fisher), additionally supplemented with 1× penicillin-streptomycin (Thermo Fisher), 10 mM HEPES (Thermo Fisher), and 10% fetal bovine serum (VWR Seradigm). All cells were maintained at confluency below 80%.
All transfections were performed with Lipofectamine 3000 (Thermo Fisher). Cells were plated 16-20 hours prior to transfection to ensure 90% confluency at the time of transfection. For 96-well plates, cells were plated at 20,000 cells/well. For each well on the plate, transfection plasmids were combined with OptiMEM I Reduced Serum Medium (Thermo Fisher) to a total of 10 μL.
fRNA scaffold backbones were cloned into a pUC19-based human U6 expression backbone and human codon-optimized Fanzor proteins were cloned into pCMV-based or pCAG-based destination vector by Gibson Assembly. Then 50 ng of protein expression construct, 50 ng of the corresponding guide construct and an optionally 20 ng of luciferase reporter were transfected in one well of a 96-well plate using lipofectamine 3000 transfection reagent. After 48 hours, reporter DNA was harvested by washing the cells once in 1×DPBS (Sigma Aldrich) and resuspended in 50 μL QuickExtract DNA Extraction Solution (Lucigen) and cycled at 65° C. for 15 min, 68° C. for 15 min then 95° C. for 10 min to lyse cells. 2.5 μL of lysed cells were used as input into each PCR reaction. For library amplification, target reporter regions were amplified with a 12-cycle PCR using NEBNext High Fidelity 2×PCR Master Mix (NEB) with an annealing temperature of 63° C. for 15 s, followed by a second 18-cycle round of PCR to add Illumina adapters and barcodes. The libraries were gel extracted and subject to single-end sequencing on an Illumina MiSeq with Read 1 220 cycles, Index 1 8 cycles, Index 2 8 cycles and Read 2 80 cycles. Insertion/deletion (indel) frequency was analyzed using CRISPResso2. All sequencing primers are listed in Table 6. Guides used for genomic target are listed in Table 7.
The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.
This invention was made with government support under EB031957 awarded by National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63578625 | Aug 2023 | US | |
| 63510866 | Jun 2023 | US | |
| 63507968 | Jun 2023 | US | |
| 63450947 | Mar 2023 | US |
