GENOMIC EDITING WITH SITE-SPECIFIC RETROTRANSPOSONS

Abstract

Genome editing tools for use in systems designed to deliver large genetic elements are disclosed herein. A genome editing system is described, which includes i) an R2 element enzyme or other non-LTR site specific retrotransposon element and ii) a payload RNA, wherein the payload RNA comprises an insertion region and optionally one or more of a 5′ homology region, a 3′ homology region, and a protein binding element, wherein the insertion region comprises a template for a small or large nucleic acid insertion into the genome, and wherein the R2 element enzyme or other non-LTR site specific retrotransposon element comprises a targeting domain, a reverse transcriptase domain, and a nickase domain. Also disclosed are cells edited using such a genome editing system, methods for editing a genome, and compositions comprising cells edited with this genomic editing system.

Description

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on Oct. 18, 2022, is named 733337_083474-024PC_SL.xml and is 66,696,250 bytes in size.

BACKGROUND

Genome editing systems have developed as a promising technology for the development of therapeutic tools. Systems such as CRISPR/Cas9, TALEN, and zinc finger proteins have been used to alter the genomes of organisms. However, these systems are limited by a number of factors, including size, cargo capacity, and targeting ability.

Retrotransposons are mobile elements that insert themselves into the genome of a host through an RNA intermediate. This is in contrast to the mechanism of most DNA transposons, which directly insert themselves into a host genome. Retrotransposons are categorized as long terminal repeat (LTR) retrotransposons and non-LTR retrotransposons.

Non-LTR retrotransposons are among the most frequently occurring transposable elements in the eukaryotic genome. They can be either randomly inserting or site-specific. Site-specific non-LTR retrotransposons are generally characterized by the presence of specific activity—reverse transcriptase activity, DNA nicking activity, and nucleic acid binding activity. The genetic loci for these activities are found in either a single open reading frame (ORF) or split between two ORFs. The DNA nicking activity of single-ORF systems is found with restriction-like endonuclease (RLE) domains. Multiple non-LTR retrotransposon families, such as the R2, R4, R5, R8, R9, Dong and Cre families, are categorized as RLE containing non-LTR retrotransposons.

Of the known non-LTR retrotransposons, the most well studied is the R2 element. The R2 element is comprised of R2 RNA and the R2 protein. The R2 element contains a single open reading frame (ORF), which encodes a reverse transcriptase, an endonuclease, and includes DNA binding regions and zinc finger motifs. R2 element. R2 inserts itself into a host genome through a mechanism known as Target Primed Reverse Transcription (TPRT), which is a stepwise reaction including a first nick of host DNA, reverse transcription of the R2 RNA into the first strand, a second nick of host DNA, and synthesis of a second strand.

The mechanism by which the R2 element inserts into a host genome, being independent of endogenous cellular repair pathways, as well as the capacity to carry an RNA molecule of varying sizes to a host genome, makes the R2 element a potentially powerful genome editing system. However, the R2 element specifically inserts itself into either the 28S or 18S ribosomal RNA locus. Therefore, it lacks the ability to target insertions to a particular locus, which is a critical aspect for viable genome editing systems. Other site-specific retrotransposons are similarly limited to particular loci. There remains an unmet need for a genome editing system that is capable of directed insertion of large nucleic acids into a host genome.

BRIEF SUMMARY

The present disclosure is directed to a genome editing system comprising: i) an R2 element enzyme; and ii) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5′ homology region, a 3′ homology region, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome, and wherein the R2 element enzyme comprises a reverse transcriptase domain, and a nickase domain.

In some embodiments the R2 element enzyme further comprises a targeting domain. In some embodiments the targeting domain is a natural targeting domain or an engineered targeting domain. In some embodiments, the nucleic acid insertion into the genome is a DNA or RNA insertion template. In some embodiments, the R2 element enzyme is a modified R2 element enzyme. In some embodiments, the coding sequence of the R2 element enzyme is modified. In some embodiments, wherein the modified R2 element enzyme is modified by an N-terminal or C-terminal truncation of the R2 element enzyme sequence. In some embodiments, the modified R2 element enzyme comprises a linker. In some embodiments the linker is an XTEN linker.

In some embodiments, the genome editing system targets a genomic locus. In some embodiments, the genome editing system targets a genomic locus other than the 28S rRNA locus. In some embodiments, an N-terminal zinc finger domain of the R2 element enzyme is modified to target a genomic locus other than the 28S rRNA locus. In some embodiments, a non-naturally occurring targeting region is fused to the N-terminus of the R2 element enzyme or inserted into the R2 element enzyme.

In some embodiments, the modified R2 element enzyme is a fusion protein. In some embodiments, the modified R2 element is fused to a Cas9 protein that is fully active, catalytically dead (H840A/D10A for SpCas9), or functioning as a nickase (H840A or D10A for SpCas9). In some embodiments, the modified R2 element is fused to a Cas12 protein that is fully active, catalytically dead, or functioning as a nickase. In some embodiments, the modified R2 element is fused to a TALEN protein, zinc finger protein, argonaute, or meganuclease protein.

In some embodiments, the genome editing system further comprises a guide RNA. In some embodiments, the 5′ homology region of the payload RNA is engineered to target a genomic locus other than the 28S rRNA locus. In some embodiments, the 5′ homology region, the 3′ homology region, or both the 5′ and 3′ homology region target an exogenously introduced landing sequence.

In some embodiments, the insertion region is introduced into the genome of a specific cell type. In some embodiments, the specific cell type is a post-mitotic cell. In some embodiments, the genome editing system functions in post-mitotic cells. In some embodiments, the genome editing system functions independently from intrinsic nucleic acid repair systems.

In some embodiments, the payload RNA template further comprises a 5′ untranslated region (UTR), a 3′ UTR, or both a 5′ UTR and a 3′ UTR. In some embodiments, the 5′ homology region and the 3′ homology region are located between the 5′ UTR and 3′ UTR. In some embodiments, the 5′ homology region and the 3′ homology region are located outside the 5′ UTR and 3′ UTR. In some embodiments, the payload RNA further comprises a 5′ untranslated region (UTR), a 3′ UTR, or both a 5′ and a 3′ UTR, wherein the UTRs are truncated. In some embodiments, the payload RNA does not comprise a 5′ UTR. In some embodiments, the payload RNA does not comprise a 3′ UTR.

In some embodiments, the payload RNA further comprises a nuclear retention element. In some embodiments, the payload RNA further comprises a Cas9 or Cas12 guide RNA, wherein the Cas9 or Cas12 guide RNA comprises an extension with a 5′ homology sequence, a 3′ homology sequence, a 5′ untranslated region (UTR), a 3′ UTR, an insertion template, or any combination thereof. In some embodiments the nucleic acid insertion template is a sequence of greater than 1000 base pairs.

In some embodiments, the R2 element enzyme comprises a nuclear localization signal (NLS).

In some embodiments, the insertion region comprises a template for a reporter gene, a transcription factor gene, a transgene, an enzyme gene, or a therapeutic gene.

The present disclosure is also directed to a method of inserting a large nucleic acid into a genome within a cell using a Cas9 or Cas12 fusion protein, wherein the method comprises supplying a Cas9 or Cas12 fusion protein to a cell, wherein the Cas9 or Cas12 fusion protein is supplied with a payload RNA template, wherein the RNA template is reverse transcribed by the Cas9 or Cas12 fusion protein prior to being inserted into the genome of the cell; and wherein the large nucleic acid is inserted into the genome of the cell.

In some embodiments, the Cas9 fusion protein comprises a Cas9 portion and an R2 element portion. In some embodiments, the Cas9 fusion protein comprises a targeting domain, a reverse transcriptase domain, and a nickase domain. In some embodiments, the Cas12 fusion protein comprises a Cas12 portion and an R2 element portion.

The disclosure is also directed to a method of inserting an exogenous nucleic acid into the genome of a post-mitotic cell, wherein the method comprises subjecting the genome of the post-mitotic cell to a modified Cas9 protein that inserts the exogenous nucleic acid into the genome of the post-mitotic cell. In some embodiments, the modified Cas9 protein is fused to an R2 element enzyme. In some embodiments, the modified Cas9 fusion protein targets an endogenous landing site. In some embodiments, the Cas9 fusion protein targets an exogenously introduced landing site in the genome of the post-mitotic cell.

The disclosure is also directed to a method of editing a genome comprising subjecting the cell to the genome editing systems described above.

The disclosure is also directed to a composition comprising a cell edited by the genome editing systems or methods of editing genomes described above.

The disclosure is also directed to a genome editing system comprising: i) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5′ homology region, a 3′ homology region, and a protein binding element, wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome; ii) a non-LTR site specific retrotransposon element enzyme; wherein the non-LTR site specific retrotransposon element enzyme comprises a reverse transcriptase domain and, optionally, a nuclease or nickase domain, and wherein if the non-LTR-site specific retrotransposon element enzyme does not comprise the optional nuclease or nickase domain, the genome editing system further comprises iii) a nuclease or nickase enzyme. In some embodiments, the nuclease or nickase enzyme is a programmable nuclease or nickase. In some embodiments, the non-LTR site specific retrotransposon element enzyme further comprises a targeting domain. In some embodiments, the targeting domain is a natural targeting domain or an engineered targeting domain.

The disclosure is also directed to a genome editing system where the non-LTR site specific retrotransposon comes from the R1, R2, R4, R5, R6, R7, R8, R9, CRE, NeSL, HERO, or Utopia families, or from the 9 family classifications established for RLE domain containing nLTR retrotransposons (FIG. 24C).

In some embodiments, the nucleic acid insertion into the genome is a DNA or RNA insertion template.

In some embodiments, the non-LTR site specific retrotransposon element enzyme is a modified non-LTR site specific retrotransposon element enzyme. In some embodiments, the coding sequence of the non-LTR site specific retrotransposon element enzyme is modified. In some embodiments, the modified non-LTR site specific retrotransposon element enzyme is modified by an N-terminal or C-terminal truncation of the non-LTR site specific retrotransposon element enzyme sequence.

In some embodiments, the modified non-LTR site specific retrotransposon element enzyme comprises a linker. In some embodiments, the linker is an XTEN linker.

The genome editing system of the disclosure targets a genomic locus. In some embodiments, the genome editing system targets a genomic locus other than the 28S rRNA locus. In some embodiments, an N-terminal zinc finger domain of the non-LTR site specific retrotransposon element enzyme is modified to target a genomic locus other than the 28S rRNA locus. In some embodiments, a non-naturally occurring targeting region is fused to the N-terminus of the non-LTR site specific retrotransposon element enzyme or inserted into the non-LTR site specific retrotransposon element enzyme.

In some embodiments, the modified non-LTR site specific retrotransposon element enzyme is a fusion protein. In some embodiments, the modified non-LTR site specific retrotransposon element is fused to a Cas9 protein that is fully active, catalytically dead (H840A/D10A for SpCas9), or functioning as a nickase (H840A or D10A for SpCas9). In some embodiments, the modified non-LTR site specific retrotransposon element is co-delivered with a Cas9 protein that is fully active, catalytically dead (H840A/D10A for SpCas9), or functioning as a nickase (H840A or D10A for SpCas9). In some embodiments, the modified non-LTR site specific retrotransposon element is fused to a Cas12, IscB, IsrB, or TnpB protein that is fully active, catalytically dead, or functioning as a nickase. In some embodiments, the modified non-LTR site specific retrotransposon element is delivered in trans with a Cas12, IscB, IsrB, or TnpB protein that is fully active, catalytically dead, or functioning as a nickase. In some embodiments, the modified non-LTR site specific retrotransposon element is fused to a TALEN protein, zinc finger protein, argonaute, or meganuclease protein.

In some embodiments, the disclosure further comprises a guide RNA. In some embodiments, the disclosure further comprises multiple guide RNA.

In some embodiments, the genome editing system of the disclosure comprises a payload wherein the 5′ homology region, the 3′ homology region, or both the 5′ and 3′ homology region of the payload RNA is engineered to target a genomic locus other than the 28S rRNA locus. In some embodiments, the 5′ homology region, the 3′ homology region, or both the 5′ and 3′ homology region target an exogenously introduced landing sequence.

In some embodiments, the insertion region is introduced into the genome of a specific cell type. In some embodiments, the specific cell type is a post-mitotic cell, a non-dividing cell, or a quiescent cell. In some embodiments, the genome editing system functions in post-mitotic cells, non-dividing cells, or quiescent cells. In some embodiments, the genome editing system functions independently from intrinsic nucleic acid repair systems.

In some embodiments, the payload RNA template further comprises a 5′ untranslated region (UTR), a 3′ UTR, or both a 5′ UTR and a 3′ UTR. In some embodiments, the 5′ homology region and the 3′ homology region are located between the 5′ UTR and 3′ UTR. In some embodiments, the 5′ homology region and the 3′ homology region are located outside the 5′ UTR and 3′ UTR. In some embodiments, the payload RNA further comprises a 5′ untranslated region (UTR), a 3′ UTR, or both a 5′ and a 3′ UTR, wherein the UTRs are truncated. In some embodiments, the payload RNA does not comprise a 5′ UTR. In some embodiments, the payload RNA does not comprise a 3′ UTR. In some embodiments, the payload RNA further comprises a nuclear retention element. In some embodiments, the payload RNA further comprises a Cas9 or Cas12 guide RNA, and wherein the Cas9 or Cas12 guide RNA comprises an extension with a 5′ homology sequence, a 3′ homology sequence, a 5′ untranslated region (UTR), a 3′ UTR, an insertion template, or any combination thereof.

In some embodiments, the nucleic acid insertion template is a sequence of greater than 1000 base pairs.

In some embodiments, the genome editing system targets a genome for a deletion. In some embodiments, the deletions are between 1 and 150 bases.

In some embodiments, the non-LTR site specific retrotransposon element enzyme comprises a nuclear localization signal (NLS).

In some embodiments, the insertion region comprises a template for a reporter gene, a transcription factor gene, a transgene, an enzyme gene, or a therapeutic gene.

The disclosure is also directed to a method of inserting a large nucleic acid into a genome within a cell using a Cas9 or Cas12 fusion protein, wherein the method comprises supplying a Cas9 or Cas12 fusion protein to a cell, wherein the Cas9 or Cas12 fusion protein is supplied with a payload RNA template, wherein the RNA template is reverse transcribed by the Cas9 or Cas12 fusion protein prior to being inserted into the genome of the cell; and wherein the large nucleic acid is inserted into the genome of the cell. In some embodiments, the Cas9 fusion protein comprises a Cas9 portion and a non-LTR site specific retrotransposon element portion. In some embodiments. the Cas9 fusion protein comprises a targeting domain, a reverse transcriptase domain, and a nickase domain. In some embodiments, the Cas12 fusion protein comprises a Cas12 portion and a non-LTR site specific retrotransposon element portion.

The disclosure is also directed to a method of inserting an exogenous nucleic acid into the genome of a post-mitotic cell, wherein the method comprises subjecting the genome of the post-mitotic cell to a modified Cas9 protein that inserts the exogenous nucleic acid into the genome of the post-mitotic cell. In some embodiments, the modified Cas9 protein is fused to a non-LTR site specific retrotransposon element enzyme. In some embodiments, the modified Cas9 fusion protein targets an endogenous landing site. In some embodiments, the Cas9 fusion protein targets an exogenously introduced landing site in the genome of the post-mitotic cell.

The disclosure is also directed to a method of editing a genome comprising subjecting the cell to the genome editing system as described herein. The disclosure is also directed to a composition comprising the cell edited by the genome editing methods described herein.

The disclosure is also directed to a method of correcting a genetic mutation related to disease or human pathology, wherein the method comprises making small nucleotide changes or small nucleotide insertions (1-100 bp) in a human genome using the genome editing system of claim 1 or claim 47.

In some embodiments, the genome editing system is delivered via single or multi vector A/AV, adenovirus, lentivirus, herpes simplex virus, PEG10 viral like particles, PNMA viral like particles, gag-like viral like particles, nanoblades, gesicles, or Friend murine leukemia virus (FMLV) viral like proteins.

In some embodiments, the components of the genome editing system are delivered as all RNA in lipid nanoparticles or another RNA delivery reagent. In some embodiments, wherein the non-LTR site specific retrotransposon is delivered as mRNA. In some embodiments, the guide RNAs are delivered as synthetic RNA. In some embodiments, the payload is delivered as mRNA.

The disclosure is also directed to a genome editing system targets and edits the genome at more than one site.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a visual depiction of PCR products isolated on an agarose gel following amplification from isolated DNA from HEK293FT cells which were transfected with two plasmids, showing insertion of R2 into the human genome. Lane 1 displays a molecular weight marker. Lane 2 displays PCR products from cells transfected with an R2 plasmid, encoding an R2 derived from the zebra finch (Taeniopygia guttata) R2 element (R2Tg) with an eGFP payload. Lane 3 displays the PCR products from cells transfected with R2Tg alone. Lane 4 displays the PCR products from cells transfected with eGFP payload alone. Lanes 5 and 6 display the PCR products from cells transfected with R2 orthologs from Geospiza fortis (Gfo) and a long Gfo payload (Lane 5) or short Gfo payload (Lane 6). Lane 7 displays PCR product from cells transfected with an R2 ortholog from Geospiza fortis alone. Lane 8 displays PCR product from cells transfected with only long Gfo payload. Lane 9 displays PCR product from cells transfected with only short Gfo payload.

FIG. 2 is a graphical depiction of luminescence readout from HEK293FT cells transfected with 3 separate plasmids: the first containing an R2 protein encoding region, the second containing an inactive luciferase reporter region (containing the promoter region and a first of two artificial and inactive luciferase exons followed by a chimeric intron) with R2 landing sites (the landing site is placed in an intronic region that is spliced out after insertion of the payload carrying the second of two artificial exons) of variable length, and the third containing a luciferase portion of a payload, 5′ and 3′ UTRs as well as regions homologous to the landing sites. The x-axis labels represent variable landing sites, named according to the number of base pairs (bp) present on the landing site on either side of the insertion; 38/10 therefore, represents 38 bp upstream of the insertion site and 10 bp downstream of the insertion site. Columns 11 and 12 display the luminescence readout of two negative controls, A/AVS1_target (non-target) and CFTR_target (non-target).

FIG. 3A is a graphical depictions of the tolerability of mutations of the landing sites with respect to R2 integration in HEK293FT cells. HEK293FT cells were transfected with 3 separate plasmids: the first containing an R2 protein encoding region, the second containing a luciferase reporter region with mutated or wild type R2 (28S) landing sites in the intronic region that follows the first of two luciferase exons, and the third (payload) plasmid containing the second exon necessary for luciferase signal after insertion and splicing of the reporter plasmid. Additionally to a small intronic sequence upstream of the second of two artificial luciferase exons (minimal cargo necessary for luciferase signal), the payload plasmid has 5′ and 3′ UTR sequences as well as 5′ and 3′ homologies (100 bp homology to the 28S locus on either side). FIG. 3A displays the location of certain mutations within the region flanking the insertion on the insertion region plasmid. Figure discloses SEQ ID NOS 33523-33534, respectively, in order of appearance. FIG. 3B is a readout of luminescence from HEK293FT cells transfected as above. The y-axis represents the specific plasmids containing altered landing sites introduced into the specific cell, with each name representing the number of base pairs (bp) present on the landing site on either side of the insertion; 37/23 therefore, represents 37 bp upstream of the insertion site and 23 bp downstream of the insertion site. A 115/115 negative control (transfected cell with no plasmid expressing R2).

FIG. 4A is a graphical depictions of the tolerability of mutations of landing sites with respect to R2 integration in HEK293FT cells. HEK293FT cells were transfected with 3 separate plasmids: the first containing an R2 protein encoding region, the second containing a luciferase reporter region with mutated or wild type R2 landing sites and the third (payload) plasmid containing the second exon necessary for luciferase signal after insertion and splicing of the reporter plasmid. Additionally to a small intronic sequence upstream of the second of two artificial luciferase exons (minimal cargo necessary for luciferase signal), the payload plasmid has 5′ and 3′ UTR sequences as well as 5′ and 3′ homologies (100 bp homology to the 28S locus on either side). FIG. 4A displays the location of certain mutations within the region flanking the insertion on the insertion region plasmid. Figure discloses SEQ ID NOS 33535-33546, respectively, in order of appearance. FIG. 4B is a readout of luminescence from HEK293FT cells transfected as above. Target_37_23_mut_10 (red box) has full mutations of all three, predicted zinc finger binding sites.

FIG. 5 is a graphical depiction of the effect of aphidicolin on the integration of a luciferase payload into a target region. HEK293FT cells were transfected with 3 separate plasmids: the first containing an R2 protein encoding region, the second containing a luciferase reporter region with R2 landing sites, and the third (payload) plasmid containing the second exon necessary for luciferase signal after insertion and splicing of the reporter plasmid. Additionally to a small intronic sequence upstream of the second of two artificial luciferase exons (minimal cargo necessary for luciferase signal), the payload plasmid has 5′ and 3′ UTR sequences as well as 5′ and 3′ homologies (100 bp homology to the 28S locus on either side)Cells were then treated] with either Dimethyl Sulfoxide (DMSO) or aphidicolin at a concentration of 1 μm, 5 μm, or 25 μm. Homologous sequences in the insertion region were either 60 bp or 40 bp long. Columns 9-12 are cells treated with either DMSO or aphidicolin and transfected with negative control plasmids.

FIG. 6 is a graphical depiction of the effect of aphidicolin on the integration of a luciferase payload into a target region. HEK293FT cells were transfected with 3 separate plasmids: the first containing an R2 protein encoding region, the second containing a luciferase reporter region with R2 landing sites, and the third (payload) plasmid containing the second exon necessary for luciferase signal after insertion and splicing of the reporter plasmid. Additionally to a small intronic sequence upstream of the second of two artificial luciferase exons (minimal cargo necessary for luciferase signal), the payload plasmid has 5′ and 3′ UTR sequences as well as 5′ and 3′ homologies (100 bp homology to the 28S locus on either side. Cells were then treated with either Dimethyl Sulfoxide (DMSO) or aphidicolin at a concentration of 1 μm, 5 μm, or 25 μm. The insertion regions of the plasmids are flanked by either 300 bp, 200 bp, or 100 bp. Columns 13-16 contain a 300 bp flanking sequence in the insertion region and were simultaneously transfected with a plasmid without an active R2 enzyme. Columns 17-20 were solely transfected with a Cas9 μlasmid.

FIG. 7 is a visual depiction of a heatmap showing the luminescence readout of HEK293FT cells transfected with 3 separate plasmids. The first plasmid contained an R2 protein encoding region, the second plasmid contained a luciferase reporter precursor region with R2 landing sites, and the third (payload) plasmid containing the second exon necessary for luciferase signal after insertion and splicing of the reporter plasmid. Additionally to a small intronic sequence upstream of the second of two artificial luciferase exons (minimal cargo necessary for luciferase signal), the payload plasmid has 5′ and 3′ UTR sequences as well as 5′ and 3′ homologies of different length (from 0 to 100 bp homology in steps of 20 bp).

FIG. 8 is a graphical depiction of the effect of modification of UTRs on the luminescence readout of transfected HEK392FT cells. HEK293FT cells were transfected with 3 separate plasmids. The first plasmid contained an R2 protein encoding region, the second plasmid contained a splice luciferase reporter region with R2 landing sites 26/22 bp, and the third (payload) plasmid containing the second exon necessary for luciferase signal after insertion and splicing of the reporter plasmid. Additionally to a small intronic sequence upstream of the second of two artificial luciferase exons (minimal cargo necessary for luciferase signal), the payload plasmid has 5′ and 3′ UTR sequences that are truncated in different ways as well as 5′ and 3′ homologies. Column 1 represents a positive control. Column 2 represents a negative control. Columns 3-8 represent truncations from the left of the 5′UTR. Columns 9-15 represent truncations from the right of the 5′ UTR. Columns 16-22 represent truncations from the left of the 3′ UTR. Columns 23-29 represent truncations from the right of the 3′UTR.

FIG. 9A is a graphical depiction exhibiting the effect that altered homology regions have on integration. HEK293FT cells were transfected with 3 separate plasmids: the first containing an R2 protein encoding region, the second containing a luciferase reporter region with 26/22 bp R2 landing sites and the third (payload) plasmid containing the second exon necessary for luciferase signal after insertion and splicing of the reporter plasmid. Additionally to a small intronic sequence upstream of the second of two artificial luciferase exons (minimal cargo necessary for luciferase signal), the payload plasmid has 5′ and 3′ UTR sequences as well as 5′ and 3′ homologies. Here, the 3′ homologies have different lengths: PBS13 (13 bp) and 3′ homology (100 bp). HDV is an HDV ribozyme, which cleaves the insertion region directly after the 3′ UTR and mHDV is a mutated HDV ribozyme that is non-functional. FIG. 9B is a visual representation of each 3′ modification.

FIG. 10 is a graphical depiction of the effect of linker insertion site on integration efficiency of the R2 protein. Linkers were inserted into various domains at specific insertion sites of an R2 derived from the zebra finch (Taeniopygia guttata) R2 element (R2Tg) with an eGFP or msfGFP payload. Positions for linkers were identified using Emboss gamier to identify potential linker regions, of which 12 were chosen. Linkers for eGFP, for example, were GSGGGSGS (SEQ ID NO: 33377)-EGFP-GSGGGGSG (SEQ ID NO: 33378). Columns 1 and 2 are wild-type R2Tg without a linker region.

FIG. 11 is a graphical depiction of editing efficiency in the short 28S landing site in an exogenous plasmid. HEK293FT cells were transfected with 3 separate plasmids: the first either containing an R2 protein encoding region or no R2 protein encoding region, the second containing a luciferase reporter region with 26/22 (26 upstream/22 downstream) R2 landing sites and the third (payload) plasmid containing the second exon necessary for luciferase signal after insertion and splicing of the reporter plasmid. Additionally, to a small intronic sequence upstream of the second of two artificial luciferase exons (minimal cargo necessary for luciferase signal), the payload plasmid has 5′ and 3′ UTR sequences and 100 bp 5′ and 3′ homologies to the 28S target site. Percent editing is measured by digital droplet PCR (ddPCR) using primers that recognize the payload.

FIG. 12 is a graphical depiction of R2 insertion efficiency within the endogenous Beta actin locus of HEK293FT cells transfected with 4 separate plasmids: the first containing an R2 protein encoding region, the second containing an insertion region with a pMAX gene flanked by 5′ and 3′ UTRs and homology regions to the 28S locus, the third a prime editor encoding region, and the fourth a prime editing guideRNA to introduce a 26/22 R2 target site at the ACTB locus. From left to right, the samples are 1) wild-type R2 protein, 2) R2 protein fused to a nuclear localization signal, 3) no R2 protein with Prime editing molecule, 4) R2 protein without prime editing molecule. Percent integration is measured by ddPCR.

FIG. 13A is a visual depiction of the integration a payload comprised of an R2 protein attached at the C-terminus to eGFP. FIG. 13B is graphical depiction is a luminescence readout of the effect of addition of a nuclear localization signal to the N and C-terminus of the R2 protein on reporter expression. Either wild-type R2 (column 1) or NLS-appended R2 (column 2) were transfected into HEK293FT cells with a stably integrated splice reporter. A negative control is shown in column 3.

FIG. 14A is a visual depiction of HEK293FT cells transfected with either an R2 expression plasmid (FIGS. 14A, 14B) or an R2 negative plasmid (FIGS. 14C, 14D) at either 20 hours post transfection (FIGS. 14A, 14C) or 36 hours post transfection (FIGS. 14B, 14D). The R2 template inserts a second GFP exon into the stably transfected splice receptor, which contains the promoter and a first exon, allowing for GFP expression following integration.

FIG. 15A is a graphical depiction of the percentage of GFP positive cells as determined by flow cytometry following transfection of specific plasmids. FIG. 15A is a graph depicting fluorescent readout of cells transfected with plasmids with wild-type R2 (column 1), a negative control (no R2 protein; column 2), 300 ng of R2 with a nuclear localization signal (column 3), 200 ng of R2 with a nuclear localization signal (column 4), 100 ng of R2 with a nuclear localization signal (column 5), 50 ng of R2 with a nuclear localization signal (column 5), and untransfected cells as a percentage of all cells in each sample. FIG. 15B is a graph depicting fluorescent readout of cells transfected with plasmids with wild-type R2 (column 1), a negative control (no R2 protein; column 2), 300 ng of R2 with a nuclear localization signal (column 3), 200 ng of R2 with a nuclear localization signal (column 4), 100 ng of R2 with a nuclear localization signal (column 5), 50 ng of R2 with a nuclear localization signal (column 5), and untransfected cells as a percentage of the number of transfected cells in each sample.

FIG. 16A is a graphic depiction exhibiting the effect that N-terminal truncations of the R2 protein have on integration. HEK293FT cells were transfected with 3 separate plasmids: the first containing an R2 protein encoding region, in which the R2 protein has been truncated from the N-terminus, the second containing a luciferase reporter region with 26/22 bp R2 landing sites, and the third (payload) plasmid containing the second exon necessary for luciferase signal after insertion and splicing of the reporter plasmid. Additionally to a small intronic sequence upstream of the second of two artificial luciferase exons (minimal cargo necessary for luciferase signal), the payload plasmid has 5′ and 3′ UTR and 5′ and 3′ homologies to the 28S target site. Wild-type R2 (column 1) and negative control (column 2) are also depicted. FIG. 16B is a visual representation of the N-terminal truncations of the R2 protein. Each horizontal bar represents the R2 protein expressed, with further N-terminal regions being removed as the numbers go from 1 to 10.

FIG. 17A is a graphic depiction exhibiting the effect that C-terminal truncations of the R2 protein have on integration. HEK293FT cells were transfected with 3 separate plasmids: the first containing an R2 protein encoding region, in which the R2 protein has been truncated from the C-terminus, the second containing a luciferase reporter region with 26/22 bp R2 landing sites, and a third (payload) plasmid containing the second exon necessary for luciferase signal after insertion and splicing of the reporter plasmid. Additionally to a small intronic sequence upstream of the second of two artificial luciferase exons (minimal cargo necessary for luciferase signal), the payload plasmid has 5′ and 3′ UTR sequences and 100 bp 5′ and 3′ homologies to the 28S target site. Wild-type R2 (column 1) and negative control (column 2) are also depicted. FIG. 17B is a visual representation of the N-terminal truncations (Nt_1-Nt_10 from FIG. 16) as well as the C-terminal truncations (Ct 1-Ct 6) of the R2 protein. Each horizontal bar represents the R2 protein expressed, with further N or C-terminal regions being removed as the numbers get larger.

FIG. 18 is a graphical representation of the luminescence readout of HEK293FT cells transfected with three separate plasmids. HEK293FT cells were transfected with 3 separate plasmids. The first plasmid either contained an R2 protein encoding region, no R2 protein encoding region, or an R2 protein with a catalytically inactive restriction-like endonuclease (RLE) domain, which should ablate insertion activity. The second plasmid contained a luciferase reporter region with 26/22 (26 upstream/22 downstream) R2 landing sites, and the third (payload) plasmid contained the second artificial exon necessary for luciferase signal after insertion and splicing of the reporter plasmid. Additionally to a small intronic sequence upstream of the second of two artificial luciferase exons (minimal cargo necessary for luciferase signal), the payload plasmid has 5′ and 3′ UTR sequences as well as 5′ and 3′ homologies.

FIG. 19 is a graphical representation of the luminescence readout of HEK293FT cells transfected with three separate plasmids. HEK293FT cells were transfected with 3 separate plasmids. The first plasmid either contained an R2 protein encoding region, no R2 protein encoding region, or an R2 protein lacking one of several specific R2 protein domains. The second plasmid contained a luciferase reporter region with 26/22 (26 upstream/22 downstream) R2 landing sites. The third plasmid contained an insertion region with a luciferase insertion as well as modified or unmodified UTRs. Columns 1-3 display the results when the transfected R2 protein is an R2 protein in which the −1 domain, which is an RNA interaction domain, has been deleted. Columns 4-6 display the results when the transfected R2 protein is an R2 protein in which the −1 and the 0 domain, which is also an RNA interaction domain, has been deleted. Columns 7-9 display the results when the transfected R2 protein is an R2 protein in which the 0 domain has been deleted. Columns 10-12 display the results when the transfected R2 protein is an R2 protein in which the 0 domain has been replaced by an eGFP domain. Columns 13-15 display the results when the transfected R2 protein is an R2 protein in which the 0 domain has been replaced by an MS2 coat protein (MCP) domain, which binds to MS2 binding sites. Columns 16-18 display the results when the transfected R2 protein is an R2 protein with the N-terminal 6_2 truncation, and the MCP domain has been fused to the new N-terminus. Columns 19-21 display the results when the transfected R2 protein is an R2 protein with the N-terminal 6_2 truncation, MCP domain fused to the new N-terminus, and the zinc finger domain has been deleted. Columns 22-24 display the results when the transfected R2 protein includes a c-terminal MCP fusion. Columns 25-27 display wild-type R2, and columns 28-30 display the negative control. Orange bars have a payload which includes a wild-type luciferase with 5′ and 3′ UTRs. Blue bars indicate payloads in which the 5′ UTR is replaced by extended MS2 regions. Green bars indicate payloads in which both the 5′ and 3′ UTR have been replaced by MS2 regions.

FIG. 20 is a graphical depiction exhibiting the effect that altered payloads have on integration. HEK293FT cells were transfected with 3 separate plasmids: the first containing an R2 protein encoding region, the second containing a luciferase reporter region with 26/22 bp R2 landing sites, and the third (payload) plasmid containing the second artificial exon necessary for luciferase signal after insertion and splicing of the reporter plasmid. Additionally to a small intronic sequence upstream of the second of two artificial luciferase exons (minimal cargo necessary for luciferase signal), the payload plasmid has 5′ and 3′ UTR sequences as well as 5′ and 3′ appended at the 3′ end with a number of different nuclear retention elements, as named on the x-axis. Figure discloses “atcTgtcaGtaAGCCCcatgGaA/A” as SEQ ID NO: 33547.

FIG. 21 is a graphical depiction exhibiting the effect that altered payloads have on integration. HEK293FT cells were transfected with 3 separate plasmids: the first containing an R2 protein encoding region, the second containing a luciferase reporter region with 26/22 bp R2 landing sites and the third (payload) plasmid containing the second artificial exon necessary for luciferase signal after insertion and splicing of the reporter plasmid. Additionally to a small intronic sequence upstream of the second of two artificial luciferase exons (minimal cargo necessary for luciferase signal), the payload plasmid has 5′ and 3′ UTR sequences and modifications thereof as named on the x-axis, as well as 5′ and 3′ homologies.

FIG. 22A is a graphical depiction of luminescence readout of HEK293FT cells transfected with three separate plasmids, indicating cleavage by Cas9. HEK293FT cells were transfected with 3 separate plasmids. The first plasmid either contained modified R2/Cas9 fusion protein, linked together by an XTEN sequence. The second plasmid contained a luciferase reporter region for Cas9 cleavage. The third plasmid a single guide RNA. Columns 1-3 display the results when the transfected R2 protein is an R2 protein in which the −1 domain, which is an RNA interaction domain, has been deleted. Gray bars indicate an R2 protein with a nuclear localization signal, while orange bars indicate an R2 protein without a nuclear localization signal. The x-axis lists the individual R2/Cas9 fusion proteins tested, as well as PDY0044 and a positive control. FIG. 22B is a visual representation of the modified fusion proteins used in FIG. 20A. Vertical lines where in the R2 protein the Cas9 portion is linked to the R2 portion by the XTEN linker.

FIG. 23A is a visual representation exhibiting the integration of a 20 bp sequence to trigger the expression of GFP using a modified Cas9/R2 protein. FIGS. 23A-N represent modified fusion proteins of Cas9 fused at the N-terminus to R2 at varying locations. The fusion proteins of FIGS. 23A-N exhibit the ability to insert a missing 20 bp region into an eGFP precursor (FIG. 23Q), leading to GFP expression. FIG. 23O is a negative control and FIG. 23P is a positive control.

FIG. 24A is a schematic of computational pipeline used to discover and classify site-specific non-LTR retrotransposon systems. Figure discloses SEQ ID NOS 33548-33553 and 33553-33554, respectively, in order of appearance. FIG. 24B-C is a visual representation of a Phylogenetic tree of single-ORF non-LTR retrotransposons. Associations with putative target sites, including tandem repeats and conserved RNA families are shown. Full length ORF size is shown in the outermost ring with associated domains shown in inner rings. Labels of specific retrotransposons orthologs used in this study as well as previously described orthologs are listed above the outer ring with associated symbols labeled on the tree. Tandem repeat GC content percentage is shown as a color scale. Protein domains are colored according to different CDD/Pfam domains analyzed. Putative Myb and zinc finger domains from Prosite and Pfam (ZF) are colored according to the different configurations detected. The 9 families of RLE-containing non-LTR retrotransposons are shaded in different colors and labeled. SL1, corresponds to SL1 spliced-leader RNA. LSU, corresponds to large subunit rRNA (28S). SSU, corresponds to small subunit rRNA (18S). ZF motif labels correspond to different pfam IDs. CDD labels correspond to different CDD IDs.

FIG. 25 is a visual representation of the Size distribution of the ORFs from the first methionine for each of the 9 families of RLE containing non-LTR retrotransposons.

FIG. 26A is a schematic of chimeric non-LTR (nLTR) retrotransposon systems with flanking homologies targeting different insertion sites. E) Gaussia luciferase (Gluc) production via payload insertion of a synthetic exon 2 by selected non-LTR retrotransposons into a 28S plasmid reporter, normalized to a Cypridina luciferase (Cluc) control. FIG. 26B is a schematic of typical non-LTR retrotransposon insertion sites with target sites consistent on both sides of the retrotransposon.

FIG. 27A is a visual analysis of results from a multiple sequence alignment of different non-LTR retrotransposons using MUSCLE, with Pfam domain schematic above as determined by HHpred. FIG. 27B is a visual analysis of sequence identity similarity of chosen non-LTR retrotransposon family members using the MUSCLE protein alignment from E.

FIG. 28 is a visual analysis of the 5′ end of the R10Mbr locus with the microsatellite repeat region and alignment to the human 28S rDNA region highlighted. Figure discloses SEQ ID NOS 33555-33557, respectively, in order of appearance.

FIG. 29A is an analysis of Gaussia luciferase (Gluc) production via payload insertion of a synthetic exon 2 by selected non-LTR retrotransposons into a 28S plasmid reporter, normalized to a Cypridina luciferase (Cluc) control. FIG. 29B is a schematic of payload homology and target sites used to evaluate R10Mbr insertion. Figure discloses SEQ ID NOS 33558-33562, respectively, in order of appearance. FIG. 29C is a visual analysis of the results of an experiment analyzing Gluc payload insertion by R10Mbr into a panel of luciferase reporters, as quantified by luciferase production, with R2Tg targeting the R2 28S sequence as control. Reporters with either similarity to the R2 28S region, or with similarity to the 28S homology region in the R10Mbr locus are used for evaluation of alternative insertion sites.

FIG. 30A is an analysis of EGFP payload insertion by wild type and domain inactivated mutants of R2Tg at the endogenous human 28S locus, analyzed at 5′ and 3′ junctions via gel electrophoresis. Mutants tested were D1274A (RLE inactivation), D877A/D878A/D884A (RT domain inactivation), and ZF2 domain inactivation (replacement of residues 262-275 with NCp7 ZF FNCGKEGHTARN (SEQ ID NO: 33379) (Rocquigny, et al., (1997) J. Biol. Chem. 272, 30753-30759) Red triangles denote faint insertion bands. Schematic above shows insert with the payload denoted in blue, UTRs denoted in black, 28S homology arms denoted red, and 28S locus denoted grey. Black primers are used to readout the left junction and gold primers are used to readout the right junction. FIG. 30B is an analysis of EGFP payload insertion by wild type and domain inactivated mutants of R2Tg into the endogenous 28S locus, quantified by next-generation sequencing. FIG. 30C is an analysis of Gluc production by wild type and domain inactivated mutants of R2Tg into a 28S plasmid reporter, normalized to a Cluc control.

FIG. 31A is graphical analysis of Gaussia luciferase exon 2 (Gluc) payload insertion by wild type and domain inactivated mutants of R2Tg into a 28S plasmid reporter, with editing outcomes profiled by next generation sequencing at the upstream (left) junction. Mutants tested are WT R2Tg and R2TgD1274A, R2TgD877A, D878A, D884A, and R2TgZF2mut, and outcomes are classified as perfect insertions, insertions with indels, or WT locus indels. FIG. 31B is a graphical analysis of Gluc payload insertion by wild type and domain inactivated mutants of R2Tg into a 28S plasmid reporter, with editing outcomes profiled by next generation sequencing at the downstream (right) junction. Mutants tested are WT R2Tg and R2TgD1274A, R2TgD877A, D878A, D884A, and R2TgZF2mut, and outcomes are classified as perfect insertions, insertions with indels, or WT locus indels. FIG. 31C are representative edits at the 5′-insertion junction, showing examples of indels in the outcome insertion products. Figure discloses SEQ ID NOS 33563-33565, respectively, in order of appearance.

FIG. 32A is a schematic of example N- and C-terminal R2Tg truncations for evaluating domain functionality. Not all truncations shown. FIG. 32B is a graphical analysis of Gluc payload insertion by wild type and N- or C-terminal truncations of R2Tg into a 28S plasmid reporter, quantified by next-generation sequencing.

FIG. 33A is a schematic of Cas9H840A-R2Tg insertion at the 28S target, allowing for rescue of R2TgZF2mut activity. FIG. 33B is a graphical analysis of guide-programmed Gluc payload insertion by SpCas9^H840A-R2TgZF2mut into a 28S plasmid reporter, in combination with paired guides or single guides, quantified by next generation sequencing. Perfect insertions, insertions with indels, and pure indel outcomes of Cas9H840A-R2TgZF2mut fusion are compared to SpCas9^H840A. FIG. 33C is a graphical analysis of Gluc payload insertion by WT R2Tg into a 28S plasmid reporter, with editing outcomes profiled by next generation sequencing. Outcomes are classified as perfect insertions, insertions with indels, or WT locus indels.

FIG. 34A is a graphical analysis of a Gluc payload insertion by dead SpCas9D10A, H840A-R2Tg and mutants with targeting and non-targeting guides into a 28S plasmid reporter, as quantified by luciferase production. FIG. 34B is a graphical analysis of a Gluc payload insertion by domain inactivated versions of SpCas9^H840A-R2Tg into a 28S plasmid reporter and quantified by luciferase production and normalized to the corresponding SpCas9^H840A guide condition. SpCas9^H840A-R2Tg is combined with either dual, single, or nontargeting sgRNA combinations. Variants tested are R2TgD1274A and R2TgZF2mut. FIG. 34C is a graphical analysis of a Gluc payload insertion by wild type and domain inactivated mutants of SpCas9^H840A-R2Tg fusion into a 28S plasmid reporter, quantified by luciferase production and normalized to SpCas9^H840A.

FIG. 35A is a schematic for homology length titration of R2Tg payloads, with varying 5′ and 3′ homology lengths (red). The Gluc cargo is shown in blue. Hairpins denote the 5′ and 3′ UTRs. FIG. 35B is a graphical analysis of a Gluc payload insertion by R2Tg into a 28S plasmid reporter with payloads of different 5′ or 3′ homology lengths, profiled by next generation sequencing. Editing outcomes are quantified as perfect insertions, insertions with indels, and pure indels. FIG. 35C is a schematic for R2Tg insertion outcomes at the 28S target site, either with or without scars, with junction amplification primers for Sanger sequencing and gel readouts shown. Black and gold primers are used for 5′ and 3′ junction analyses, respectively. Schematic shows payload denoted in blue, UTRs denoted in black, 28S homology arms denoted red, and 28S locus denoted grey.

FIG. 36A is a schematic of R2Tg scarless payload designs, with permuted and deleted UTR domains. FIG. 36B Sanger sequencing of 5′ and 3′ insertion junctions at the 28S target for additional selected payload designs after R2Tg integration. Payload numbers correspond to those in FIG. 36A. Figure discloses SEQ ID NOS 33566-33567, respectively, in order of appearance. FIG. 36C is a visual depiction of Sanger sequencing of 5′ and 3′ insertion junctions at the 28S target for selected payload designs after R2Tg integration. Payload numbers correspond to those in 36A. Figure discloses SEQ ID NOS 33566, 33568-33569, 33568-33569, 33567, 33569, and 33567, respectively, in order of appearance.

FIG. 37A is a visual representation of edits at the 5′ insertion junction, showing examples of indels in the outcome insertion products. Figure discloses SEQ ID NOS 33563-33565, respectively, in order of appearance. FIG. 37B is a visual depiction of indels at the 5′ junction for R2Tg insertion at the 28S target for selected payloads. Non-templated Cs from reverse transcription in the bottom strand (G in the top strand) are highlighted with red boxes. Figure discloses SEQ ID NOS 33570-33571, 33564, 33572, 33571, 33564, 33582, and 33571, respectively, in order of appearance. FIG. 37C is a visual depiction of a size analysis by gel of 5′ and 3′ insertion junctions at the 28S target reporter for selected payload designs after R2Tg integration. Payload numbers correspond to those in FIG. 36A.

FIG. 38A is a graphical depiction of integration efficiency of R2Tg at the 28S target reporter with different payload designs. Integration is profiled by next-generation sequencing as perfect insertions, insertions with indels, or WT locus indels. Payload numbers correspond to those in FIG. 36A. FIG. 38B is a visual depiction of example indels at the WT 28S locus target for selected payloads. Non-templated Cs from reverse transcription in the bottom strand (G in the top strand) are highlighted with red boxes. Figure discloses SEQ ID NOS 33563, 33565, 33564, 33571, 33564, 33573, 33571, 33564-33565, and 33573, respectively, in order of appearance. FIG. 38C is a schematic representation of additional payload variant with internal homology arms against the 28S target. FIG. 38D is a graphical representation of the Gaussia luciferase exon 2 (Gluc) payload insertion by wild type R2Tg into a 28S plasmid reporter with payload variants shown in part B, with editing outcomes profiled by next generation sequencing at the upstream (left) junction. Outcomes are classified as perfect insertions, insertions with indels, or WT locus indels.

FIG. 39A is a schematic for reprogramming of a R2Tg payload for insertion at the A/AVS1 site with scarless insertion. FIG. 39 B is a graphical depiction of a payload insertion by SpCas9^H840A-R2Tg into the endogenous NOLC1 and A/AVS1 loci, mediated by either single, dual guides, or non-targeting guides and quantified by next generation sequencing. FIG. 39C is a schematic of A/AVS1 targeting payload variations used in FIG. 39D. Payload is shown in blue, homology arms are shown in gold, 5′ 28S homology is shown in red, and UTRs are shown as hairpins. FIG. 39D is a graphical depiction of a Gluc payload insertion, with variations on UTR, 28S homology, and A/AVS1 homology (100 nt), by SpCas9^H840A-R2Tg at endogenous A/AVS1 locus, using a single bottom strand nicking guide. Integration is profiled by next-generation sequencing as perfect insertions, insertions with indels, or indels.

FIG. 40A is a schematic of SpCas9^H840A fused to N- and C-terminal truncations of R2Tg at different amino acid positions. Not all tested constructs are shown. FIG. 40B is a graphical depiction of a Gluc payload insertion by different SpCas9^H840A-R2Tg fusions, according to the schematic in A, into the endogenous A/AVS1 locus quantified by next generation sequencing. FIG. 40C is a graphical depiction of the payload insertion by SpCas9^H840A-R2Tg fusion, SpCas9D10A,_H840A-R2Tg fusion, and SpCas9^H840A and R2Tg in trans. Payloads are inserted at either A/AVS1 or NOLC1 loci, with insertion at A/AVS1 quantified by next generation sequencing and insertions at NOLC1 quantified by ddPCR.

FIG. 41A is a graphical depiction of a Gluc payload insertion by SpCas9^H840A-R2Tg at the endogenous A/AVS1 target site with a panel of dual and single guides, compared with SpCas9^H840A. Payloads have 100 nt of homology to the target site. Editing outcomes are quantified as perfect insertions, insertions with indels, and indels at the unmodified target site. The optimized payload design is used with a 5′ 28S homology arm, truncated 5′ R2Tg UTR, and internal A/AVS1 homology arms. FIG. 41B is a graphical depiction of the integration of Gluc payload at the endogenous A/AVS1 locus by the SpCas9^H840A-R2Tg fusion with a payload containing 50 nt homology arms.

FIG. 42A is a graphical depiction of a Gluc payload insertion into a 28S plasmid reporter by selected non-LTR retrotransposons fused to SpCas9^H840A, with either targeting or non-targeting guides, quantified by Gluc production normalized to a control Cluc. Data is shown as ratio of targeting signal to non-targeting signal. FIG. 42B is a schematic of A/AVS1 insertion with optimized payloads containing the cognate 5′ UTR corresponding to each non-LTR retrotransposon ortholog being evaluated. FIG. 42C is a graphical depiction of a Gluc payload insertion into the endogenous A/AVS1 locus by selected non-LTR retrotransposons fused to SpCas9^H840A, with either targeting or non-targeting guides, quantified by next generation sequencing. FIG. 42D Gluc payload insertion into the endogenous A/AVS1 locus by selected non-LTR retrotransposons fused to SpCas9^H840A, with either targeting or non-targeting guides, profiled by next generation sequencing. Editing outcomes are quantified as perfect insertions, insertions with indels, and indels at the unmodified WT target site.

FIG. 43A is a graphical depiction of EGFP payload insertion (50 nt homology arms) by STITCHR with SpCas9^H840A-R2Toc into the endogenous NOLC1 locus, with combinations of single and dual guides, compared to SpCas9^H840A and quantified by digital droplet PCR (ddPCR). Editing outcomes are quantified as total insertions, integrations with indels, and WT locus indels. FIG. 43B is a graphical depiction of a Gluc payload insertion by STITCHR with SpCas9^H840A-R2Toc into the endogenous SERPINA1 locus (left homology 100 nt and right homology 50 nt), with combinations of single and dual guides, compared to SpCas9^H840A and profiled by next generation sequencing. Editing outcomes are quantified as perfect insertions, insertions with indels, and WT locus indels. FIG. 43C is a graphical depiction of an EGFP payload insertion by STITCHR with SpCas9^H840A-R2Toc into the endogenous NOLC1 locus, with combinations of single and dual guides, compared to a non-targeting guide control and quantified by digital droplet PCR (ddPCR). FIG. 43D is a graphical depiction of an EGFP payload insertion by STITCHR with SpCas9^H840A-R2Toc into the endogenous NOLC1 locus, with combinations of single and dual guides, compared to a non-targeting guide control and profiled by next generation sequencing. Editing outcomes are quantified as perfect insertions, insertions with indels, and WT locus indels.

FIG. 44A is a graphical depiction of an EGFP payload insertion by STITCHR with SpCas9^H840A-R2Toc into the endogenous NOLC1 locus, with a panel of payloads with 50 nt homology arms targeting NOLC1 or A/AVS1 targets, or without homology. Payloads are evaluated with single, dual, or non-targeting guides and are compared to SpCas9^H840A. Editing is quantified by ddPCR. N denotes the NOLC1 target. A denotes the A/AVS1 target. FIG. 44B is a graphical depiction of an EGFP payload insertion by STITCHR with SpCas9^H840A-R2Toc into the endogenous NOLC1 locus, with a panel of payloads with varying homology arm lengths. Payloads are evaluated with dual or non-targeting guides and are compared to SpCas9^H840A. Editing is quantified by ddPCR. FIG. 44C is a graphical evaluation of gene integration at the A/AVS1 locus with SpCas9^H840A-R2Toc and SpCas9^H840A using payloads of varying sized homology arms (100 nt, 75 nt, 50 nt, and 30 nt). Integration is evaluated with dual guides, single guides, and non-targeting guides. FIG. 44D is a graphical evaluation of gene integration at the SERPINA1 locus with SpCas9^H840A-R2Toc and SpCas9^H840A using payloads of varying sized homology arms (100 nt, 75 nt, 50 nt, and 30 nt). Integration is evaluated with dual guides, single guides, and non-targeting guides.

FIG. 45A is a schematic of STITCHR using SpCas9^H840A-R2Toc to insert EGFP as a scarless in-frame fusion at the N-terminus of the human NOLC1 gene. The EGFP template is transcribed in a reverse complement manner to minimize background expression in the absence of insertion with 50 nt homology arms. FIG. 45B is an immunohistochemical analysis of STITCHR-mediated EGFP tagging of NOLC1, visualized by confocal microscopy, and compared to immunofluorescence staining of NOLC1. White scale bar denotes 10 μm. FIG. 45C is a graphical depiction of therapeutically relevant payload insertion by STITCHR with SpCas9^H840A-R2Toc into the endogenous A/AVS1 locus, with sizes and identities of payload panel members shown and 100 nt homology arms. Integration is quantified by next generation sequencing and compared to SpCas9^H840A. FIG. 45D is a graphical depiction of therapeutically relevant payload insertion by STITCHR with SpCas9^H840A-R2Toc into the endogenous A/AVS1 locus, compared to SpCas9^H840A. Integration is profiled by next-generation sequencing as perfect insertions, insertions with indels, or WT locus indels.

FIG. 46A is a graphical depiction of EGFP payload insertion (50 nt homology arms) by STITCHR with SpCas9^H840A-R2Toc into the endogenous NOLC1 locus in cells treated with varying concentrations of aphidicolin. Integration is quantified by ddPCR and compared to SpCas9^H840A. FIG. 46B is a graphical depiction of SpCas9-mediated HDR editing of the EMX1 gene in cells treated with varying concentrations of aphidicolin. Genome editing is quantified by next generation sequencing. FIG. 46C is a graphical depiction of EGFP payload insertion efficiencies at endogenous NOLC1 locus by homology-directed repair (HDR), using SpCas9, at different concentrations of the cell cycle inhibitor aphidicolin or DMSO control.

FIG. 47A is a graphical depiction of multiplexed gene integration by STITCHR with SpCas9^H840A-R2Toc at NOLC1 and A/AVS1 sites. EGFP payload insertion at NOLC1 is quantified by ddPCR, and Gluc insertion at A/AVS1 is quantified by next generation sequencing. Targeting conditions are compared to non-targeting guide controls. FIG. 47B is a graphical depiction of multiplexed gene integration by STITCHR with SpCas9^H840A-R2Toc at NOLC1 and A/AVS1 sites, profiled by next generation sequencing. Total insertion for NOLC1 is quantified by ddPCR. Editing outcomes are quantified as perfect insertions, insertions with indels, and WT locus indels. N denotes NOLC1, whereas A denotes A/AVS1.

FIG. 48 is a schematic representation of STITCHR, enabling programmable and modular scarless gene insertion with site-specific non-LTR (nLTR) retrotransposons.

FIG. 49 is a graphical representation of the results of an experiment in which an EGFP payload was inserted (50 nt homology arms) by STITCHR with SpCas9^H840A-R2Toc into the endogenous NOLC1 locus, with a single fixed guide, compared to SpCas9^H840A and quantified by digital droplet PCR (ddPCR). Homology arms on the templates are separated by 0, 50, 100, or 150 bp on the genome causing a deletion to occur followed by simultaneous insertion of the STITCHR EGFP payload. The payload arms are also shifted to match the locations of the single nicking guide and the desired end of the deletion to enable the deletion and subsequent insertion.

FIG. 50A is a graphical representation of payload insertion (50 nt homology arms) by STITCHR with SpCas9^H840A-R2Toc into the endogenous NOLC1 locus, with dual guides N4 and N8, compared to SpCas9^H840A and quantified by next generation sequencing. The introduced edit is either a mismatch to the genome to demonstrate single base corrections or are small insertions as noted in the x-axis of the plot. FIG. 50B is a graphical representation of payload insertion (50 nt homology arms) by STITCHR with SpCas9^H840A-R2Toc into the endogenous NOLC1 locus, with dual guides N4 and N8, compared to SpCas9^H840A and quantified by next generation sequencing. The introduced edit is either a mismatch to the genome to demonstrate single base corrections or are small insertions as noted in the x-axis of the plot. Cargo is driven by either the U6 promoter or the CAG promoter, showing that the CAG promoter expression of the cargo results in slightly higher editing.

FIG. 51 is a graphical representation of the results of an experiment in which EGFP payload was inserted (50 nt homology arms) by STITCHR with SpCas9^H840A-R2Toc into the endogenous NOLC1 locus, with dual guides N4 and N8, compared to SpCas9^H840A and quantified by digital droplet PCR (ddPCR). STITCHR insertion is also compared to SpCas9^H840A and R2Toc being expressed separately (in trans).

FIG. 52 is a heatmap chart representation of nLTR families with diverging target preferences, with counts of co-occurring divergent Rfam annotation target pairs.

FIG. 53 are loci of nLTR system families with divergent target preferences as determined via Rfam analysis. Families are clustered by ORF identity.

FIG. 54A is a schematic representation of the insertion by non-LTR retrotransposons at the natural 28S target site, depicting initial nicking and strand invasion, target-primed reverse transcription, first strand synthesis, nicking-initiated second strand synthesis, and insertion of a payload sequence into the genome. 28S homology, UTR sequences, and payload sequence are indicated. FIG. 54B is a schematic representation of Gaussia luciferase (Gluc) production via payload insertion of a synthetic Gluc exon 2 by 12 selected non-LTR retrotransposons into a 28S plasmid reporter containing a synthetic Gluc exon 1, normalized to a constitutive Cypridina luciferase (Cluc) control. FIG. 54C is a schematic representation of Gluc exon 2 payload insertion by R2Tg into a 28S plasmid reporter with payloads of different 5′ or 3′ UTR deletions and homology site permutations, profiled by next generation sequencing. Schematic shows the payload design used with UTRs, 5′ 28S homology arms, 3′ 28S homology arms, and the Gluc exon 2 insert.

FIG. 55A are gel electrophoresis images of the analysis of 5′ and 3′ insertion junctions at the 28S target reporter using payload designs with permuted UTR and homology positions after R2Tg integration. Payload numbers correspond to those in FIG. 54C. FIG. 55B is a schematic representation of the Gluc exon 2 payload insertion by WT R2Tg, R2Tg^D1274A or the RT domain deletion R2T^gA(874-884) into a 28S plasmid reporter with payloads containing 28S or A/AVS1 targeting homology arms, profiled by next generation sequencing. FIG. 55C is a graphical representation of the EGFP payload insertion at the NOLC1_target using R2Tg, R2Tg^D1274A, or R2Tg^RTmut and a payload containing the 5′ UTR and 50 nt NOLC1 homology arms, quantified by next-generation sequencing.

FIG. 56A is a schematic representation of the reprogramming of a R2Tg payload for insertion at a novel site with scarless insertion using SpCas9^H840A. FIG. 56B is a graphical representation of the payload insertion by SpCas9^H840A-R2Tg or SpCas9^H840A-R2Tg^D1274A into the endogenous NOLC1 locus, mediated by dual guides or non-targeting guides and quantified by ddPCR.

FIG. 57 is a schematic representation of the EGFP payload insertion, with variations on 5′ and 3′ UTR sequence by SpCas9^H840A-R2Tg at the endogenous NOLC1 locus, using dual guides. Integration is quantified by ddPCR. Schematic of payload variations used with the payload, homology arms, 5′ and 3′ UTRs are illustrated.

FIG. 58A is a graphical representation of the EGFP payload insertion by SpCas9^H840A-R2Tg (WT), SpCas9^H840A-R2Tg^{F875A/A876L/iD877A/D878A/L879A/V880A/L881A} (RTmut), and SpCas9^H840A-R2Tg^Δ(874-884) (Δ(874-884)), and SpCas9^H840A at the endogenous NOLC1 target site with dual guides. FIG. 58B is a schematic representation of A/AVS1 insertion with optimized payloads containing the cognate 5′ UTR corresponding to each non-LTR retrotransposon ortholog being evaluated. Gluc payload insertion into the endogenous A/AVS1 locus by selected non-LTR retrotransposons fused to SpCas9^H840A, with either targeting or non-targeting (NT) guides, is quantified by next generation sequencing. The heatmaps correspond to Gluc integration efficiency (top) and the associated indels generated at the A/AVS1 locus (bottom).

FIG. 59A is a schematic representation of the EGFP payload insertion (50 nt homology arms) by STITCHR with SpCas9^H840A-R2Toc into the endogenous A/AVS1, LMNB1, EMX1, and NOLC1 loci, with combinations of single and dual guides, compared to SpCas9^H840A-R2TocRTmut and wild-type SpCas9. The left heatmap shows integration rate of the EGFP payload, whereas the right heatmap corresponds to indels detected at the corresponding loci. FIG. 59B is a schematic representation of different STITCHR edits evaluated ranging from single-base variants, small insertions, and large insertions. FIG. 59C is a graphical representation of the evaluation of different sized edits using STITCHR at the NOLC1 locus using either SpCas9^H840A-R2Toc or SpCas9^H840A

FIG. 60A is a schematic representation of STITCHR-replace methodology involving replacement of a region of the genome while inserting the STITCHR payload. FIG. 60B is a graphical representation of the evaluation of STITCHR-replace at the NOLC1 locus using a single guide and homology arms spaced 50-150 bp apart on the genome.

FIG. 61 is a schematic representation of the natural reprogramming of RLE-containing non-LTR retrotransposons, incorporating flexible internal priming and UTR deletions that might occur during the process.

FIG. 62A is a graphical representation of the distribution of distances from candidate retrotransposons to detected Rfam annotation or tandem repeat targets for each of the 9 families of RLE containing non-LTR retrotransposons. FIG. 62B is a graphical representation of the distribution of the predicted 5′ and 3′ UTR sizes for all non-LTR RLE-containing retrotransposons. UTR sizes are predicted based on the distance from the ORF and nearest predicted target site. Box plots are shown with the median, 25th percentile, 75th percentile, and whiskers that are 1.5× the interquartile range. All outliers are shown as individual points. FIG. 62C is a graphical representation of the distribution of the lengths of observed non-coding conservation regions flanking the 5′ and 3′ ends of the retrotransposon ORF. Box plots are shown with the median, 25th percentile, 75th percentile, and whiskers that are 1.5× the interquartile range. All outliers are shown as individual points.

FIG. 63 is the phylogenetic tree representation of 9 families of RLE-containing nLTR systems showing majority of detected Rfam targets in the vicinity of the nLTR ORF.

FIG. 64A-E are the DNA sequence alignments of nLTR families with divergent target preferences in the noncoding areas surrounding the nLTR ORFs. Identified Rfam annotations in the surrounding locus are highlighted.

FIG. 65A is the graphical representation of the Gluc payload insertion by R2Tg reverse transcriptase domain deletions, RLE inactivation mutants (R1274A) and reverse transcriptase mutations (R2Tg^{F875A/A876L/D877A/D878A/L879A/V880A/L881A}, RTmut), at the 28S locus luciferase reporter, as quantified by luciferase. FIG. 65B is the graphical representation of the Gluc payload insertion by R2Tg reverse transcriptase domain mutations, including R2Tg^{F875A/A876L/D877A/D878A/L879A/V880A/L881A} (RTmut) and RLE inactivation mutants (R1274A), at the 28S locus luciferase reporter, as quantified by luciferase.

FIG. 66A is a schematic representation of the secondary structure analysis of the 5′ UTR of R2Tg, including the full length, 15 nt truncated variant, and the 15 nt truncated variant with the 50 nt 28S homology sequence upstream. Figure discloses SEQ ID NOS 33574-33576, respectively, in order of appearance. FIG. 66B is a graphical representation of the validation of the 3-primer NGS assay for analysis of A/AVS1 integration via the left insertion junction. Standards consist of edited and WT amplicons that are mixed in the listed ratios (xaxis) and the measured editing is determined by the 3-primer NGS assay (y-axis). FIG. 66C is the schematic and graphical representation of the Gluc integration at the endogenous A/AVS1 locus via the SpCas9^H840A-R2Tg fusion using payloads with the full length or 15-nt truncated 5′ UTR, an upstream 28S 50 nt sequence, and internal A/AVS1 homology arms. Integration is quantified by next-generation sequencing.

FIG. 67A is a schematic representation of SpCas9^H840A fused to N- and C-terminal truncations of R2Tg at different amino acid positions. Not all tested constructs are shown. FIG. 67B is a graphical representation of the Gluc payload insertion by different SpCas9^H840A-R2Tg fusions, according to the schematic in FIG. 67A, into the endogenous A/AVS1 locus quantified by next generation sequencing. FIG. 67C is a graphical representation of the Gluc integration at the endogenous A/AVS1_target by SpCas9^H840A-R2Tg, SpCas9^H840A-R2Tg^{F875A/A876L/D877A/D878A/L879A/V880A/L881A}, and SpCas9^H840A-R2Tg^Δ(874-884), and SpCas9^H840A alone.

FIG. 68 is a schematic representation of the Gluc payload insertion into the endogenous A/AVS1 locus by selected non-LTR retrotransposons fused to SpCas9^H840A, with either targeting or nontargeting guides, profiled by next generation sequencing. Editing outcomes are quantified as perfect insertions, insertions with indels, and indels at the unmodified WT target site.

FIG. 69A is a graphical representation of the Gluc payload insertion by STITCHR with SpCas9^H840A-R2Toc into the endogenous A/AVS1 locus, with combinations of single and dual guides, compared to a non-targeting guide control and quantified by next generation sequencing. FIG. 69B is a graphical representation of the EGFP payload insertion by STITCHR with SpCas9^H840A-R2Toc into the endogenous LMNB1 locus, with combinations of single and dual guides, compared to a non-targeting guide control and SpCas9^H840A alone. Editing was quantified by digital droplet PCR (ddPCR). FIG. 69C is a graphical representation of the EGFP payload insertion by STITCHR with SpCas9^H840A-R2Toc into the endogenous EMX1 locus, with combinations of single and dual guides, compared to a non-targeting guide control and SpCas9^H840A alone. Editing was quantified by digital droplet PCR (ddPCR).

FIG. 70A is a graphical representation of the Gluc payload insertion by SpCas9^H840A-R2Toc (WT), SpCas9^H840AR2Toc^{F811A, A812L, D813A, D814A, L815A, V816A, L817A}, SpCas9^H840A-R2Toc^Δ(811-814), SpCas9^H840A-R2Toc^Δ(810-820), and SpCas9^H840A at the endogenous A/AVS1 target site. Editing is quantified by next generation sequencing. FIG. 70B is a graphical representation of the EGFP payload insertion by SpCas9^H840A-R2Toc (WT), SpCas9^H840AR2Toc^{F811A, A812L, D813A, D814A, L815A, V816A, L817A}, SpCa9^H840A-R2Toc^Δ(875-878), SpCas9^H840A-R2Toc^Δ(874-884), and SpCas9^H840A at the endogenous NOLC1 target site. Editing is quantified by ddPCR. FIG. 70C is a graphical representation of the GFP payload insertion by SpCas9^H840A-R2Toc (WT), SpCas9^H840A-R2Toc^D1210A and SpCas9^H840A at the endogenous NOLC1 target site. Editing is quantified by ddPCR.

FIG. 71A is a graphical representation of the GFP payload insertion by STITCHR with SpCas9^H840A-R2Toc into the endogenous NOLC1 locus in HepG2 cells, compared to SpCas9^H840A. Editing is quantified by ddPCR. FIG. 71B is a graphical representation of STITCHR EGFP payload insertion at endogenous EMX1, NOLC1 and two A/AVS1 loci in Huh-7 cells by SpCas9^H840A-R2Toc compared to SpCas9^H840A-R2TocRTmut. Insertion is quantified by ddPCR. FIG. 71C is a graphical representation of STITCHR EGFP payload insertion at endogenous EMX1 and NOLC1 loci in HepG2 cells by SpCas9^H840A-R2Toc compared to SpCas9^H840A-R2TocRTmut. Insertion is quantified by ddPCR.

FIG. 72 is a graphical representation of the installation of small edits and insertions using STITCHR at the NOLC1 locus, using a U6 promoter for payload expression.

FIG. 73 are sequencing reads of the EGFP insertion site at NOLC1 for STITCHR replace, showing the desired 50-150 bp deletions. Figure discloses SEQ ID NOS 33577-33578, 33577, 33577, 33577, 33579, 33579, 33579, 33579-33580, 33580, 33580, 33580-33581, 33581, 33581, and 33581, respectively, in order of appearance.

FIG. 74A is a graphical representation of the EGFP payload insertion (50 nt homology arms) by STITCHR with SpCas9^H840A-R2Toc into the endogenous A/AVS1 locus in cells treated with cell cycling inhibitor Mirin or double thymidine. Integration is quantified by next-generation sequencing and compared to SpCas9^H840A. FIG. 74B is a graphical representation of the SpCas9-mediated HDR editing of the EMX1 gene in cells treated with cell cycling inhibitor Mirin or double thymidine. Genome editing is quantified by next generation sequencing.

FIG. 75 is a graphical representation of 10 orthologs sampled from various nLTR families (1, 4, 5, 6, 7, 9) compared to R2Toc for programmed insertion at the A/AVS1 locus. Orthologs were synthesized with mammalian codon optimization, and putative 5′ and 3′ UTR regions were cloned surrounding a luciferase payload. Protein and payload constructs were transfected along with a SpCas9 μlasmid and guide plasmid into HEK293FT cells, and 3 days later cells were harvested and efficiency of insertion were quantified by next generation sequencing.

FIG. 76A-C are tables showing plasmid vectors for genome editing.

FIG. 77 is a heatmap of 28S luciferase reporter assay, testing integration by R2Bm, R2Tg, R2Mes and R2TgRTmut (x axis) using RNA payloads containing UTRs from different retrotransposon ortholog systems (y axis).

FIG. 78A is a depiction of long-read sequencing of EGFP payload insertion by SpCas9^H840A-R2Toc at the endogenous NOLC1 locus. Shown is read alignments to scarless EGFP insertion at NOLC1. FIG. 78B Circos plots depicting genome-wide insertion sites of payloads by SpCas9^H840A-R2Toc using sgRNAs and payload homologies to b) A/AVS1 (chr19) and c) NOLC1 (chr10). Counts are defined as the number of mapped reads occurring within a 5kb window.

FIG. 79A-B are bar graphs and western blots examining the insertion of Gluc payload with (A) or without (B) UTRs into 28S DNA target+/−payload RNA, +/−28S DNA, +/−R2 protein, +/−Mg2+ and +/−dNTPs, as indicated. Above, NGS quantitation of insertion efficiency and schematic of the used RNA payloads. Arrows on the gels indicate the specific TPRT products.

FIG. 80A is a bar graph depicting the results of a biochemical assay assessing TPRT by R2Tg into 28S DNA using RNA payloads with 100 bp, 60 bp, 30 bp and 0 bp 28S homology and no RNA payload control. Insertion frequency is quantified by NGS. FIG. 80B is an image of a western blot depicting the results of a Biochemical assay assessing TPRT by R2Tg into 28S DNA using RNA payloads with or without 5′ cap and/or 3′ poly-A tail modifications as well as no RNA payload control. FIG. 80C is a bar graph depicting the NGS insertion quantification of TPRT shown in 80B.

FIG. 81A is an image of a western blot result of a biochemical assay assessing TPRT by R2Tg into 28S DNA with decreasing concentrations of protein using the 28S RNA payload with full UTRs and 100 bp homology. Concentrations tested were (left to right): 500 nM, 166 nM, 55 nM, 19 nM, 6 nM and 0 nM. FIG. 81B is an image of a western blot assessing the timecourse of biochemical TPRT by R2Tg into 28S DNA with or without RNA payloads with different incubation times, as indicated. FIG. 81C is a bar graph depicting the NGS insertion quantification of TPRT shown in FIG. 81B.

FIG. 82A depicts the R2Tg retrotransposition of synthetic RNA payload into top- and bottom-strand labeled 28S DNA (top is Cy5 labeled, red, and bottom is FAM labeled, green), +/−payload RNA, +/−dNTPs and +/−R2 protein, as indicated. Schematics on the side of the gel indicate the expected identify of each band, including the TPRT product. FIG. 82B is a bar graph and western blot depicting the results of an assay assessing biochemical insertion of the Gluc RNA payload into 28S DNA by wild type R2Tg, RT inactivated, and RLE inactivated proteins and a 1:1 mixture of RT and RLE mutant proteins. Above, NGS quantification of insertion efficiency and a schematic of the RNA payload used. Mutants tested were D1274A (RLE inactivation) and A(875-878) (RT domain inactivation). Arrow on the gel indicates the specific TPRT product. FIG. 82C is an immunoblot depicting the results of a Primer extension assay by WT R2Tg, RLE inactivated R2Tg^D1274A, and no protein, where 28S RNA payload and complementary primer were hybridized and extended by reverse transcription activity of the R2Tg protein.

FIG. 83A is an immunoblot depicting a size analysis by gel electrophoresis of 5′ and 3′ insertion junctions at the 28S target reporter for payload designs from FIG. 38C-D after R2Tg integration. FIG. 83B depicts the integration percentage of Gluc exon 2 payload insertion by WT R2Tg, R2TgD1274A, or the RT domain deletion R2Tg^Δ(874-884) into a 28S plasmid reporter with payloads containing 28S or A/AVS1 targeting homology arms, profiled by next generation sequencing.

FIG. 84A depicts the results of a biochemical primer extension assay, using IVT-transcribed RNA payloads for NOLC1 or 28S sequences and a primer complementary to the 28S and NOLC1 sequences (U) or to the NOLC1 sequence only (N). Arrow on the gel indicates the specific TPRT product. FIG. 84B depicts the results of an assay assessing biochemical retrotransposition of an RNA payload into the NOLC1 DNA target+/−Cas9-assisted nicking and with withdrawal of R2Tg protein, RNA, dNTPs, DNA target, or MgCl2, as indicated. Labels on the gel indicate the specific TPRT product, DNA target band, Cas9 produced nicked fragments, and R2Tg produced nicked fragments. Unless otherwise specified, paired NOLC1 guides 4 and 8 are used. s1, single NOLC1 guide 4. s2, single NOLC1 guide 8. FIG. 84C depicts the editing percentage as determined by NGS quantification of insertion data shown in FIG. 84B.

FIG. 85 is a bar graph depicting the integration percentage of an EGFP payload insertion at human endogenous NOLC1 locus by natural reprogrammed wild-type R2Tg as well as R2TgD1274A and R2TgRTmut.

FIG. 86A is an image depicting the reprogrammed biochemical retrotransposition of an IVT-transcribed RNA payload containing the optimized 5′ and 3′ UTR and homology regions into the A/AVS1 DNA target by R2Tg+/−DNA target, +/−RNA, +/−Cas9-assisted nicking, and +/−R2Tg, as indicated. Black arrow on the gel indicates the specific TPRT product. The blue arrow denotes the cleaved DNA band generated by R2Tg protein alone reprogrammed by its payload RNA. FIG. 86B is a bar graph depicting the NGS quantification of insertion data shown in FIG. 86A. FIG. 86C is an image depicting the reprogrammed biochemical retrotransposition by R2Tg into the NOLC1 DNA target, using a homologous IVT-transcribed NOLC1 payload (N) with +/−5′ cap and 3′ tail modifications compared to EMX1 (E)- or 28-homologous (28S) payloads (i.e. non-homologous to NOLC1). Integration is quantified by NGS.

FIG. 87A is a bar graph depicting the integration efficiencies, quantified by NGS, of reprogrammed biochemical TPRT of an RNA payload by R2Tg into varying amounts of NOLC1 DNA target compared to no RNA controls. FIG. 87B is a bar graph depicting the integration efficiencies, quantified by NGS, of reprogrammed biochemical TPRT by R2Tg using NOLC1 RNA payloads incorporating either different single-base mismatches or insertions into the NOLC1 DNA, as indicated. Either in vitro transcribed mRNA or synthetic RNA templates were used as the payloads.

FIG. 88A is a bar graph depicting the payload retrotransposition into the human endogenous NOLC1 genomic locus by SpCas9^H840A-R2Tg fusion, SpCas9^H840A only, SpCas9^H840A-R2TgRTmut, SpCas9^H840A-R2TgD1274A, and complementing SpCas9^H840A-R2TgD1274A+SpCas9^H840A-R2TgRTmut. Integration is quantified by NGS. Inset shows payload design and locus schematic with homology arms colored and top guide in red and bottom guide in blue. FIG. 88B is a bar graph depicting Gluc integration at the endogenous A/AVS1_target by SpCas9^H840A-R2Tg, SpCas9^H840A-R2TgF875A/A876L/D877A/D878A/L879A/V880A/L881A(RTmut), and SpCas9^H840A-R2Tg^Δ(874-884), and SpCas9^H840A alone. FIG. 88C is a bar graph depicting TPRT activity in HEK293FT cells with SpCas9^H840A alone or fused to R2Tg, R2TgF875A/A876L/D877A/D878A/L879A/V880A/L881A (RTmut), or R2TgA874-884 into the NOLC1 genomic target with dual guides. EGFP payload contains the full 5′ and 3′ UTRs for R2Tg.

FIG. 89A and FIG. 89B are bar graphs depicting TPRT activity in HEK293FT cells by R2Tg into the NOLC (FIG. 89A) and A/AVS1 (FIG. 89B) genomic targets, with in trans complementation of R2TgRLEmut and R2TgRTmut, quantified by NGS.

FIG. 90A is a pair of images depicting the results of an assay in which EGFP payload insertion by wild type and domain inactivated mutants of R2Tg at the endogenous human 28S locus in HEK293FT cells was assessed, analyzed at 5′ and 3′ junctions via gel electrophoresis. Mutants tested were RLE inactivated, RT domain inactivated, and ZF2 domain inactivated (replacement of residues 262-275 with NCp7 ZF FNCGKEGHTARN37). Payload was expressed from a plasmid template. FIG. 90B is a bar graph depicting the results of Gluc payload insertion by R2Tg reverse transcriptase domain deletions, RLE inactivation mutants (R1274A), and reverse transcriptase mutations (R2TgF875A/A876L/D877A/D878A/L879A/V880A/L881A, RTmut), at the 28S locus luciferase reporter target, as quantified by luciferase activity. Luciferase activity was assayed in HEK293FT cells. FIG. 90C is a bar graph depicting the results of Gluc payload insertion by R2Tg RT domain mutations, including R2TgF875A/A876L/D877A/D878A/L879A/V880A/L881A (RTmut), R2TgD877R, D878R, and R2TgD877H, D878H, and the RLE inactivation mutant (R1274A) at the 28S locus luciferase reporter, as quantified by luciferase. Luciferase activity was assayed in HEK293FT cells.

FIG. 91A depicts a pair of images assessing payload insertion into a 28S integration site. Above, RNA payload insertion into a 28S plasmid reporter by wild type R2Tg, RLE inactivated, RT inactivated, and complemented RT and RLE inactivated proteins+/−RNA payload, as indicated. RNA templates used were IVT transcribed with a 5′ cap and a poly A tail. Expected band size=260 bp. NT, non-targeting RNA templates that have homology to the NOLC1_target instead of the 28S locus. Below, R2Tg insertion into human 28S endogenous locus with payloads containing 100, 50, 30 or 0 homology to the 28S target site. RNA templates used were IVT transcribed with a 5′ cap and a poly A tail. Expected band size=374 bp. FIG. 91B is a bar graph depicting the results of a Luciferase assay of Gluc insertion of an IVT-transcribed RNA payload with variable 3′ tail length into a 28S reporter target by WT R2Tg and RLE-inactivated R2TgD1274A. Luciferase activity was assayed in Huh-7 cells.

FIG. 92A is a bar graph and image depicting the results of a biochemical assay editing percentage in an A/AVS1 site. Top: Biochemical retrotransposition of an RNA payload into A/AVS1 DNA by R2Toc with and without SpCas9^H840A-assisted nicking, +/−RNA payload, +/−DNA target, +/−R2Toc protein, as indicated. Bottom, NGS quantification of R2Toc insertion. Black arrow on the gel indicates the specific TPRT product. The blue arrow denotes the cleaved DNA band generated by the R2Toc protein alone reprogrammed by its payload RNA. FIG. 92B is a bar graph depicting STITCHR payload insertions at human endogenous genomic NOLC1 loci by SpCas9^H840A-R2Toc, SpCas9^H840A only, SpCas9^H840A-R2TocRTmut, R2TocRTmut alone, SpCas9-R2TocRTmut and complemented R2TocRTmut+SpCas9-R2TocRLEmut. Integration is quantified by ddPCR. Inset shows payload design and locus schematic with homology arms colored and top guide in red and bottom guide in blue. FIG. 92C is a bar graph depicting STITCHR payload insertions at human endogenous genomic A/AVS1 loci by SpCas9^H840A-R2Toc, SpCas9^H840A only, SpCas9^H840A-R2TocRTmut, R2TocRTmut alone, SpCas9-R2TocRTmut and complemented R2TocRTmut+SpCas9-R2TocRLEmut. Integration is quantified by NGS. Inset shows payload design and locus schematic with homology arms colored and bottom guide in blue. FIG. 92D is a bar graph depicting protein expression of wild type and mutant versions of different R2Tg orthologs (indicated on x-axis), quantified by luciferase signal.

FIG. 93A is a bar graph depicting STITCHR 20 bp payload insertion on a luciferase reporter plasmid from a synthetic RNA lacking the 5′ UTR and containing a Cas9 guide scaffold. Integration is quantified by NGS. FIG. 93B is a bar graph depicting Gluc reconstitution by correction of a 20 bp deletion by delivery of plasmid or synthetic RNA payloads, quantified by Gluc expression normalized to control Cluc expression. The synthetic RNA template is an extension of the Cas9 sgRNA. FIG. 93C-D are bar graphs depicting Gluc reconstitution by R2Tg mutants with synthetic RNA payloads extended off the guide RNA as quantified by NGS (FIG. 93C) and Gluc (FIG. 93D) expression normalized to control Cluc.

FIG. 94A is a bar graph depicting STITCHR 38 bp payload insertion at the endogenous LMNB1 locus from a synthetic RNA lacking the 5′ UTR and containing a Cas9 guide scaffold. Integration is quantified by NGS. FIG. 94B is a bar graph depicting STITCHR 700 bp EGFP payload insertion at the endogenous NOLC1 locus from an in vitro transcribed mRNA containing a full 5′ and 3′ UTR and either with or without a 5′ cap or polyA tailing. Integration is quantified by NGS. Inset shows payload design and locus schematic with homology arms colored and top guide in red and bottom guide in blue. FIG. 94C is a bar graph depicting STITCHR 700 bp EGFP payload insertion in the Huh-7 hepatocellular carcinoma line at the endogenous NOLC1 locus from an in vitro transcribed mRNA containing either a truncated R2Tg 5′ UTR (s) or full R2Tg 5′ and 3′ UTRs. Insertion is tested with either SpCas9^H840A-R2Tg or SpCas9^H840A-R2TgRTmut. Integration is quantified by NGS. Inset shows payload design and locus schematic with homology arms colored and top guide in red and bottom guide in blue. FIG. 94D is a bar graph depicting insertion of a GFP payload delivered as an IVT-transcribed mRNA with UTRs and other variable modifications, as indicated, into the human endogenous NOLC1 locus by SpCas9^H840A-R2Toc in HEK293FT cells.

FIG. 95A is an image depicting the result of a PCR junctional analysis of STITCHR 700 EGFP bp payload insertion at the endogenous NOLC1 locus from an in vitro transcribed mRNA containing either or truncated 5′ UTR (s) or a full 5′ and 3′ UTR (f). Insertion is tested with either SpCas9^H840A-R2Toc or SpCas9^H840A-R2TocRTmut. FIG. 95B is a bar graph depicting STITCHR 700 bp EGFP payload insertion in the Huh-7 hepatocellular carcinoma line at the endogenous NOLC1 locus from an in vitro transcribed mRNA containing either a truncated R2Tg 5′ UTR (s) or full R2Tg 5′ and 3′ UTRs. Insertion is tested with either SpCas9^H840A-R2Tg or SpCas9^H840A-R2TgRTmut. Integration is quantified by NGS. Inset shows payload design and locus schematic with homology arms colored and top guide in red and bottom guide in blue. FIG. 95C is a bar graph depicting the results of an assay assessing SpCas9^H840A-R2Toc integration of an RNA payload containing an intron at the human endogenous NOLC1 locus. Shown is NGS quantification of the spliced insertion (i.e., without intron).

FIG. 96A is a diagram depicting guide positions of FIG. 59A, depicted as loci schematics indicating the top (red) or bottom (blue) SpCas9 guide positions relative to the colored homology region for each gene. FIG. 96B is a bar graph depicting the results of EGFP payload insertion by STITCHR with SpCas9^H840A-R2Toc into the endogenous A/AVS1 locus, with combinations of single and dual guides, compared to SpCas9^H840A alone, SpCas9^H840A-R2TocRTmut, and SpCas9. Insertion is quantified by ddPCR.

FIG. 97A is a bar graph depicting the results of EGFP payload insertion at endogenous NOLC1 by STITCHR, delivered by adenovirus to HEK293FT cells at different viral amounts. Shown is a comparison of insertion efficiency when delivering STITCHR machinery with one vector and guides and template with the other, compared to delivery of guides and template only as a control. FIG. 97B is a bar graph depicting the results of EGFP payload insertion by SpCas9^H840A-R2Toc at NOLC1_target in quiescent primary human hepatocyte cells compared to SpCas9^H840A control. Total viral amount used was 1.4e11 viral copies in the dual vector condition and half of that for the single vector payload only condition. FIG. 97C is a bar graph depicting the results of EGFP payload insertion by STITCHR at the NOLC1 endogenous locus in HEK293FT cells, comparing editing efficiencies with two insertion designs: one that removes the endogenous PAM sequence and one that maintains it. FIG. 97D is a bar graph depicting the results of STITCHR insertion of EGFP payload at the endogenous NOLC1 locus by SpCas9^H840A-R2Toc compared to SpCas9D10A-R2Toc.

FIG. 98A is an image showing the results from the retargeting of R2 substrate nicking via reprogramming of homology arms independent of Cas9 toward a A/AVS1 target. FIG. 98B is a schematic representation of the profiling with next-generation sequencing of adaptors ligated to free DNA ends. FIG. 98C is a graph that shows that no appreciable cleavage could be observed with the majority reads corresponding to the 5′ or 3′ ends of the uncleaved target.

FIG. 99A is an image showing the results from the reprogramming of R2Tg towards a NOLC1 target. FIG. 99B is a graph showing the cleavage sites when using R2Tg combined with RNA. FIG. 99C is a graph showing the cleavage sites when using R2Tg not combined with RNA.

DETAILED DESCRIPTION

The present disclosure is directed to site specific non-Long Terminal Repeat (LTR) retrotransposons and systems incorporating these non-LTR retrotransposons for inserting large nucleic acids at targeted locations within a genome. The present disclosure is also directed to site-specific non-LTR retrotransposons and related systems for performing small nucleotide changes in a genome. In some embodiments, a small nucleotide change comprises a point mutation. In some embodiments, a small nucleotide change comprises a small nucleotide insertion.

The present disclosure is also directed to modified R2 fusion proteins for inserting large nucleic acids at targeted locations within a genome. The present disclosure is also directed to Cas9 fusion proteins for inserting large nucleic acids at targeted locations within a genome, which includes Cas9-R2 fusion proteins. In some embodiments, the genome is a human genome.

The present disclosure is also directed to the insertion of exogenous R2 landing sites within a genome, such that a R2 protein, modified R2 protein, or R2 fusion protein that may target a non-28S locus for insertion of a large genetic element. In some embodiments, the R2 fusion protein is an R2-Cas9 fusion protein. In some embodiments, the R2 fusion protein is a Cas12-R2 fusion protein. In some embodiments, the R2 fusion protein is a TALEN-R2 fusion protein.

Definitions

Unless stated otherwise, terms and techniques used within this application have the meaning generally known to one of skill in the art.

The term “about” as used herein is understood to modify the specified value. Unless explicitly stated otherwise, the term about is understood to modify the specified values+/−10%. As used herein, the term about applied to a range modifies both endpoints of the range. By way of example, a range of “about 5 to 10” is understood to mean “about 5 to about 10.”

Unless explicitly stated otherwise, the term “payload” as used herein means at least a nucleic acid that may be integrated into a host genome. Thus, “payload RNA” will be understood to comprise an RNA molecule comprising at least an insertion region, wherein the insertion region can be integrated into a host genome.

As used herein, “cell-specific,” or “cell-type specific,” would be understood by one of skill in the art to mean occurring or being expressed at a higher frequency or existing at an increased level in one cell type in contrast to other cell types.

As used herein, the terms “target site” and “landing site” are used interchangeably unless specified otherwise.

Unless explicitly stated otherwise, the term “nucleic acid” is understood to refer to both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) molecules. This may include chemically synthesized nucleic acid molecules, single stranded or double stranded nucleic acid molecules, linearized nucleic acid molecules, circularized nucleic acid molecules, chemically modified nucleic acid molecules, and nucleic acids with biochemical modifications.

RLE Domain Containing non-LTR Retrotransposon Families

In addition to canonical single-ORF RLE domain containing non-LTR retrotransposons, such as R2, R4, R5, R8, R9, Dong, and Cre families, retrotransposons for use in or as part of the genome editing system described herein may also be characterized as part of a larger phylogenetic family. The retrotransposons in these larger phylogenetic families contemplated for use in or as a part of the genome editing systems described herein include the 8,248 RLE-domain containing retrotransposon uncovered as part of the computational analysis described in Example 7. These 8,248 retrotransposon-like orthologs are divided into 9 families, termed RLED1-RLED9. In some embodiments, the non-LTR retrotransposon is a member of the RLED1 family. In some embodiments, the non-LTR retrotransposon is a member of the RLED2 family. In some embodiments, the non-LTR retrotransposon is a member of the RLED3 family. In some embodiments, the non-LTR retrotransposon is a member of the RLED4 family. In some embodiments, the non-LTR retrotransposon is a member of the RLED5 family. In some embodiments, the non-LTR retrotransposon is a member of the RLED6 family. In some embodiments, the non-LTR retrotransposon is a member of the RLED7 family. In some embodiments, the non-LTR retrotransposon is a member of the RLED8 family. In some embodiments, the non-LTR retrotransposon is a member of the RLED9 family. In some embodiments, the non-LTR retrotransposon is a member of the R1 family. In some embodiments, the non-LTR retrotransposon is a member of the R2 family. In some embodiments, the non-LTR retrotransposon is a member of the R4 family. In some embodiments, the non-LTR retrotransposon is a member of the R5 family. In some embodiments, the non-LTR retrotransposon is a member of the R6 family. In some embodiments, the non-LTR retrotransposon is a member of the R7 family. In some embodiments, the non-LTR retrotransposon is a member of the R8 family. In some embodiments, the non-LTR retrotransposon is a member of the R9 family. In some embodiments, the non-LTR retrotransposon is a member of the Cre family. In some embodiments, the non-LTR retrotransposon is a member of the NeSL family. In some embodiments, the non-LTR retrotransposon is a member of the HERO family. In some embodiments, the non-LTR retrotransposon is a member of the Utopia family.

R2 Element Enzymes

Without limiting the instant disclosure to any one particular theory, R2 retrotransposons are thought to work via a mechanism known as target-primed reverse transcription, or “TPRT.” TPRT is a mechanism by which an endonuclease creates a nick in a first DNA strand at a specific location, creating a “primed” 3′ hydroxyl end for reverse transcription. After the initial DNA nick, an mRNA molecule is reverse transcribed by the reverse transcriptase.

In some embodiments, the R2 element enzyme is modified. In some embodiments, the R2 element enzyme is modified by an N-terminal truncation of the R2 element enzyme sequence, a C-terminal truncation of the R2 element enzyme sequence, or both an N-terminal and a C-terminal truncation of the R2 element enzyme sequence.

In some embodiments, the R2 element enzyme is a fusion protein. In some embodiments, the R2 element enzyme comprises a fusion of an R2 protein with a Cas9 protein. In some embodiments, the R2 element enzyme comprises a fusion of an R2 protein with a Cas12 protein. In some embodiments, the R2 element enzyme comprises a fusion of an R2 protein with a Cas9 protein, wherein the Cas9 portion and the R2 protein portion are connected by a linker. In some embodiments, the R2 element enzyme comprises a fusion of an R2 protein with a Cas12 protein, wherein the Cas12 portion and the R2 protein portion are connected by a linker.

Protein Binding Elements

Protein binding elements of the disclosure can come in a multitude of forms. In one embodiment, a protein binding element may be an endogenous nucleic acid sequence. In one embodiment, a protein binding element may be an exogenous or introduced nucleic acid sequence. In one embodiment, the protein binding element may be a synthesized nucleic acid sequence.

Guide Elements

In some embodiments the genome editing system comprises a guide RNA. In some embodiments, the genome editing system comprises multiple guide RNAs. In some embodiments, the genome editing system comprises paired guide RNAs.

Genomic Insertion Sites and Targets

The R2 element naturally targets the 28S rRNA locus. The instant disclosure contemplates the insertion of payloads into either the 28S rRNA locus or into other genomic loci. In some embodiments, the insertion site is a targeted genomic insertion site. In some embodiments, the insertion site is targeted by a targeting domain in a fusion protein. In some embodiments, the insertion site has been exogenously introduced to the genome. In some embodiments, the insertion site has been exogenously introduced by a site-directed genome editing system that is not capable of delivering large genetic insertions. In some embodiments, the targeted genomic site is targeted for a point mutation. In some embodiments, the targeted genomic site is targeted for a small nucleotide insertion.

The instant disclosure also contemplates additional non-LTR site-specific retrotransposons for use in or as part of the genome editing system described herein that do not target the 28S rRNA locus. In some embodiments, the genome is targeted for a large genetic insertion. In some embodiments, the insertion site is a targeted genomic insertion site. In some embodiments, the insertion site is targeted by a targeting domain in a fusion protein. In some embodiments, the insertion site has been exogenously introduced to the genome. In some embodiments, the insertion site has been exogenously introduced by a site-directed genome editing system that is not capable of delivering large genetic insertions. In some embodiments, the targeted genomic site is targeted for a point mutation. In some embodiments, the targeted genomic site is targeted for a small nucleotide insertion.

Payloads

Payloads of the instant disclosure may encode proteins, such as enzymes. In some embodiments, the payload may act as a regulatory element. Thus, if an embodiment of the disclosure states, by way of example, that “the payload comprises a therapeutic protein,” it is generally understood that the payload comprises a template that, upon insertion, will lead to expression of a therapeutic protein encoded by the template. Exemplary vectors for expression are shown in FIG. 76.

In some embodiments, the insertion region comprises a template for a reporter gene. In some embodiments, the reporter gene encodes a fluorescent protein. In some embodiments, the reporter gene encodes a green fluorescent protein. In some embodiments, the reporter gene encodes eGFP.

In some embodiments, the insertion region comprises a template for a transcription factor gene.

In some embodiments, the insertion region comprises a template for a transgene.

In some embodiments, the insertion region comprises a template for an enzyme gene, or a therapeutic gene. In some embodiments, the therapeutic protein can be used in conjunction with another therapeutic.

In some embodiments, the payload comprises a protein that is capable of converting one cell type to another.

In some embodiments, the payload comprises a protein that is capable of killing a specific cell type. In some embodiments, the payload comprises a protein that is capable of killing a tumor cell. In some embodiments, the payload comprises an immune modulating protein.

In some embodiments, the payload comprises a 5′UTR. In some embodiments, the payload comprises a 3′UTR. In some embodiments, the payload comprises a 5′UTR and a 3′ UTR. In some embodiments, the payload consists of a 5′UTR. In some embodiments, the payload consists of a 3′UTR. In some embodiments, the payload comprises a 5′UTR and a 5′ homology region. In some embodiments, the payload comprises a 3′UTR and a 3′ homology region. In some embodiments, the payload comprises a 5′UTR, a 5′ homology region, a 3′UTR and a 3′ homology region. In some embodiments, the payload comprises a 5′ homology region, a 3′UTR and a 3′ homology region. In some embodiments, the payload comprises a 5′UTR, a 5′ homology region, and a 3′ homology region. In some embodiments, the payload comprises a 5′ homology region and a 3′ homology region. In some embodiments, the 3′ homology region comprises less than 30 base pairs. In some embodiments the 3′ homology region comprises less than 20 base pairs. In some embodiments, the 3′ homology region comprises less than 10 base pairs. In some embodiments, the 3′ homology region comprises less than 5 base pairs.

Programmable Nucleases, Nickases, and DNA Binding Proteins

The instant disclosure contemplates programmable nucleases or nickases for use in or as a part of the genome editing systems described herein. In some embodiments, the programmable nuclease or nickase is a Cas9 protein. In some embodiments, the programmable nuclease or nickase is a Cas12 protein. In some embodiments the programmable nuclease or nickase is IscB. In some embodiments, the programmable nuclease or nickase is IsrB. In some embodiments, the programmable nuclease or nickase is TnpB. In some embodiments, the programmable nuclease or nickase is a TALEN nuclease. In some embodiments, the programmable nuclease or nickase is fused to the non-LTR site-specific retrotransposon element. In some embodiments, the programmable nuclease or nickase is non-covalently linked to the non-LTR site-specific retrotransposon element. In some embodiment, the programmable nuclease or nickase acts in cis with the non-LTR site-specific retrotransposon element. In some embodiments, the programmable nuclease or nickase acts in trans with the non-LTR site-specific retrotransposon element.

Therapeutic Gene Insertions

In some embodiments, the payload results in the insertion of a therapeutic gene into a host genome. In some embodiments, the therapeutic gene is intended to treat a neurological disorder or a neurodegenerative disorder. In some embodiments, the therapeutic gene is intended to treat cancer. In some embodiments, the therapeutic gene is intended to treat an autoimmune disorder.

In some embodiments, the payload results in the insertion of a therapeutic gene for treating a genetically inherited disease. In some embodiments, the genetically inherited disease is Meier-Gorlin syndrome. In some embodiments, the genetically inherited disease is Seckel syndrome 4. In some embodiments, the genetically inherited disease is Joubert syndrome 5. In some embodiments, the genetically inherited disease is Leber congenital amaurosis 10. In some embodiments, the genetically inherited disease is Charcot-Marie-Tooth disease, type 2. In some embodiments, the genetically inherited disease is leukoencephalopathy. In some embodiments, the genetically inherited disease is Usher syndrome, type 2C. In some embodiments, the genetically inherited disease is spinocerebellar ataxia 28. In some embodiments, the genetically inherited disease is glycogen storage disease type III. In some embodiments, the genetically inherited disease is primary hyperoxaluria, type I. In some embodiments, the genetically inherited disease is long QT syndrome 2. In some embodiments, the genetically inherited disease is Sjögren-Larsson syndrome. In some embodiments, the genetically inherited disease is hereditary fructosuria. In some embodiments, the genetically inherited disease is neuroblastoma. In some embodiments, the genetically inherited disease is amyotrophic lateral sclerosis type 9. In some embodiments, the genetically inherited disease is Kallmann syndrome 1. In some embodiments, the genetically inherited disease is limb-girdle muscular dystrophy, type 2L. In some embodiments, the genetically inherited disease is familial adenomatous polyposis 1. In some embodiments, the genetically inherited disease is familial type 3 hyperlipoproteinemia. In some embodiments, the genetically inherited disease is Alzheimer's disease, type 1. In some embodiments, the genetically inherited disease is metachromatic leukodystrophy. In some embodiments, the genetically inherited disease is cancer. In some embodiments, the genetically inherited disease is Uveitis. In some embodiments, the genetically inherited disease is SCA1. In some embodiments, the genetically inherited disease is SCA2. In some embodiments, the genetically inherited disease is FUS-Amyotrophic Lateral Sclerosis (ALS). In some embodiments, the genetically inherited disease is MAPT-Frontotemporal Dementia (FTD). In some embodiments, the genetically inherited disease is Myotonic Dystrophy Type 1 (DM1). In some embodiments, the genetically inherited disease is Diabetic Retinopathy (DR/DME). In some embodiments, the genetically inherited disease is Oculopharyngeal Muscular Dystrophy (OPMD). In some embodiments, the genetically inherited disease is SCA8. In some embodiments, the genetically inherited disease is C9ORF72-Amyotrophic Lateral Sclerosis (ALS). In some embodiments, the genetically inherited disease is SOD1-Amyotrophic Lateral Sclerosis (ALS). In some embodiments, the genetically inherited disease is SCA6. In some embodiments, the genetically inherited disease is SCA3 (Machado-Joseph Disease). In some embodiments, the genetically inherited disease is Multiple system Atrophy (MSA). In some embodiments, the genetically inherited disease is Treatment-resistant Hypertension. In some embodiments, the genetically inherited disease is Myotonic Dystrophy Type 2 (DM2). In some embodiments, the genetically inherited disease is Fragile X-associated Tremor Ataxia Syndrome (FXTAS). In some embodiments, the genetically inherited disease is West Syndrome with ARX Mutation. In some embodiments, the genetically inherited disease is Age-related Macular Degeneration (AMD)/Geographic Atrophy (GA). In some embodiments, the genetically inherited disease is C9ORF72-Frontotemporal Dementia (FTD). In some embodiments, the genetically inherited disease is Facioscapulohumeral Muscular Dystrophy (FSHD). In some embodiments, the genetically inherited disease is Fragile X Syndrome (FXS). In some embodiments, the genetically inherited disease is Huntington's Disease. In some embodiments, the genetically inherited disease is Glaucoma. In some embodiments, the genetically inherited disease is Acromegaly. In some embodiments, the genetically inherited disease is Achromatopsia (total color blindness). In some embodiments, the genetically inherited disease is Ullrich congenital muscular dystrophy. In some embodiments, the genetically inherited disease is Hereditary myopathy with lactic acidosis. In some embodiments, the genetically inherited disease is X-linked spondyloepiphyseal dysplasia tarda. In some embodiments, the genetically inherited disease is Neuropathic pain (Target: CPEB). In some embodiments, the genetically inherited disease is Persistent Inflammation and injury pain (Target: PABP). In some embodiments, the genetically inherited disease is Neuropathic pain (Target: miR-30c-5p). In some embodiments, the genetically inherited disease is Neuropathic pain (Target: miR-195). In some embodiments, the genetically inherited disease is Friedreich's Ataxia. In some embodiments, the genetically inherited disease is Uncontrolled gout. In some embodiments, the genetically inherited disease is Inflammatory pain (Target: Nav1.7 and Nav1.8). In some embodiments, the genetically inherited disease is Choroideremia. In some embodiments, the genetically inherited disease is Focal epilepsy. In some embodiments, the genetically inherited disease is Alpha-1 Antitrypsin deficiency (A/ATD). In some embodiments, the genetically inherited disease is Androgen Insensitivity Syndrome. In some embodiments, the genetically inherited disease is Opioid-induced hyperalgesia (Target: Raf-1). In some embodiments, the genetically inherited disease is Neurofibromatosis type 1. In some embodiments, the genetically inherited disease is Stargardt's Disease. In some embodiments, the genetically inherited disease is Dravet Syndrome. In some embodiments, the genetically inherited disease is Retinitis Pigmentosa. In some embodiments, the genetically inherited disease is Hemophilia A (factor VIII). In some embodiments, the genetically inherited disease is Hemophilia B (factor IX). In some embodiments, the genetically inherited disease is Parkinson's Disease.

Linkers

In some embodiments, the linker is a polypeptide linker. In some embodiments, the linker is a non-peptide linker. In some embodiments, the linker comprises a polypeptide portion and a non-peptide portion. In some embodiments, the linker comprises an extended recombinant polypeptide (XTEN). In some embodiments, the linker comprises the amino acid sequence (Gly₄Ser)_n (SEQ ID NO: 33380), where n is an integer. In some embodiments, the linker comprises the amino acid sequence (Gly₄Ser)_n, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO: 33381). In some embodiments, the linker comprises the amino acid sequence (Gly₄Ser)_n, wherein n is greater than 10 (SEQ ID NO: 33382). In some embodiments, the linker comprises a synthetic portion. In some embodiments, the linker comprises polyethylene glycol (PEG). In some embodiments, the linker is a synthetic linker. In some embodiments (Gly₂Ser)_n, wherein n is an integer. In some embodiments, the linker comprises the amino acid sequence (Gly₂Ser)_n, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO: 33383). In some embodiments, the linker comprises the amino acid sequence (Gly₂Ser)_n, wherein n is greater than 10 (SEQ ID NO: 33384). In some embodiments, the linker comprises the amino acid sequence (Ser-Gly-Gly-Ser)_n (SEQ ID NO: 33385), where n is an integer. In some embodiments, the linker comprises the amino acid sequence (Ser-Gly-Gly-Ser)_n, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO: 33386). In some embodiments, the linker comprises the amino acid sequence (Ser-Gly-Gly-Ser)_n, wherein n is greater than 10 (SEQ ID NO: 33387). In some embodiments the linker comprises the amino acid sequence (Glu-Ala-Ala-Ala-Lys). (SEQ ID NO: 33388), wherein n is an integer. In some embodiments, the linker comprises the amino acid sequence (Glu-Ala-Ala-Ala-Lys)_n, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO: 33389). In some embodiments, the linker comprises the amino acid sequence (Glu-Ala-Ala-Ala-Lys)_n, wherein n is greater than 10 (SEQ ID NO: 33390). In some embodiments, the linker comprises a proline linker.

Methods of the Disclosure

The present disclosure relates to a method of editing a genome using a genome editing system. The present disclosure also relates to the method of editing a genome using a genome editing system, wherein the genome editing system comprises i) an R2 element enzyme, and ii) a payload RNA; wherein the payload RNA comprises one or more of a 5′ homology region, a 3′ homology region, a protein binding element, and an insertion region; wherein the insertion region comprises a template for a small or large nucleic acid insertion into the genome; and wherein the R2 element enzyme comprises a targeting domain, a reverse transcriptase domain, and a nickase domain.

In some embodiments, the target genome is in a eukaryotic cell. In some embodiments, the targeted genome is in a mammalian cell. In some embodiments, the targeted genome is in a dividing mammalian cell. In some embodiments, the targeted genome is in a non-dividing cell. In some embodiments, the targeted genome is in a quiescent cell.

In some embodiments, the genome editing system targets a genomic position for deletion rather than editing. In some embodiments, the genome editing system targets a genomic site for deletion that is between 1 and 150 nucleotides. In some embodiments, the genome editing system comprises a payload RNA with a 5′ homology region and a 3′ homology region, wherein the 5′ homology region and the 3′ homology region, wherein the 5′ homology region and the 3′ homology region are positioned to delete the genomic target. In some embodiments, the genome editing system is capable of deleting a genomic target and inserting a novel nucleic acid region into the genome concurrently.

Compositions

The present disclosure relates to compositions, wherein the composition comprises a cell, and wherein the cell comprises a genome that has been edited using a genome editing system.

Delivery Systems

The present disclosure relates to genome editing systems comprising an R2 element enzyme and a template. In some embodiments, the genome editing system is a delivery system comprising several components. In some embodiments, the components comprise an R2 element enzyme or a molecule that encodes an R2 element enzyme. In some embodiments, the components comprise a template. In some embodiments, the delivery system components comprise an R2 element enzyme or a molecule that encodes an R2 element enzyme and a template. In some embodiments, the R2 element enzyme and the template are delivered together. In some embodiments, the R2 element enzyme and the template are delivered in a designed delivery system. In some embodiments, the R2 element enzyme is delivered as an RNA molecule that is suitable for in vivo translation to an R2 element enzyme. In some embodiments, the R2 element enzyme is delivered in its proteinaceous form. In some embodiments, the template is delivered as an RNA molecule. In some embodiments, the template is delivered as a DNA molecule suitable for in vivo transcription to an RNA molecule. In some embodiments, the R2 element enzyme is delivered as a DNA molecule that is suitable for in vivo transcription and translation to an R2 element enzyme. In some embodiments, the R2 element enzyme is delivered as an RNA molecule and the template is delivered as an RNA molecule. In some embodiments, the designed delivery system comprises a lipid nanoparticle. In some embodiments, the designed delivery system comprises a cationic molecule. In some embodiments, the R2 element enzyme and the template are iected directly into a cell as naked RNA. In some embodiments, the components of the delivery system can be delivered by lipofection. In some embodiments, the components of the delivery system can be delivered in lipid nanoparticles. In some embodiments, the components of the delivery system can be delivered by a viral delivery system.

Sequences

Exemplary sequences of payload UTRs and target homologies are provided in Table 1.

TABLE 1
Exemplary payload UTRs and target homologies.
Name                  Sequence
28S 5′ sequence
50 bp                 ATTCAATGAAGCGCGGGTAAACGGCGGGAGTAACTATGAC
                      TCTCTTAAGG (SEQ ID NO: 33391)
30 bp                 ACGGCGGGAGTAACTATGACTCTCTTAAGG (SEQ ID NO:
                      33392)
5′ UTRs
Full length 5′ UTR    GTCTAGTTACAACTGGGCATCGCTGCAGAGATCGCACCTC
R2Tg                  CTCGTGGTCCCGCTGGTAGCCCTTCGAAGGGTGACTAAGT
                      CGATCTCTGCCCCAGGTACGGAGCCGTTGGGACTCACCAG
                      TCCAACGTAACTCCTGCCTAAATTCGGTGAAACAAATTCCT
                      CGGTAAAAAGCCCC (SEQ ID NO: 33393)
truncated 5′ UTR      GTCTAGTTACAACTG (SEQ ID NO: 33394)
R2Tg
Full length 5′ UTR    TCTAGTTACAACTGGGCATAGCTGCAGAGATCTCACCTCCT
R2Toc                 CGTGGTCCCGCTGGTAAGCCCTTAACAGGGTGACTAAGTA
                      GATCTCTGCCCCAGTCAAGGAGCCGCTGGGAATCACCAGC
                      CCAGCGATTCCTTTCAAATTTAGGTGAAACAAATTTCTCGG
                      TGTGGGTCGCAAGACTTACTACCTAAAACCTGGCCCCACG
                      GTCTGACAGGGGCAACGGGTTCGGAGAT (SEQ ID NO:
                      33395)
truncated 5′ UTR      TCTAGTTACAACTGG (SEQ ID NO: 33396)
R2Toc
Full length 5′ UTR    TCGGCGATGCTGAACCACCTCCTCGTGGTGCCGACTGGGC
nLTR1Mbr              AGCTTTGGAGAAATCCTAAGCTGGCTAAGAGTTCAGCAAC
                      TCCTG (SEQ ID NO: 33397)
28S/5′ UTR truncation gttgacgcgatgtgatttctgcccagtgctctg
1                     aatgtcaaagtgaagaaattcaatgaagcgcg
                      (SEQ ID NO: 33398)
28S/5′ UTR truncation attcaatgaagcgcgggtaaacggcgggagtaa
2                     ctatgactctcttaaggtctagttacaactgg
                      (SEQ ID NO: 33399)
28S/5′ UTR truncation atgactctcttaaggtctagttacaactgggca
3                     tcgctgcagagatcgcacctcctcgtggtccc
                      (SEQ ID NO: 33400)
28S/5′ UTR truncation cgctgcagagatcgcacctcctcgtggtcccgc
4                     tggtagcccttcgaagggtgactaagtcgatc
                      (SEQ ID NO: 33401)
28S/5′ UTR truncation gtagcccttcgaagggtgactaagtcgatctct
5                     gccccaggtacggagccgttgggactcacca
                      g (SEQ ID NO: 33402)
28S/5′ UTR truncation cccaggtacggagccgttgggactcaccagtcc
6                     aacgtaactcctgcctaaattcggtgaaacaa
                      (SEQ ID NO: 33403)
28S/5′ UTR truncation gactcaccagtccaacgtaactcctgcctaaat
7                     tcggtgaaacaaattcctcggtaaaaagcccc
                      (SEQ ID NO: 33404)
Target homologies
AAVS1 cargo 100 bp    AGGGCCGGTTAATGTGGCTCTGGTTCTGGGTACTTTTATCT
5′ homology           GTCCCCTCCACCCCACAGTGGGGCCACTAGGGACAGGATT
                      GGTGACAGAAAAGCCCCAT (SEQ ID NO: 33405)
AAVS1 cargo 100 bp    CCTTAGGCCTCCTCCTTCCTAGTCTCCTGATATTGGGTCTA
3′ homology           ACCCCCACCTCCTGTTAGGCAGATTCCTTATCTGGTGACAC
                      ACCCCCATTTCCTGGAGC (SEQ ID NO: 33406)
NOLC1 cargo 50 bp 5′  TCCTGAGTCGTGCTGCGTCGACAACGGTAGTGACGCGTAT
homology              TGCCTGGAGG (SEQ ID NO: 33407)
NOLC1 cargo 50 bp 3′  GCGGACGCCGGCATTCGCCGCGTGGTTCCCAGCGACCTGT
homology              ATCCCCTCGT (SEQ ID NO: 33408)
LMNB1 cargo 50 bp 5′  GCCATGGCGACTGCGACCCCCGTGCCGCCGCGGATGGGCA
homology              GCCGCGCTGG (SEQ ID NO: 33409)
LMNB1 cargo 50 bp 3′  CGGCCCCACCACGCCGCTGAGCCCCACGCGCCTGTCGCGG
homology              CTCCAGGAGA (SEQ ID NO: 33410)
EMX1 cargo 50 bp 5′   GAGGACATCGATGTCACCTCCAATGACTAGGGTGGGCAAC
homology              CACAAACCCA (SEQ ID NO: 33411)
EMX1 cargo 50 bp 3′   CGAGGGCAGAGTGCTGCTTGCTGCTGGCCAGGCCCCTGCG
homology              TGGGCCCAAG (SEQ ID NO: 33412)

Exemplary sequences of Cas9 guides are provided in Table 2.

TABLE 2
Exemplary Cas9 guides.
Name	Sequence	SEQ ID NO:
AAVS1_guide_1 A1	gAAGGAGGAGGCCTAAGGATG	33413
AAVS1_guide_2 A2	gCTGTCCCCTCCACCCCACAG	33414
AAVS1_guide_3 A3	gATATCAGGAGACTAGGAAGG	33415
AAVS1_guide_4 A4	gAGGGCCGGTTAATGTGGCTC	33416
AAVS1_guide_5 A5	gCTAGTGGCCCCACTGTGGGG	33417
AAVS1_guide_6 A6	GAAGGAGGAGGCCTAAGGAT	33418
AAVS1_guide_7 A7	GGAAGGAGGAGGCCTAAGGA	33419
AAVS1_guide_8 A8	GTCCCCTCCACCCCACAGTG	33420
AAVS1_guide_9 A9	gACTAGGAAGGAGGAGGCCTA	33421
NOLC1_guide_1 N1	GAGTCGTGCTGCGTCGACAA	33422
NOLC1_guide_2 N2	gCGGTAGTGACGCGTATTGCC	33423
NOLC1_guide_3 N3	gTAGTGACGCGTATTGCCTGG	33424
NOLC1_guide_4 N4	GACGCGTATTGCCTGGAGGA	33425
NOLC1_guide_5 N5	GCGTATTGCCTGGAGGATGG	33426
NOLC1_guide_6 N6	GCCTGGAGGATGGCGGACGC	33427
NOLC1_guide_7 N7	GCCGGCGTCCGCCATCCTCC	33428
NOLC1_guide_8 N8	GGGAACCACGCGGCGAATGC	33429
NOLC1_guide_9 N9	gACAGGTCGCTGGGAACCACG	33430
NOLC1_guide_10	gACGAGGGGATACAGGTCGCT	33431
N10
NOLC1_guide_11	gCACGAGGGGATACAGGTCGC	33432
N11
NOLC1_guide_12	gAGCCGAGCACGAGGGGATAC	33433
N12
LMNB1 gl	GCTGTCTCCGCCGCCCGCCA	33434
LMNB1 g2	gCTGCGACCCCCGTGCCGCCG	33435
LMNB1 g3	GACCCCCGTGCCGCCGCGGA	33436
LMNB1 g4	gACCCCCGTGCCGCCGCGGAT	33437
LMNB1 g5	gCCGCGGATGGGCAGCCGCGC	33438
LMNB1 g6	gCGGATGGGCAGCCGCGCTGG	33439
LMNB1 g7	gTGGGGCTCAGCGGCGTGGTG	33440
LMNB1 g8	gCGTGGGGCTCAGCGGCGTGG	33441
LMNB1 g9	GACAGGCGCGTGGGGCTCAG	33442
LMNB1 g10	GGAGCCGCGACAGGCGCGTG	33443
LMNB1 g11	gTGGAGCCGCGACAGGCGCGT	33444
LMNB1 g12	gCCTTCTCCTGGAGCCGCGAC	33445
EMX1 gl	GGGCAACCACAAACCCACGA	33446
EMX1 g2	gAAGCAGCACTCTGCCCTCGT	33447
EMX1 g3	gCAAGCAGCACTCTGCCCTCG	33448
EMX1 g4	gCTTGGGCCCACGCAGGGGCC	33449
EMX1 g5	gTCCAGCTTGGGCCCACGCAG	33450
EMX1 g6	GTCCAGCTTGGGCCCACGCA	33451
EMX1 g7	GAGTGGCCAGAGTCCAGCTT	33452

Exemplary sequences of NGS, gel primers, and Sanger primers are provided in Table 3.

TABLE 3
Exemplary NGS, gel primers, and Sanger primers.
Name	Sequence	SEQ ID NO:
GLuc cargo reporter 28S 5′	ACACTCTTTCCCTACACGACGCTCTT	33453
junction stagger 1 Forward	CCGATCTCCAGGTAAGTATCAAGGT
	TACAAGACAGG
GLuc cargo reporter 28S 5′	ACACTCTTTCCCTACACGACGCTCTT	33454
junction stagger 2 Forward	CCGATCTACCAGGTAAGTATCAAGG
	TTACAAGACAGG
GLuc cargo reporter 28S 5′	ACACTCTTTCCCTACACGACGCTCTT	33455
junction stagger 3 Forward	CCGATCTGACCAGGTAAGTATCAAG
	GTTACAAGACAGG
GLuc cargo reporter 28S 5′	ACACTCTTTCCCTACACGACGCTCTT	33456
junction stagger 4 Forward	CCGATCTTGACCAGGTAAGTATCAA
	GGTTACAAGACAGG
GLuc cargo reporter 28S 5′	GTGACTGGAGTTCAGACGTGTGCTC	33457
junction in 5′ Tg UTR	TTCCGATCTCTGGTGAGTCCCAACG
Reverse	GCTC
GLuc cargo reporter 28S 5′	GTGACTGGAGTTCAGACGTGTGCTC	33458
junction scarless Reverse	TTCCGATCTCACAGATCGACCTGTG
	GAGAGAAAG
GLuc cargo reporter 28S 5′	GTGACTGGAGTTCAGACGTGTGCTC	33459
junction non-inserted Reverse	TTCCGATCTGAGGGATCTGCGGCCG
	CTT
Genomic AAVS1 5′ junction	ACACTCTTTCCCTACACGACGCTCTT	33460
Forward	CCGATCTCCGAGCTGGGACCACCTT
	ATATTC
Genomic AAVS1 5′ junction	GTGACTGGAGTTCAGACGTGTGCTC	33461
scarless Reverse	TTCCGATCTCGTTGGCAAGCCCTTTG
	AGGCA
Genomic AAVS1 5′ junction	GTGACTGGAGTTCAGACGTGTGCTC	33462
non-inserted Reverse	TTCCGATCTCCCTCCCAGGATCCTCT
	CTGGC
Genomic NOLC1 5′ junction	ACACTCTTTCCCTACACGACGCTCTT	33463
Forward	CCGATCTCAATGACGTAACACAGGC
	CCGC
Genomic NOLC1 5′ junction	GTGACTGGAGTTCAGACGTGTGCTC	33464
scarless Reverse	TTCCGATCTTCCTGTCGCTTTGGCGA
	ACTTATTG
Genomic NOLC1 5′ junction	GTGACTGGAGTTCAGACGTGTGCTC	33465
non-inserted Reverse	TTCCGATCTCGAGCACGAGGGGATA
	CAGGTC
pMax cargo genomic 28S 5′	CCCACCCCACGTCTCGTCGCG	33466
junction gel Forward
pMax cargo genomic 28S 5′	CCGAAGTGGTAGAAGCCGTAGC	33467
junction gel Reverse
pMax cargo genomic 28S 3′	GCCCGCACCTTCAGCCTGCGC	33468
junction gel Forward
pMax cargo genomic 28S 3′	TCCGATCTGCCGGGGGCCTCCCACT	33469
junction gel Reverse	TATT
GLue cargo reporter 28S 5′	CCAGGTAAGTATCAAGGTTACAAGA	33470
junction Forward	CAGG
GLuc cargo reporter 28S 5′	CCACCTGGCCCTGGATCTTGCTGGC	33471
junction Reverse	AAAG
GLuc cargo reporter 28S 3′	TAAGGAGACCAATAGAAACTGGGC	33472
junction Forward	TTGTCGAGACAGAGAAG
GLuc cargo reporter 28S 3″	CACCGGCCTTATTCCAAGCGGCTTC	33473
junction Reverse	GGC

Exemplary sequences of ddPCR primers and probes are provided in Table 4.

TABLE 4
Exemplary ddPCR primers and probes.
Name	Sequence	SEQ ID NO:
NOLC1 Endogenous locus	TGGAGCCCACCCTTTCCGT	33474
ddPCR Forward
LMNB1 Endogenous locus	TCCTTATCACGGTCCCGCTCG	33475
ddPCR Forward
EMX1 Endogenous locus	GCATTGCCACGAAGCAGG	33476
ddPCR Forward
EGFP R2 cargo ddPCR	GAACTCCACGCCGTTCA	33477
Reverse
EGFP R2 cargo ddPCR FAM	/56-FAM/CC ATG AAG A/ZEN/T CGA	33478
probe	GTG CCG CAT CA/3IABKFQ/

Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples. The following examples are included solely for purposes of illustration and are not considered limiting embodiments. All patents and publications referred to herein are expressly incorporated by reference.

EXAMPLES

Example 1. Insertion of Non-Human R2 into the 28S Locus of the Human Genome

To determine the ability of animal R2 elements to integrate into the human genome, HEK293FT cells were transfected with specific plasmids containing the zebra finch (Taeniopygia guttata) R2 element (R2Tg), a payload, or both the R2tg plasmid and a payload plasmid. Following isolation of DNA from transfected cells, those cells transfected with an R2Tg plasmid and an eGFP payload (eGFP flanked by UTR regions and 100 bp homology to the human R2 locus), showed a distinct PCR product (FIG. 1, lane 1), indicating integration of the eGFP payload into the human genome through R2Tg. When cells were transfected with R2Tg without a payload (FIG. 1, lane 2), payload alone with no R2 (FIG. 1, lanes 3, 8, 9), or other R2 orthologs from Geospiza fortis (Gfo; FIG. 1, lane 3-5) with or without payloads, no PCR product is identified These results demonstrate that R2Tg can successfully integrate payloads into the human genome.

Example 2. Modification of the Target Landing Site

Following successful insertion of an eGFP payload, the features of the R2 system that could increase integration efficiency were examined. In the following experiments, unless otherwise stated, three plasmids are used. The first plasmid contains at least an R2 protein. The second plasmid contains at least a portion of a payload reporter. The third plasmid contains at least R2 landing sites.

The R2 landing site plasmids contain R2 landing sites of variable size. This size is indicated in the format 26/3 (FIG. 2), where the first number indicates the number of base pairs upstream of the insertion site, and the second number indicates the number of base pairs downstream of the insertion site.

Following transfection of these three plasmids with varying length of R2 landing sites, integration was measured by luminescence, indicating integration of the luminescent payload (FIG. 2). of an artificial luciferase exon (introduced only with the payload) that allows the inserted reporter to splice and reconstitute a functional luciferase gene (FIG. 2). The payload, which is RNA, is transcribed from a DNA (payload plasmid template) where an artificial luciferase exon is flanked by 5′ and 3′ UTRs as and 5′ and 3′ homologies. Two negative controls (FIG. 2, lanes 11-12) exhibited little luminescence. The landing site which proved to be the most efficient for integration was 26/6 (FIG. 2, lane 6; 26 bp upstream, 6 bp downstream of the insertion site). Given that the normal target site at the 28S locus in the human genome is hundreds of base pairs, it is unexpected that the shorter landing sites tested here provided such efficient integration.

Next, the tolerability of mutations within the R2 landing sites was tested. FIG. 3A displays the predicted zinc finger binding sites (red) within the R2 landing sites and the mutations tested (orange, lowercase bases). FIG. 3 B shows that there is a great deal of tolerability within the R2 landing sites that still allows for integration. FIG. 4 shows additional mutations that may be tolerated. However, mutation of all three, predicted zinc finger binding sites results in abrogated insertion efficiency (FIG. 4B, target_37_23_mut_10). Based on this evidence, a great degree of tolerability for mutations away from the traditional R2 landing sites is found and can help in the development of exogenous landing sites.

Example 3. Modification of the Payload Homology Regions

After determining that short landing sites could provide for efficient integration, the effect of insertion homology length (to the landing sites) on integration efficiency was evaluated. To test the effect of homology length on integration efficiency, HEK293FT cells were transfected with three separate plasmids. The first plasmid contained an R2 protein encoding region, the second plasmid encoded a partial (inactive) luciferase reporter region and R2 landing sites, and the third plasmid encoded a luciferase insertion as well as regions of homology of varying number of base pairs homologous to the R2 landing site in the second plasmid. Cells were then treated with aphidicolin, which blocks cell division and thus also stops Homology Directed Repair (HDR). Without being bound to any one theory, by blocking HDR, integration is more likely to occur due to an R2 related mechanism.

When treated with 1 μm, 5 μm, or 25 μm aphidicolin (or DMSO control) (FIG. 5), plasmids with either 60 base pair homology (FIG. 5, columns 1-4) or 40 base pair homology (FIG. 5, columns 5-8) still exhibited successful integration, indicating that the integration of these payloads occurs by an HDR-independent mechanism.

When flanking regions (UTR and additional homology region) were increased in size to 100 bp (FIG. 6, columns 1-4), 200 bp (FIG. 6, columns 5-8), or 300 bp (FIG. 6, columns 9-12) and treated with aphidicolin at 1 μm, 5 μm, or 25 μm (or DMSO control), a significant improvement in integration efficiency is exhibited with longer flanking regions (FIG. 6). When transfected with Cas9 only, no integration was seen. Cells were also transfected with a 300 bp flanking template and no R2 protein (FIG. 6, lanes 13-16) to measure the level of HDR in the system.

An overview of the role homology of the payload plays in integration efficiency (as measured by luminescent readout) is seen in FIG. 7. Greater 5′ homology (y-axis) to the R2 landing site is associated with more efficient integration. This is not the case for 3′ homology (x-axis), which is less clear, but indicates that shorter homology results in more efficient integration in some cases.

Also, the effect of truncations of the 5′ and 3′UTRs from the payload portion (FIG. 8) on integration efficiency was examined. Three plasmids were transfected into HEK293FT cells. The first plasmid contained a partial luciferase reporter with wild-type R2 landing sites (wtR2) of 26/22 bp. The second plasmid encoded an R2 protein. The third plasmid contained a luciferase payload with the UTR modifications listed along the x-axis. Generally, 3′ UTR (FIG. 8, columns 16-29) truncations resulted in greater integration efficiency (as measured by luminescence readout) than 5′ UTR truncations (FIG. 8, columns 3-15). The greatest increases in integration efficiency were seen in truncations greater than 90 base pairs. Nearly all truncations, however, retained some form of integration activity.

Next, we evaluated how solely altering the 3′ homology regions would affect integration efficiency. In this experiment, HEK293FT cells were transfected with 3 μlasmids. The first plasmid contained an R2 protein encoding region. The second plasmid contained a partial luciferase reporter with wtR2 landing sites. The third plasmid contained a luciferase insertion with alterations to the 3′ UTR, as named on the x-axis (FIG. 9A) and described visually in FIG. 9B. HDV is an HDV ribozyme, which cleaves the insertion region directly after the 3′ UTR. mutHDV is an inactive HDV, incapable of cleaving the homology region just beyond the 3′UTR. All modifications retained significant activity, except for the HDV only modification This indicates that cleavage directly beyond the 3′UTR in the homology region (i.e., no further homology region remains), dramatically decreased integration efficiency (FIG. 9A, column 3). This is in concert with the discoveries above, where a minimal (but not absent) 3′ homology region is required for significant integration efficiency.

Example 4. Modification of the R2 Enzyme

Next, we evaluated whether modifications to the R2 protein could increase integration efficiency. First, permissible domains within the R2 protein into or onto which various additional moieties could be fused were identified. As before, three plasmids were introduced into HEK293FT cells. The first plasmid contained an R2 protein which contained different GFP variants at different points along the R2 protein. The second plasmid encoded a partial (inactive) luciferase reporter region and R2 landing sites, and the third plasmid encoded a luciferase insertion as well as regions of homology to the R2 landing site in the second plasmid.

These variant R2 proteins were modified by inserting GFP variants throughout the length of the protein, beginning from the N-terminus. By example, LNK1_1 is located closer to the N-terminus than is LNK1_7. LNK_nt indicates a fusion to the N-terminus, while LNK_ct indicates a fusion to the C-terminus. As seen in FIG. 10, an N-terminal fusion of eGFP resulted in the greatest integration efficiency, suggesting that this fusion may be ideal for additional fusion molecules. However, multiple “permissive insertion sites” were identified in FIG. 10, including R2Tg_LNK1_1, R2Tg_LNK1_2, R2Tg_LNK1_3, R2Tg_LNK1_4, R2Tg_LNK1_5, R2Tg_LNK2_1, R2Tg_LNK2_3, R2Tg_LNK2_9 and R2Tg_LNK2_10 (FIG. 10).

The matter of whether R2 could integrate a payload using a “short target”-truncated R2 landing sites (26/3 bp) was investigated. FIG. 11 exhibits the ability of R2 to deliver a payload even given this short landing site (FIG. 11, column 1).

Also, the matter of whether the addition of a nuclear localization signal would increase integration efficiency at the Beta-actin locus of HEK293FT cells (FIG. 12) was examined. HEK293FT cells were transfected with four separate plasmids. The first plasmid encoded an R2 protein. The second plasmid contained pMAX as a payload (including 5′ and 3′ UTRs, as well as 5′ and 3′ homologies) for R2-dependent insertion. The third plasmid encoded a prime editor protein, and the fourth plasmid expressed a prime editing guideRNA. The prime editor first inserts a 48 bp (28S) target site in ACTB to then, in a second step, R2-dependent insertion of the pMAX payload.

After determining the ability of a nuclear localization signal to boost integration, the primary localization of transfected R2 proteins into HEK293FT cells was evaluated. FIG. 13A shows that R2 does not primarily localize to the nucleus of the cell. However, when HEK293Ft cells were transfected with two plasmids (the first an R2 protein, the second a payload protein) into cells that had been stably transfected to integrate a portion of the splice reporter, addition of a nuclear localization signal to the N- and C-terminus of the R2 protein dramatically increased payload insertion efficiency (FIG. 13B).

Thus, modifying the R2 protein portion can allow for greater integration efficiency. To further study integration efficiency, a fluorescent GFP reporter responsive to R2 activity (FIG. 14) was developed. The R2 reporter that was developed has a single GFP exon and promoter that is not activated until the R2 payload, with a second GFP exon, is integrated (FIG. 14A, B). Thus, R2 integration can be read by a fluorescent readout.

Using this fluorescent readout approach, the efficiency of integration was evaluated using flow cytometry. HEK293FT cells were transfected with specific plasmids. These samples were wild-type R2 (FIG. 15A, column 1), a negative control (FIG. 15A, no R2 protein; column 2), 300 ng of R2 with a nuclear localization signal (FIG. 15A, column 3), 200 ng of R2 with a nuclear localization signal (FIG. 15A, column 4), 100 ng of R2 with a nuclear localization signal (FIG. 15A, column 5), 50 ng of R2 with a nuclear localization signal (FIG. 15A, column 5), and untransfected cells as a percentage of all cells in each sample. The results shown in FIG. 15A clearly demonstrate the increased integration efficiency of R2 proteins with a nuclear localization signal compared to wild type R2 without a nuclear localization signal. This increase persists when the GFP+cells are normalized to only those cells that were successfully transfected (FIG. 15B).

Next, the matter of whether the truncation of the R2 protein resulted in alteration of integration efficiency was studied. the N-terminal portion of the R2 protein was serially truncated, as indicated by the vertical lines in FIG. 16B. These truncations did not result in any significant drop in integration efficiency until reaching NT 7, which demarcates the current limit of truncation for a single R2 protein to maintain integration efficiency.

Also, the matter of whether C-terminal truncations of the R2 protein may result in viable R2 proteins that sustain integration efficiency (FIG. 17) was evaluate. However, even the shortest C-terminal truncation resulted in a drastic decrease in integration efficiency, highlighting the importance of the C-terminal domains in contrast to the somewhat expendable N-terminal domains (FIG. 17A, 17B).

The issue that ablation of the restriction-like endonuclease (RLE) domain would affect integration activity was then studied. HEK293FT cells were transfected by three plasmids. The first plasmid contains a partial luciferase reporter with wtR2 landing sites (26/22 bp). The second plasmid encodes either a wild type R2 protein or an RLE deficient R2 protein. The third plasmid encodes a luciferase payload. Absence of the RLE domain in the R2 protein almost completely abolishes the integration efficiency of a wild-type R2 protein (FIG. 18, column 3).

Finally, the matter of whether certain other domains of the R2 protein could be removed or modified without adverse effect. was evaluated. FIG. 19. Displays the results of an experiment in which HEK293FT cells were transfected with 3 μlasmids. The first plasmid encoded a partial luciferase reporter with wtR2 landing sites. The second plasmid encoded a luciferase payload. The third plasmid encoded an R2 protein with various modifications, including to the −1 domain, 0 domain, zinc finger domains, or to add C- or N-terminal fusions. Three payloads were examined for each modified group of plasmids. A wild type luciferase payload (orange), a luciferase payload in which the MS2 binding site replaces the 5′UTR, and a luciferase payload in which the 5′ and 3′UTRs are replaced with MS2 binding sites. Deletion of the −1 domain (FIG. 19, columns 1-3), of the −1 and 0 domains (FIG. 19, columns 4-6) and of the 0 domain alone (FIG. 19, columns 7-9) significantly impaired integration efficiency. Further, replacing the 0 domain with an eGFP (FIG. 19, columns 10-12) or with an MCP domain (FIG. 19, columns 13-15) also significantly decrease integration efficiency, as did deleting a zinc finger domain (FIG. 19, columns 19-21). However, fusing the truncated N-terminus of the R2 protein to an MCP domain (i.e., a targeting domain) did show some integration efficiency, though not to the level of wild type. This experiment helps to define the indispensable domains for the R2 protein, and where modifications may be made.

Example 5. Modification of Payloads

This Example tested whether the payload itself could be modified to sustain nuclear localization. FIG. 20 sets out the relative insertion efficiency of payloads with various nuclear retention elements appended to the payload. Nuclear retention signals have varying levels of effect on the integration efficiency of the R2 payloads, indicating that nuclear localization may be important for optimal integration activity.

Further, whether the UTR elements of the payload were necessary for their integration, or if they may be modified, was studied. In this experiment, HEK293FT cells were transfected with three plasmids. The first plasmid encoded an R2 protein. The second plasmid encoded a partial luciferase reporter and wtR2 landing sites. The third plasmid contained the luciferase payload and any of many UTR modifications (FIG. 21). UTRs were replaced by MS2 binding sites (FIG. 21, columns 1, 2, and 4), the 3′UTR was deleted (FIG. 21, column 3), the 5′ UTR replaced by an MS2 binding site while the 3′UTR is deleted (FIG. 21, column 5), the 5′ and 3′ UTR were both deleted (FIG. 21, column 6), the 5′UTR is deleted and the 3′UTR is replaced with an MS2 binding site (FIG. 21, column 7), as well as positive and negative controls (FIG. 21, columns 8 and 9, respectively). In each situation some integration activity was confirmed. Importantly, this also occurred in which both the 5′ and 3′ UTRs were deleted without any replacement (FIG. 21, column 6).

Example 6. Linkers and Fusion Proteins

Also, the evaluation of R2 fusion proteins and fusion proteins with linkers were viable for use in genome editing was carried out. In this experiment, HEK293FT cells were transfected with 3 μlasmids. The first plasmid contained an R2 protein (with or without an NLS) fused to a Cas9 protein connected by an XTEN linker (16 amino acids in length) at various points through the N-terminal portion of the R2 protein (see FIG. 22B). The second plasmid contains a luciferase reporter that is designed to indicate cleavage by Cas9. The third plasmid expresses a single guide RNA. Multiple Cas9-R2 fusion proteins exhibited the ability to cleave the Cas9 target protein, either with or without the nuclear localization signal (FIG. 22A).

Lastly, the determination of whether these Cas9-R2 fusion proteins were capable of editing human genomes was carried out. HEK293FT cells were stably transfected with a eGFP precursor gene with a 20 bp deletion. As such, the reporter is inactive until the 20 base pairs are inserted into the precursor. FIG. 23A-N exhibit integration and editing efficiency based on the expression of eGFP in these cells. This indicates that the large-scale insertion mechanism of R2 can function in concert with the targeted editing enzyme Cas9 for editing a human genome.

Example 7. Computational Analysis of Single-ORF Retroelements

The ORFs of 4,464 eukaryotic assemblies (animals and protists) from GenBank for RT, CCHC zinc finger, and RLE domains of known retroelements were also examined. Using a computational pipeline (FIG. 24A) we searched for single protein site-specific non-LTR retrotransposons based on their stereotyped architecture of C-terminal RLE and RT domains as a signature. We identified 8,248 RLE-domain containing representative orthologs (FIG. 24) with diverse RT domains, ZF motifs, and predicted insertion preferences, which clustered into 9 families based on phylogenetic analysis and the presence or absence of flanking rRNA sequences, microsatellite repeats, tRNA genes, or splicing RNAs (Table 1).

We found that families varied in length, with the longer family 3 and 5 ORFs having mean lengths of 1,390 and 1,280 residues, respectively, and family 4 containing shorter ORFs, with a mean length of 966 residues (FIG. 24B, 24C; FIG. 25; Table 1).

The distance to the predicted insertion site, which is indicative of UTR length, also varied substantially, with family 1, 8, and 9 having the least distance between up and downstream annotations and ORF, suggesting shorter UTR lengths (FIG. 62A). While the 5′ and 3′ UTR lengths based on distance to nearest predicted target site show wide variability between 181-932 bp and 101-647 bp (inter-quartile ranges), respectively, the UTRs predicted based on non-coding sequence conservation flanking the ORFs are much smaller and have tighter length distributions (FIG. 62B-C). Using non-coding conservation, 5′ and 3′ UTR lengths vary between 115-327 bp and 82-172 bp (interquartile ranges), respectively (FIG. 62C). While families 1, 3, 5, 6, 7, 8, and 9 associate with previously identified orthologs and subfamilies, families 2 and 4 had no association to known subfamilies (Table 5).

TABLE 5
Summary of characteristics of non-LTR RLE containing retrotransposon families
		Mean
	Mean Length	Length for	Mean	Association
	for full	ORF	distance to	with			5′ and 3′
	ORF	starting at	target	known	Integration	RT	homology
Family	(bp)	Methioine	(UTR) (bp)	orthologs?	target	architecture	agree?
1	1108	1055	91	✓	28S, 18S	Non-LTR	X
2	1027	983	778	X	unknown	Non-LTR	✓
3	1558	1390	818	✓	5S, leader	RT-like	✓
4	988	966	666	X	Tandem	RT-like	✓
					repeat
5	1403	1280	704	✓	snRNA,	Non-LTR	X
					tRNA
6	1103	1016	845	✓	Tandem	Non-LTR	✓
					repeat
7	1185	1138	584	✓	tRNA	Non-LTR	✓
8	1262	1220	351	✓	28S	Non-LTR	✓
9	1199	1200	411	✓	Tandem	Non-LTR	✓
					repeat

We next examined the preferred integration sites for these families. Family 1 exhibited a preference for integrating into 28S and 18S rRNA gene sites; family 3 exhibited a preference for integrating into 5S and likely spliced leader sequences; families 4, 6, and 9 exhibited a preference for integrating into tandem repeats and microsatellites, including novel repeat sequences; family 5 exhibited a preference for integrating into snRNA gene loci and some tRNA preferences; family 7 exhibited a preference for integrating into tRNA; and family 8 exhibited a preference for integrating into 28S loci (Table 1). Family 2 has an unknown integration site preference. Accordingly, the zinc finger motifs across these different families are divergent (FIG. 24B, 24C).

Clusters showed two reverse transcriptase (RT) architectures, with families 3 and 4 containing broad RT-like domains, and all other families containing more specific non-LTR retrotransposon RT domains (FIG. 24A, 24B). In contrast to previous efforts profiling RLE-containing non-LTR retrotransposon diversity (Kojima, et al., PLoS One. 11, e0163496 (2016); Eickbush et al., PLoS One. 8, e66441 (2013); Luchetti, et al., PLoS One. 8, e57076 (2013)), our computational expansion covers an order of magnitude more orthologs than the 418 surveyed before (Kojima et al., Genes Genet. Syst. 94, 233-252 (2020); Bao et al., Mob. DNA. 6, 11 (2015)), allowing discovery of families with multiple integration preferences, such as in family 1, where 5S rDNA preferences are interspersed between 28S preferences, or families with discordance between 5′ and 3′ site predictions, such as in family 5.

We next investigated retroelements which had discordant 5′ and 3′ homologies. We found multiple instances of discordant homologies, including in family 1, which has members with 5′ small subunit rRNA preferences and 3′ large subunit rRNA preferences, and family 5 which contains systems with 5′ SL1 splicing leader preferences and 3′ U2 small nuclear RNA (snRNA) target preferences (FIG. 26). Our analysis revealed two broad classes of insertions, which we termed Class 1 and Class 2 based on the nature of surrounding elements (FIG. 26A, 26B). Class 1 insertions include ORFs with a canonical target site on one side and a different target gene on the other side. In contrast, Class 2 insertions involve canonical integration into a target site flanking the ORF retrotransposon, with additional putative insertion targets nearby.

To elucidate divergent target preferences, Rfam annotations were made around all members (FIGS. 24A-B, 62, 63) and the results show 188 systems clustering into 39 groups with divergent target preferences (FIG. 52, 53). Many of these divergent protein families show one dominant target site preference with a subset of member systems displaying new preferences such as a change from a 5S to tRNA site preference (FIG. 53).

We also heterologously reconstituted site-specific retrotransposition in human cells to model integration preferences and retargeting in eukaryotic genomes. We synthesized a panel of 12 retrotransposon ORFs from our computational exploration, selecting a sample that included R2 elements with demonstrated activity in mammalian cells (R201) (A. Kuroki-Kami, et al. 2019 Mob. DNA. 10, 23; Su, et al., 2019. RNA. 25, 1432-1438), experimentally characterized retrotransposon groups without proven activity in mammalian cells (R2Bm), previously computationally described retrotransposons (R2Ci, R2Tg, R2Is, R2Pap, R2Dr, R2Tsp, HeroDr) (Kojima et al., 2016 PLoS One. 11, e0163496), and novel retrotransposons (R10Mbr, R2Toc, R2Mes) (Table 6), which all ranged in sequence similarity between 13%-67% (FIG. 27, 64A-E). R10Mbr, which occurs in the genome of Myotis brandtii, appeared to integrate in GTA microsatellites (FIG. 28) and lacked similarity to integration preferences to other families. As such it was designated as a putative R10 family member.

To evaluate the native targeting capacity of these candidates for the 28S loci, we developed a plasmid reporter containing 200 bp of the 28S target with upstream expression of the N-terminus of Gaussia luciferase (Gluc) and delivered a payload containing an exon with 28S homology, predicted UTRs for corresponding orthologs, and a C-terminal Gluc fragment. This system enabled readout of insertion efficiency by luciferase production, and we found that only a limited subset (R2Bm, R2Tg, and R2Mes) had native activity from insertion of this heterologous Gluc cargo in HEK293FT cells (FIG. 29A) as measured by luciferase reporter reconstitution. Interestingly, R201, which has previously been demonstrated to be active for 28S genome insertion (Su, et al., 2019. RNA. 25, 1432-1438), did not have activity in the luciferase reconstitution assay (FIG. 29A). As suggested by the predicted R10Mbr preference for microsatellites, we did not see any production of luciferase in these conditions. To confirm that R10Mbr did not integrate into any putative 28S sites, we evaluated a panel of 28S sites known to have insertion by various site-specific retroelements and found no insertion at the tested 28S targeting sites, including in a 28S region with similarity to sequence flanking the R10Mbr locus (FIG. 29B-C, 65A-B), validating our assignment to a novel R10 family that prefers GTA microsatellites, which do not occur in the human genome (Subramanian et al., 2003. Genome Biol. 4, R13). R10Mbr and R2Toc, which occurs in the Talpa occidentalis genome, are both found in mammalian genomes.

To evaluate cross compatibility between payloads with UTRs from different orthologs and orthlogs R2Bm, R2Tg, and R2Mes, we utilized a 28S luciferase reporter assay to ascertain whether these three orthologs could perform insertion with the other templates. For R2Tg and R2Mes their cognate UTR payload was most efficiently integrated. For R2Bm the payload with R2Toc UTRs was most efficiently integrated, with 6.4× higher insertion efficiency than the cognate R2Bm payload. (FIG. 77).

TABLE 6
Exemplary non-LTR retrotransposon orthologs
			SEQ		SEQ		SEQ
Host			ID	Predicted	ID	Predicted	ID
Name	Abvn	Protein	NO:	5′ UTR	NO:	3′ UTR	NO:	Accession
Danio	Hero	MTTHRAEVTTSGKTQEE	33479	TTCAAGCCTG	33491	TGATCAACCC	33501	LR812084.1
rerio	DR	PGPEATHSAQSLLVSPT		GCGCAGCCAG		CGGCTGGGTC
		PAAGRSP		TGACTCCTAG		ACCTGGGTGA
		ATQSCPQVTAAHNSPQS		GAATAGACTA		GAGTGTATGA
		PQSQQVAVTRSDCVPLA		GGTGGCAACC		TGTTGAGAGA
		QPRIQWP		AAGAATAGTTT		CCCGAAACAC
		QSSKKAEWLQFDKDVNQ		GGTCGACTAC		TCAATGATCC
		ILEVTGKGGVDQRLSTM		TGGAGAGACA		CAGGATACAT
		TTLIVNI		GTTGACGGCA		CACTGATGAT
		AAERFGTVTPKPTPSTY		CGGAAAGACG		GTGTCCCAAA
		TPSHRVKEIKRLRKELK		GCACTTGGGA		TGCATCCATG
		LLKRQYK		CAGTATGGGT		AGATGTTTCTT
		AAGEVERAGLEDLRGIL		TAGCACCCCA		GCATAA
		RKQLVNLCRAEYHRKRR		GCCTGTGTCT
		RERARKR		TTCGTGAGAG
		AAFLANPFKLTKQLLGQ		AGAACCCAAA
		KRTGKLTCSKEAINNHL		CAAGCTACGG
		KATYSDP		AAAGCCCCAC
		NREQPLGPCGALLTPPE		AGAGATATAC
		PTSEFNMKEPCRSEVEE		CCCCAGGAGA
		VVRRARS		TCCCGAGAGG
		SSAPGPSGVPYKVYKNC		GGGGGAGGA
		PKLLHRLWKALKVIWRR		TGAGATCTCC
		GKIAQPW		AATCGGACGG
		RYAEGVYIPKEEKSENI		ATCAAAGGTT
		DQFRVISLLSVESKIFF		A
		SIVAKRL
		SNFLLSNKYIDTSMQKG
		GIPGVPGCLEHTGVVTQ
		LIREARE
		GRGDLAVLWLDLTNAYG
		SIPHKLVEVALEKHHVP
		QKVKDLI
		IDYYSKFSLRVSSGQLT
		SDWHQLEVGIITGCTIS
		VTLFALA
		MNMMVKAAETECRGPLS
		KSGVRQPPIRAFMDDLT
		VTTTSVP
		GARWILQGLERLVAWAR
		MSFKPAKSRSLVLRKGK
		VRDEFRF
		RLGQHQIPSVTERPVKS
		LGKAFNCSINDRDSIRE
		TSTAMEA
		WLKAVDKSGLPGRFKAW
		VYQHGILPRLLWPLLIY
		EVPMTVV
		EGFEQKVSSYLRRWLGL
		PRSLSNIALYGNTNKLK
		LPFGSVR
		EEFIVARTREHLQYSGS
		RDAKVSGAGIVIRTGRK
		WRAAEAV
		EQAETRLKHKAILGAVA
		QGRAGLGSLAATRYDSA
		SGRERQR
		LVQEEVRASVEEERTSR
		AVAMRQQGAWMKWEQAM
		ERNVTWK
		DIWTWNPLRIRFLIQGV
		YDVLPSPSNLYIWGRVE
		TPACPLC
		SKPGTLEHILSSCSKAL
		GEGRYRWRHDQVLKSIA
		EAISKGI
		KDSRYRQATAKVIQFIK
		EGQRPERTAKNCSAGLL
		STARDWV
		MTVDLERQLKIPPHITQ
		STLRPDIILVSEATKQL
		ILLELTV
		PWEERMEEAQERKRGKY
		QELVEQCRANGWRTRCM
		PVEVGSR
		GFASYTLSKAYGTLGIT
		GTNRRRALSNNVEAAEK
		ASRWLWL
		KRGEQWGQ
Myotis	R10M	MGLTTPPGFIVLVTIET	33480	TGCCGACTGG	33492	GGATCTGCAA	33502	NW_
brandtii	br	ENDISPGVPTPAYTSTQ		GCAGCTTTGG		TTTACCATTG		005337413.1
		EGRAEL		AGAAATCCTA		GTTCAACTCC
		ACGSCGKICKSKAGLVS		AGCTGGCTAA		GTGTGACGGG
		HRKVHVQGNANSQSGCP		GAGTTCAGCA		CACATCGTGC
		FTDVDR		ACTCCTG		CCCGATGTGT
		TCRICDRQFSSKSGLTQ				GAGCCTGGAC
		HKRHRHPEARNQEKLSC				TGTGGTAGCA
		MKTAGS				CTTCGGTGCA
		HWTEQESTALLRIATKL				CTTGAAGAGC
		APTCSNLRSLYCRLEHD				AATGTCAGTT
		FPGRSA				GTCCGTGTAT
		CSIKTRLRTLNWKPTRV				TCTTCATTCTT
		TLPDDVCVASQESTNND				CGAAC
		TQRIEW
		ASKTVDVAIRQLKDSPQ
		ESLRSADLLAMAESFQR
		GAIDSQ
		QLLSLLEMHAVSTFPHR
		WRMNTKGHARRANATYK
		NRKQIR
		RANYASLQALYHQRRKD
		AATAVFSGTWKDAHLST
		RGLPDN
		SDKYWQDILSAPSHCDN
		RPCRSVTPIDWSLIEPI
		HHEEVT
		SAVKQMGNTAPGLDKIR
		PAELKHYSSKALAGYFN
		LLLLSE
		GCPEHLCLSRITLVPKV
		PNPSCPSELRPIAVSSS
		IIRCFH
		KIIADRWNSRLSLPSLQ
		FAFLKRDGCLEATSTLH
		AILRHS
		CSTGSGLSVAFIDVAKA
		FDSVSHETIIRSAKAFG
		APPPLT
		QYLTTSYERAAAAISTS
		TVKCHRGVRQGDPLSPL
		LFIMAM
		DEVLSSSMPQLGYQFHD
		TLVDGFAYADDLIIMAE
		NLPRLQ
		EKLDAASVALGFAGMKI
		NAKKTKLLDIRGARKPY
		VTATCE
		TPVSFQNEEIKPLSSTE
		TLTYLGIPFTSKGKASI
		NHRRQL
		QEVLSQIRKAPLKPQQR
		LELTREHLIPKYTHTLV
		LGNAHR
		NTLKRMDNAIRQSLRDW
		LRLPPDTPTAYFHTACS
		LGGLGV
		PCLSTTIPLYKKTRMEK
		LLTATCPVLRNVVNSGS
		FKPIIK
		ELSIPIRVHGTIVTDKE
		GAREAWHEHLLSSVDGR
		GLRDVA
		KSPLSNAWLIRPERIFP
		RIFLRAVHLRCNLLRTK
		VRSARG
		GRGDQSVLCRGNCGQPE
		SLAHILQSCWVTHDARC
		ARHNRV
		AKELARRLRKLGYSVFE
		ELRVPTSHSFIKPDLIV
		VQDTSA
		FVLDVSIVGDGRMQSAW
		SEKVEKYSTEAHTAAIS
		SMLSSI
		GKPVEHVFHEPVIFSFR
		GVCYSRSVKSIIRLGLP
		RYSISD
		LCLLTIIGSLRTYDTFM
		RGTWK
Phleb-	R2Pap	METNRENIYSRDEAGVN	33481	AACTATGACG	33493	CCTACGGCGA	33503	AJVK01
otomus		SLGSRPQMRPRSQTMER		TATGTTATAG		AGTTTGCGGC		060896.1
papatasi		SIVEAG		GGAGTTTTATT		GTTACCATTCT
		CDQNEFGCDLCDRRFRT		AGTTAAGGTT		GGGTGCTAAG
		TRGLGQHFRHSHPREHN		GGGTGCGTG		ACTGAACAAG
		DRLNTD		GAGTCGGATC		GCATTGATGG
		RIKARWSPEEEYLLALE		GTTGAAGTCT		GTTCCATTGT
		EVRATSRGIRFLNQHLA		TCATTGACCT		CTAAGGTTGC
		EAFPNR		AAATGTATCG		TTTTATATTGA
		TIEAIKCHRRORTYKEL		TTGACCATCG		GGTGTGCCGT
		VANLLVRASSARESQTS		TAGCCCTTCA		CGAGCACCTG
		YAGRLG		AGTGTCCATC		GTAGCATTCA
		ETSSSVSAELVVEVNRL		TGACGCCCCC		TTCTTATGGG
		IDYLAIHPVRKYFSDEL		TCATGGCGAC		CGAAAGAATG
		VAAAHA		CGCTGGGGAT		ATAAATATGAT
		AIIGDVECDELILSWLQ		TGCTTTCGAG		GATCGCGAGA
		KAFRIRHGLRPSTSTAA		CATGAGCTAA		TCATGATCCA
		GNPGSY		GAGCAGTGGA		CTTTCTTGGC
		RSGSDRPLSNRKRRRQD		TGCGGGGGT		GTAATGTGGA
		YARVQRLWNKSVKKAAR		GGTACAGGCG		AAGTCTAGCA
		GILEGS		TATCACCCTTA		TGGTAATGGG
		DEANSGESVHPTPERML		AAAAGAAAGT		GTAAAGTTGG
		RYWSDIFKQEGPIIPDR		CGATC		GTCTCTGATC
		TNQSPR				CAGGCTATAC
		NEELKDMWEPITIDEVK				CTATGATGAG
		LARLDPGSAAGIDRISV				AAACCCTAGT
		QQFQRC				TCACTTGCTC
		PVHVRVLLFNVLLLVGH				AATTCTATTTG
		LPGRMSCARTVFLPKVE				TCGTAAGACT
		GSSDPK				TATGGAAATA
		DYRPISITSVITRQFHK				AGTGACAAAA
		ILAARLTSMHAWDERQA				CGATCTAGAC
		GFLPVD				TATTTCTGAG
		GCGENLAILNELIRFSR				GGTA
		VNRRELHLASLDISKAF
		DMVPRQ
		AIINSVAQLGAPQNLVE
		YLKGLYANNQTTLEYGG
		SELYCR
		VKRGVRQGDPLSPLLEN
		LVMESALVRLDKKLSFS
		LYGVSV
		NGLAYADDVILVASTSG
		GLQKNTESFLGALREIG
		LDLNLA
		KCKSLSLKPSGRDKRCK
		VLSESQLSIGGTSVPQV
		DLVGFW
		RYLGIWFSGPRVVSPEQ
		LSMGVYLERISKAPLKP
		QQRIRI
		LVDYLLPKYTHGSVLGR
		YTRKTYKAMDAQIRSYV
		RKWLHL
		PLDTTLGYFYAPVMSGG
		LGIPNFEMTVPLMKVER
		NRKLLS
		SARGTIRAVMHGSPLIR
		DTERTASWLLTRLPALD
		IECYKG
		YWIKSLYESSDGRDNRA
		INGVRGSIGWSRKFSNK
		LTGRDF
		VHFHQIRINALDSKART
		LRGRGVDVRCRAGCLDR
		ETPYHI
		VQRCFRSHGGRVLRHDN
		SVQLLCSEMTRKGYNVA
		VERQLQ
		TVEGMRKPDLIAVKDGR
		AAVIDMQVVSGGSMESS
		HREKVE
		KYQRIPGYTELVKEAFG
		VASVEYRAATISWRGIW
		FKPSYD
		SLTRLGVGERCLGSICC
		QVMRGSYLNFVRFKQST
		QMVWSAV
Bombyx	R2Bm	MMASTALSLMGRCNPDG	33482	GGGCGATACG	33494	GCCTTGCACA	33504	AB076841.1
mori		CTRGKHVTAAPMDGPRG		CATAATTTTAA		GTAGTCCAGC
		PSSLAG		TTTCCCGATT		GGTAAGGGTG
		TFGWGLAIPAGEPCGRV		GAAATCCAGT		TAGATCAGGC
		CSPATVGFFPVAKKSNK		CGTCTTAATCT		CCGTCTGTTT
		ENRPEASG		GGTGACCAGT		CTTCCCCGGA
		LPLESERTGDNPTVRGS		GGCGCGGTCA		GCTCGCTCCC
		AGADPVGQDAPGWTCQF		CCAGTATAGT		TTGGCTTCCC
		CERTFST		GCACAGGACG		TTATATTTAAC
		NRGLGVHKRRAHPVETN		TGAATGGCTC		ATCAGAAACA
		TDAAPMMVKRRWHGEEI		CGAGGCTGGC		GACATTAAAC
		DLLARTE		GGAGTCActcac		ATCTACTGAT
		ARLLAERGQCSGGDLFG		tataagtgtgaga		CCAATTTCGC
		ALPGFGRTLEAIKGQRR		ga		CGGCGTACGG
		REPYRALV		cgatgtcctgtgc		CCACGATCGG
		QAHLARFGSQPGPSSGG		ca		GAGGGTGGG
		CSAEPDFRRASGAEEAV		agtatacgtccaa		AATCTCGGGG
		EERCAED		cc		ATCTTCCGAT
		AAAYDPSAVGQMSPDAA		ctaacgggttaag		CCTAATCCAT
		RVLSELLEGAGRRRACR		tg		GATGATTACG
		AMRPKTA		aaattagttgctc		ACCTGAGTCA
		GRRNDLHDDRTASAHKT		ata		CTAAAGACGA
		SRQKRRAEYARVQELYK		acagggacggtgt		TGGCATGATG
		KCRSRAA		a		ATCCGGCGAT
		AEVIDGACGGVGHSLEE		cctgtttgctcgt		GAAAA
		METYWRPILERVSDAPG		ggct
		PTPEALHA		ggctatcgaatgg
		LGRAEWHGGNRDYTQLW		ac
		KPISVEEIKASRFDWRT		gggaccaatacac
		SPGPDGIR		c
		SGQWRAVPVHLKAEMFN		cccctgttagtaa
		AWMARGEIPEILRQCRT		tgg
		VFVPKVE		ggtaagagagagc
		RPGGPGEYRPILIASIP		ggtctgaaactat
		LRHFHSILARRLLACCP		gg
		PDARQRGFIC		ccgaaatcacgac
		ADGTLENSAVLDAVLGD		g
		SRKKLRECHVAVLDFAK		ccccactcctacc
		AFDTVSHE		ca
		ALVELLRLRGMPEQFCG		taacctgcacgtg
		YIAHLYDTASTTLAVNN		gt
		EMSSPVKVG		accgccgcacatt
		RGVRQGDPLSPILFNVV		g
		MDLILASLPERVGYRLE		accgatacgggag
		MELVSALAY		g
		ADDLVLLAGSKVGMQES		aggggcagcactt
		ISAVDCVGKQMGLRLNC		g
		RKSAVLSM		aatcacgtagtct
		IPDGHRKKHHYLTERTF		tgg
		NIGGKPLRQVSCVERWR		tgtagccattgcg
		YLGVDFEA		gg
		SGCVTLEHSISSALNNI		actacagccctcg
		SRAPLKPQQRLEILRAH		ta
		LIPRFQHGFV		agtgccgccttag
		LGNISDDRLRMLDVQIR		aa
		KAVGQWLRLPADVPKAY		cgcaacggggcaa
		YHAAVQDG		t
		GLAIPSVRATIPDLIVR		aggtgggccgggg
		RFGGLDSSPWSVARAAA		c
		KSDKIRKKLR		gctagcggggggg
		WAWKQLRRFSRVDSTTQ		agtaatctcccct
		RPSVRLFWREHLHASVD		gtt
		GRELRES		ggcgtgcaccgca
		TRTPTSTKWIRERCAQI		c
		TGRDFVQFVHTHINALP		tgctcccactggg
		SRIRGSRGR		gg
		RGGGESSLTCRAGCKVR		cagtgtcatccgg
		ETTAHILQQCHRTHGGR		aa
		ILRHNKIV		acaggtgggccgg
		SFVAKAMEENKWTVELE		g
		PRLRTSVGLRKPDIIAS		gcgccaccagggg
		RDGVGVIVD		ggagcaatccctc
		VQVVSGQRSLDELHREK		ct
		RNKYGNHGELVELVAGR		g
		LGLPKAE
		CVRATSCTISWRGVWSL
		TSYKELRSIIGLREPTL
		QIVPILALRGS
		HMNWTRFNQMTSVMGGG
		VG*
Mesol-	R2Mes	MGVAFDDNNELYGRTSG	33483	ATCTTGTTGCT	33495	TCGGATTCCT	33505	OU744815.1
igia		LPQEPEASTLKPVSPTA		CACGTCTGGG		GTGTCGCGGG
fur-		RSPPRSG		GATGTGTACC		CCGCCGCCG
uncula		RGEGEWTCPECSRAFRT		CTCTTTGCTG		GGCCCAGGG
		KTGLGVHKRRAHPVTAN		CCGCTATCAG		GTCGAAGCTC
		AAAAPPQ		TTAATTGGGA		CCACCTTTGG
		VKRRWLEEEGELLAQTE		AAGAGCTGTT		CTTCACCTGG
		ARLVRAGGSASTINQQL		CACAGACGTC		TTAATTTTTGT
		MRELPQLG		AATGTCCTAG		ACCAACTTCG
		RSLEAIKGYRRKEAYKS		CGATACGACT		ACCTCGCCAG
		RVQACLADLAQPPSPST		AAATGGGGGT		AGATCCCACC
		PGEANLPIR		TGATGTCGCT		GGCGTAATTT
		STPIAGTAESSTPEQPL		AGGAAACTCC		CGACAGTTAG
		VWAEPPSEVLPVSVSLQ		TATCCTTGGC		GTGACGGTAG
		SPDLSEALD		CTGCCCGTGG		GGGACGTGG
		HVKIVEDLLASSEARMA		CGCCGCTGCT		GAGTCCTGTC
		RGASDGKRRGRPRRKGQ		TCGCGAACAA		ACAACAATCT
		SPEETQIA		GGGGGGGGG		GTGATGATTA
		FARLSARKRRRMEYARV		GGGGCAATCA		TATTACGTTCA
		QELYKTCRSRAAAEVID		CTAATATATG		CTAAGACGAT
		GQTRGVSH		GACTGGATAG		GGCACTGTTC
		SLSELEAYWRPVMEAVS		CAAGGACAGT		GAGC
		DAPGLTPEVLGALQRSE		CCGTTAAAAG
		QYGGSRD		CATCTCATGG
		YSQLWTPFTSDEVKACR		TGGTAGACCC
		VDNRSGPGPEGILPGAW		TAATACAAGTT
		NTLSSAT		GAGGTGTCGC
		QAEIFNAWLMAGEVPEK		GCATACCTCC
		LRGCRTVFVPKTETPAG		CCGACTTGAC
		PGEYRPISI		CAGAAGACGC
		ASVPLRHMHSVLAKRLE		GAGTCTGATG
		ACCPPDARQRGFICADG		GTCCTAGGGG
		TLENSAVL		TACGGTGAGC
		DAVLGDCGKKLRECHVA		CTGGAGACCT
		VLDFAKAFDTVSHAALI		TGGTCACGAA
		DLLRKRGLP		ATCGGATCAA
		EGFCNYVARLYDTSETV		GGTGCGCAAA
		LVANGARSGPARVGQGV		CACGCGAAAG
		RQGDPLS		GTGCATATCC
		PLLFNMAMDVILAALPR		AGGACAGGCT
		EVGYGLEGENVSALAYA		CTGGGGGAAG
		DDLVLLAGS		GTCTCAGAGA
		KVGMQSSIDCVWRTGRM		AGCCCCGATG
		MGLFISHAKSAVLSMVP		GGAGTCCTTG
		DGKRKKV		GGCTCCAAAT
		HFLTDRTFKVGSRWLRQ		CTAGCAGCTC
		VSCVERWRYLGVDFKAS		GCATGGCTGG
		GCVTLEH		GGCGTACACG
		DVKVALNNITKAPLKPQ		AGAGCTGTGG
		QRLEILRVHLIPRFLHG		GTGAACCTGT
		FVLGIITDDRL		GGATAGGGCT
		RMLDVQIRRAVRTWLRL		AGCCACCCTA
		PKDVPVGYFHAATADGG		CCACAATAGG
		LAIPSLRT		TATTAAGGTG
		CVPDLIKKRFGRLDSSR		TTGCTTTCGAT
		WPVARAAARSERIRRKL		GATAATAATG
		QWADKQLR		AGTTGTATGG
		KFTAENPKSGERTTAMY		TCGAACCTCC
		WREALHASVDGLELREC		GGCCTCCCAC
		PRVPASTK		AGGAGCCGGA
		WMRERSMQYTGRDFVQF		GGCGTCGACC
		VHTHINALPSRVRNTRG		CTTAAACCTG
		RRTGVAS		TAAGTCCGAC
		ELNCRAGCMVRETTAHT		TGCTCGATCT
		IQQCHRTHGGRIKRHNC		CCGCCTCGGT
		VADVVCSA		CGGGGCGGG
		MEDKGWTVVKEPKVRTA		GGGAAGGCG
		LGLRKPDIIASRNGVGV		AGTGGACCTG
		IVDAQVVS		TCCGGAATGT
		GQRPLDELHREKRNKYG		AGCAGGGCAT
		NHAELVEKVADILGLPC		TCCGGACCAA
		KESVHSTS		AACAGGGTTG
		CTLSWRGVWSLASYREL		GGCGTGCACA
		KRFVGLDEGVLAGVPSL		AGCGGCGCG
		VLRGSHIN		CACACCCTGT
		WTRFNRMTTVSTESGSE		GACGGCAAAC
				GCCGCAGCC
				GCCCCACCGC
				AGGTGAAGCG
				GAGATGGCTT
				GAAGAAGAGG
				GTGAGCTTCT
				TGCGCAGACG
				GAGGCGCGTC
				TGGTTAGAGC
				CGGAGGCAGT
				GCCAGTACAA
				TTAACCAGCA
				ATTA
Ciona	R2Ci	MGEWPWVSWSLTVLVEK	33484	CGACGGTGAA	33496	TGACAGTAAT	33506	AB097122.1
intes-		WRPFTILQPYPMPGQLR		CCACCTTGTC		ATGAAAACAT
tinalis		VDVYLPR		GCGGTGTAAG		CACATCTGAC
		KTSYLMDKNIYENTTSP		AGCTTTAGTG		CGGCACAGAA
		GGGPLCGEKTHRSDVII		TCTCGAACAA		TCACCATGCC
		PPPGFAPST		GAAATAGCTT		GTAATGCACC
		DTASNTLGENVDASATT		GTGTGCTGTC		CAACTAAGGA
		SSANPLSQEPGWCESCS		CTTCTGGGCG		TTCCAATGGG
		KLFKSQR		GTGCACATAC		TAAAAAAAAAA
		GLRVHQRSKHPELYHSQ		TTCTTAACCTC		AAAAAAAAAAA
		NQPLPRSKARWSDEEMV		CCGAGGCCAT		AAAAAAAAAAA
		IFAREEIA		GCCGGCGGG		AAAAAAAAA
		NRKIRFINQHLHKVFPH		GGCTTTAGCC
		RTLESIKGLRGKNVRYA		CCCGGCAGGT
		RIMADLEAEM		TTTACCATGC
		TSQPEAATSLCTETSEN		CGGACGGGTT
		LASSNVLPQTRGWAENL		CGAGAGGTAG
		VENIDTAHL		AGGCCAAACT
		ANLGPLSQFEPGKPSSS		AAGAGTTCAC
		TKEAINTEYNDWISKWL		CAGCAGACTT
		PSGAAHRE		CGCACGCGG
		RRANPPSTKLNARATRR		CTGGCCACTG
		LQYSRIQNLYKLNRSAC		GCCGAAGTTT
		AQEVLSGA		AAACAACAGG
		WKVQSGELNLKEVQPFW		GCCGCATCTT
		EKMFRKESAKDRRKPKP		CCCAAACTCA
		TGEVLW		ATATATGGTG
		GLMEPLTIAEVGSTLKS		TTAAGTGAAC
		TTPSAPGPDKLTLDGVK		CGTGCCG
		RIPIAELVSH
		YNLWLYAGYQPEGLREG
		ITTLIPKIKGTRDPAKL
		RPITVSSFICR
		IFHRCLAQRMETSLPLG
		ERQKAFRKVDGICHNIW
		SLRSLIHNS
		KDNLKELNITFLDVRKA
		FDSISHKSLGIAAARLG
		LPPPLITYISNL
		YPNCSTKLKVNGKISKP
		IEVRRGVRQGDPLSPLL
		FNAVMDWA
		LSELDPRVGVQIGEQRI
		NHLAFADDIILVSSTKI
		GMVSSINTLSR
		HLAKSGLEISAGKEGKS
		ASMAIVVDGKKKMWTVD
		PLPRFKVN
		SQKIPALSITQQYKYLG
		INIDAQGARNDAARILT
		EGLAELSRAPL
		KPQQRLYLLRVHLLPKL
		QHGLVLSSCAKRALTYL
		DKSVRSAIR
		RWLTLPKDTPTAFYHAK
		ACDGGLGITRLEHTIPI
		LKRNRMMKL
		TLSEDPVIMELVKLTYF
		TNLLHKYSNVKLLNSWP
		VTDKDSLAR
		AEASMLHTSVDGRGLSN
		CSDVPRQSDWVTNGASL
		LSGRDFI
		GAIKVRGNLLPTKVSAA
		RGRQREITCDCCRRPES
		LGHILQTCP
		RTWGPRISRHDSLLKRV
		RNQACLKNWTPIIEPSI
		PTNIGLRRPD
		LVLAKGNIAFLVDATVV
		ADNANMQLQHEAKVEKY
		NNSDIKEWI
		KVHCPGVDEVRVTSLTA
		NWRGCLYGGSASFLTED
		LGLPKAEL
		SLLSAKINEKGYYLWCA
		HYRGTARLWNRPLRS
Ixodes	R2Is	MQCTSRLADAPRFARVG	33485	GTTCCAAAGG	33497	TAGTGTGACG	33507	GCA_
scapu-		VEGEGVGASGNGTDAQL		AAGGCACTCC		GAGTCCTCAA		016920785.2
laris		WYGCTG		TTTGGTTCGT		GCCCCCACAA
		CDEAFSSLRGLRIHAAQ		GATGAGATGT		GTGCCTGCCA
		KKHGNQDGLLRLPAGRP		TCATGGTGCT		GGTGGCAGGA
		RKRRVGKS		TGCCTAGCTG		AAGGGCAACT
		TTAGASDRVTTDPVPAP		GAGAAATCCG		ACTGGTGAGC
		VPESPGLLPGLPGPSLP		ACTCACACCT		GACCCAAGCA
		GCSDLPPG		GCACGTGGTC		AGGCGGAGC
		VLPGGWSASPGPLSWPP		CCTGCCGCCT		CAAGACCAAG
		SLDAGPLPGPSRVSPGP		GCCAGTATGC		CTGGAGCCAA
		SRPSPGK		CGAGGAAACG		GAGCAACTCC
		PTGPPSLDAGPLPGPSR		GGTGCAACTT		AGGAGGCAG
		VSPGPSRPSPGKPPGTP		AATCCGTGGA		GGGTGGATAT
		EPLPGSP		TACTGGTAGC		CAAGAGCAAC
		GGRRGVSPGQPGSRTDP		AACGTGAGCA		CCCAAGGGAC
		SSSAGAGHFVCPQCSRA		ACGGTACGGT		ACAGACCACG
		FSSKIGM		CCTTCGCGGA		GGCAACTACT
		SQHQKHAHLEEYNAGIN		CCACCCTGGG		GGTGAGCGCC
		ITRTKARWDPEETYLLA		CGTTCGGGTT		CAAGACAGGG
		RLEATLNPD		GCCAGCCCGT		GTGGATATTA
		HKNINQTLHAALPRGSC		TCGCCCGAAA		AGAACAGCCC
		RTLESIKAHRKQAAYRD		TATCTTGGCC		CACAAAGTGT
		LVTSLRSAR		CTGAAACTAA		TACCTATATTA
		ESSEAQHVPDRPLETPE		AAGAAAA		ACAATAAAGTT
		PQTPANPQRDSKQAVIE				GAAGCCTCAA
		ALQSLIGRA				CCACGCATTG
		PPGSFQGARLWDIARQA				CGGGTTAGAT
		TRGTNILPLLNSYLRDV				GGCGTGGCTT
		FTLPTKPTR				GGCCCGCCG
		KKPAVRPARSRRKQKKQ				CCATGATGAG
		EYARTQDLFRKKQSDCA				CTGGAACCCT
		RAVLDGP				CCACCTGGTG
		TSSSVPGTGAFLQTWRE				GGCCGCACGA
		IMTGPSPALEAPPLPTR				GACCACCGGC
		GEVDLFFPA				TCTTTCTACTA
		TAQEIQSAEIAVNSAAG				AGGCCGGTCT
		PDGFSARLLKSVPALLL				CCGTGACTGC
		RVMVNLLLLV				GGTTGGGATA
		RRVPAALRDARTTFIPK				AACTCCAAGC
		VPDAVDPSQFRPITVAS				ACTGAGCGGT
		VLQRLLHRIL				AAAAAAAAAAA
		AKRALEAIPLNFRQRAF				AAAAAAAAAAA
		QPVDGCAENIWLLSTAL				AAAAA
		NEARTRRRP
		LHMASVDLTKAFDRVTT
		DAILRGARRAGLSGEFI
		GYLKELYTTS
		RTLLQFQGESLLVEPTT
		GVRQGDPLSPILFNLVL
		DEYLSSLDP
		DISFVSGDLRLDAMAFA
		DDLIVFASTPAGLQDRL
		DALVEFFDP
		RGLRVNVKKSFTLSLQP
		GRDKKVKVVCDQIFTIG
		GTPLPASKV
		ATPWRYLGMTFTPQGSI
		NKGTSEQLDLLLTRTSK
		APLKPQQR
		LVVLRNYLLPRLYHRLV
		LGPWSAALLLKMDTTIR
		GAIRRWMDL
		PHDTPLGFFHAPVTEGG
		LGINSLRASIPAMVLQR
		LDGLHFSTH
		PGAEVAIQLPFLTGLHR
		RAEAAAQYQGQRLLSKA
		DVHRMWSA
		RLHGSCDGRPLRESKRV
		PAAHRWAAEGTRLLSGR
		DFISITKLK
		INALPTLERTSRGQHKD
		IQCRAGCQAVESLGHVL
		QACHRGHR
		GRIRRHDNIARYVCGRL
		TQIGWAVKWEPHYSVAG
		RTLKPDIVA
		HRGAETVVLDAQVVGTS
		MRLGFHHAQKKEKYSLP
		DLLHQVC
		EGRRDAARVSTITLNFR
		GVWAPESAQDLKSLGLT
		DNDLKLLTV
		RCLQGGAQCFRLHRRMT
		TVVKATGDEANALPAHS
		GLPPTQL
		GGRTLGPSAHNQSARTT
Trich-	R2Tsp	MSNRLANTAAAGGVPEK	33486	CTCCTGACTA	33498	TGAGGTTTTT	33508	CP032378.1
inella		TSGTLDIPGQPSSSGEK		ACCTGATTTC		GTTTTCTTTTT
spiralis		RAISYPGP		GTCCGTGCGG		TCCTTTTACCA
		FGCNSCSFTSTTWLSLE		CGGCGTTTTC		TTCTTGTTCCA
		LHFKSVHNIRDFVFLCS		TTTTCGCTCTC		TTGTTGTTATT
		KCKKSWPSI		CGCTCGTCGA		TGCTTTAATCC
		NSVASHYPRCKGSVKAA		AATTTGCTGTA		TGTATTTTACC
		VVPTSLANTCTTCGSSF		GTTGATTCGC		GCCGGCAATT
		GTFSGLQL		TTTTCTTTGCG		CCATTGTTATT
		HRKRAHPDVFAASCSKK		TTTTCTTCTAC		ATTACTGTTAC
		TKARWSNDEFTLLARLE		TTTCGCAGTTT		TGTTATTATTG
		AGLDPACK		TTTCTGCATTG		TTACTATTGTT
		NINQVLAERLMEYNITR		CCACG		TTTACTTTTAC
		GVEMIKGQRRKDQYKAL				TTACTACTGTT
		VRQLRSNS				ATTATACTTTA
		ETQQCVGLAGSMDSNVP				ATTCGTTAACT
		ANDTSSSVASEVSITYP				TACGTTATTGT
		EYGAVMSC				TACCACTACTT
		DLIKEATGMAIVDINEL				ACTTTGCTCT
		QSNLRKAFLSGRKLPMK				CTCGCAAACG
		FHGARETAQ				TTCGTTGTTGT
		KKMANPRVAKFKRFQRL				TTCTTTTGGAC
		FRSNRRKLASHIFDKAS				CAGGTTTAGA
		LEQFGGSID				GAAATCGCAC
		EASDHLEKFLSRPRLES				GCACAGCGGA
		DSYSVISGDKSIGVAHP				ACTGGACCGC
		ILAEEVELEL				TTAAGCCAGA
		KASRPTAVGPDGIALED				AATAGTAAAG
		IKKLNTYDIASLENLWL				TAACAA
		KAGDLPASVK
		ASRTIFLPKSDGTTDIS
		NCRPITIASAMYRLFSR
		IITRRLAARLEL
		NVRQKAFRPEMNGVFEN
		SAILYALIKDAKVRSRE
		ICVTTLDLAK
		AFDTVPHSRILRALRKN
		NVDPESVDLISKMLTGT
		TYAEIKGLQG
		KLIPIRNGVRQGDPLSP
		LLFSLFIDEIIGRLQAC
		GPAYDFHGEKI
		CILAFADDLTLVADSAA
		GMKILLKAACDFLEESG
		MSLNAEKCR
		TLCITRSPRSRKTFVNP
		AAKFIISDWKTGISSEI
		PSLCATDTFRF
		LGHTFDGEGKIHIDTEE
		IRSMLKSVKSAPLKPEQ
		KVALIRSHLL
		PRLQFLESTAEADSRKA
		WLIDSIIRGCVKEILHS
		VKAGMCTDIF
		YIPSRDGGMGFTSLGEF
		SLFSRQKALAKMAGSSD
		PLSKRVAE
		FFIERWNIARDPKVIEA
		ARRVYQKKRYQRFFQTY
		QSGGWNEF
		SGNTIGNAWLTNGRARG
		RNFIMAVKFRSNTAATR
		AENLRGRP
		GTKECRFCKSATETLAH
		ICQRCPANHGLVIQRHD
		AVVTFLGEV
		ARKEGYQVMIEPKVSTP
		VGALKPDLLLIKADTAF
		IVDVGIAWEG
		GRPLKLVNKMKCDKYKT
		AIPAILETFHVGHAETY
		GVILGSRGC
		WLKSNDKALASIGLNIT
		RKMKEHLSWLTFEIIFI
		TQISRIYNSFMK
		K
Taeni-	R2Tg	MASCPKPGPPVSAGAMS	33487	GTCTAGTTAC	33393	TTCAGGTTATT	33509	XR_
opygia		LESGLTTHSVLAIERGP		AACTGGGCAT		TAGATGCTTA		005978890.1
guttata		NSLANSGS		CGCTGCAGAG		GTTTTTGTACC
		DFGGGGLGLPLRLLRVS		ATCGCACCTC		TTTCTTGTTTT
		VGTQTSRSDWVDLVSWS		CTCGTGGTCC		GTTTAGGATTT
		HPGPTSK		CGCTGGTAGC		TGATAGTGTT
		SQQVDLVSLFPKHRVDL		CCTTCGAAGG		AGTATTTTTAT
		LSKNDQVDLVAQFLPSK		GTGACTAAGT		ATTTTTGTACG
		FPPNLAEN		CGATCTCTGC		ATTGCATAAT
		DLALLVNLEFYRSDLHV		CCCAGGTACG		GTTCTTTTTTA
		YECVHFAAHWEGLSGLP		GAGCCGTTGG		TACAGTTCTG
		EVYEQLAP		GACTCACCAG		TTTTAATAAAA
		QPCVGETLHSSLPRDSE		TCCAACGTAA		TAGACGATAG
		LFVPEEGSSEKESEDAP		CTCCTGCCTA		CTAGAGACGT
		KTSPPTPG		AATTCGGTGA		TAGGGCAGCC
		KHGLEQTGEEKVMVTVP		AACAAATTCCT		ACAAGCCAGT
		DKNPPCPCCGTRVNSVL		CGGTAAAAAG		TAGGTAGCGG
		NLIEHLKV		CCCC		ATAGTAGGTA
		SHGKRGVCFRCAKCGKE				GGAACAGACT
		NSNYHSVVCHFPKCRGP				TTTACTATTTC
		ETEKAPA				ATAACGCGTC
		GEWICEVCNRDFTTKIG				AATTACCACC
		LGQHKRLAHPAVRNQER				TGATTTGGAC
		IVASQPKE				CAATTCACGG
		TSNRGAHKRCWTKEEEE				GATTTGTCCA
		LLIRLEAQFEGNKNINK				AGGTGGACGG
		LIAEHITTKT				GCCACCTTTA
		AKQISDKRRLLSRKPAE				CTTAACCCGG
		EPREEPGTCHHTRRAAA				AAAAGGAACA
		SLRTEPEM				TATATAATTTA
		SHHAQAEDRDNGPGRRP				TGTGTGTTCG
		LPGRAAAGGRTMDEIRR				ATAAA
		HPDKGN
		GQQRPTKQKSEEQLQAY
		YKKTLEERLSAGALNTF
		PRAFKQVM
		EGRDIKLVINQTAQDCF
		GCLESISQIRTATRDKK
		DTVTREKHPK
		KPFQKWMKDRAIKKGNY
		LRFQRLFYLDRGKLAKI
		ILDDIECLSC
		DIPLSEIYSVFKTRWET
		TGSFKSLGDFKTYGKAD
		NTAFRELITA
		KEIEKNVQEMSKGSAPG
		PDGITLGDVVKMDPEFS
		RTMEIFNL
		WLTTGKIPDMVRGCRTV
		LIPKSSKPDRLKDINNW
		RPITIGSILLR
		LFSRIVTARLSKACPLN
		PRQRGFIRAAGCSENLK
		LLQTIIWSAK
		REHRPLGVVFVDIAKAF
		DTVSHQHIIHALQQREV
		DPHIVGLVSN
		MYENISTYITTKRNTHT
		DKIQIRVGVKQGDPMSP
		LLFNLAMDPL
		LCKLEESGKGYHRGQSS
		ITAMAFADDLVLLSDSW
		ENMNTNISI
		LETFCNLTGLKTQGQKC
		HGFYIKPTKDSYTINDC
		AAWTINGTP
		LNMIDPGESEKYLGLQF
		DPWIGIARSGLSTKLDF
		WLQRIDQAP
		LKPLQKTDILKTYTIPR
		LIYIADHSEVKTALLET
		LDQKIRTAVKEW
		LHLPPCTCDAILYSSTR
		DGGLGITKLAGLIPSVQ
		ARRLHRIAQS
		SDDTMKCFMEKEKMEQL
		HKKLWIQAGGDRENIPS
		IWEAPPSS
		EPPNNVSTNSEWEAPTQ
		KDKFPKPCNWRKNEFKK
		WTKLASQ
		GRGIVNFERDKISNHWI
		QYYRRIPHRKLLTALQL
		RANVYPTREF
		LARGRQDQYIKACRHCD
		ADIESCAHIIGNCPVTQ
		DARIKRHNYI
		CELLLEEAKKKDWVVFK
		EPHIRDSNKELYKPDLI
		FVKDARALVV
		DVTVRYEAAKSSLEEAA
		AEKVRKYKHLETEVRHL
		TNAKDVTFV
		GFPLGARGKWHQDNFKL
		LTELGLSKSRQVKMAET
		FSTVALFS
		SVDIVHMFASRARKSMV
		M
Talpa	R2Toc	MLAPRSDRGNGFGDGPA	33488	TCTAGTTACAA	33395	TGACTGTTTA	33510	NW_
occid-		THPVPVNEIGQEPIDPD		CTGGGCATAG		GAGTAGGATT		023605038.1
entalis		PFLGGENC		CTGCAGAGAT		TTTTATTTGAT
		GLPLRLFGVSVGTQTSQ		CTCACCTCCT		ATTATGTATGT
		EDLTPIPTKLAVNELDV		CGTGGTCCCG		TTTATACCTTG
		LVNFSFEVY		CTGGTAAGCC		TACTTTGTTCA
		RSDLKGYVGGVHFPVNL		CTTAACAGGG		TTTATATTGTA
		EVLEGFPEVYEHLEPQP		TGACTAAGTA		TTGGGGGGAT
		CQGDNLD		GATCTCTGCC		TTTTTGTAGCA
		PSPPDDGVQVVLGREEG		CCAGTCAAGG		TGGGATTGTT
		KKEREGAPEALPPVQRG		AGCCGCTGGG		TTTATTGTATG
		HSEQVPD		AATCACCAGC		ACCTTTTTGAT
		DIVKVTVPDKNPPCPCC		CCAGCGATTC		ATTTTTAATAA
		STRLNSVLALIDHLKGS		CTTTCAAATTT		ACTAGACGGT
		HGKRRVCFR		AGGTGAAACA		AGCTATGGGG
		CAKCGRENFNHHSTVCH		AATTTCTCGG		GTTAGGGCAC
		FAKCKGPSEEKPPVGEW		TGTGGGTCGC		GCCACAAGCC
		ICEVCGR		AAGACTTACT		AGTTAGGGCG
		DFTTKIGLGQHKRLAHP		ACCTAAAACC		CTCATAGTGA
		MVRNQERIDASQPKETS		TGGCCCCACG		GTAGGGACAG
		NRGAHKKC		GTCTGACAGG		TAATTTTAATT
		WTKEEEELLARLEVQFE		GGCAACGGGT		CACAACGCGT
		GHKNINKLIAEHITTKT		TCGGAGAT		CAATTACCAT
		NKQISDKRRQ				CTGATTCGGA
		MTRKDKGEGGAAGKLGP				CCAATCTTAC
		DTGRGNHSQAKVGNNGL				CTGACTTGTA
		GGNQLP				CTAAGTTACC
		GGPAATKDKAGCHLDKE				GGATTTGTCC
		EGNRIAISQQKKGRLQG				CAGGTGGACG
		RYHKEIKR				GGCCACCTTT
		RLEEGVINTFTKAFKQL				ACTTAACCCG
		LECQEVQPLINKTAQDC				GAAAAGGAAC
		FGLLESACHI				ATGTATTTTAT
		RTALRGKNKKETQEKPT				ATATGTGTT
		GGQCLKWMKKRAVKKGN
		YLRFQRL
		FHLDRGKLARIILDDIE
		CLSCDIAPSEIYSVFKA
		RWETPGQFAGL
		GNFKSTGKADNKAFSDL
		ITAKEIKKNVQEMSKGS
		APGPDGIAI
		GDIKGMDPGYSRTAELF
		NLWLTSGEIPDMVRGCR
		TVLIPKSTQ
		PERLKDINNWRPITIGS
		ILLRLESRIITARMTKA
		CPLNPRQRGFI
		RAAGCSENLKLLQTIIR
		TAKSEHRPLGVVFVDIA
		KAFDTVSHQH
		ILHVLQQRGVDPHIIGL
		VSNMYKDISTFVTTKKD
		THTDKIQIRVG
		VKQGDPLSPLLFNLAMD
		PLLCKLEESGNGFHRGG
		HTITAMAF
		ADDLVLLSDSWENMEKN
		IEILEAFCDLTGLKTQG
		QKCHGFYIK
		PTKDSYTVNNCAAWTIY
		GTPLNMINPGDSEKYLG
		LQIDPWTGI
		ARSNISSKLDSWLERIN
		QAPLKPLQKLDILKTYT
		IPRLTYMVDH
		SEMKAGALEALDLQIRS
		AVKDWLHLPSCTCDAIL
		YVSTKDGGL
		GVTKLAGLIPSIQARRL
		HRIAQSPDETMKAFLDK
		EQMEKQYAK
		LWVQAGGKREKIPSIWD
		ALPTPVLLTTSDTLSEW
		EAPNPKSKY
		PRPCNWRRKEFEKWTKL
		QCQGRGIQNFKGDVISN
		NWIQNYR
		RIPHRKLLTAVQLRANV
		YPTREFLGRGRGDDCVK
		FCRHCEVD
		LETCGHIISYCPVTKEA
		RIKRHNRICERLIEEAE
		KKDWVVFKEP
		HIRDAVKELFKPDLIFV
		KEDRALVVDVTVRFEAT
		TTSLEEAAIEK
		VDKYKRLETEVRSLTNA
		KDVLFMGFPLGARGKWY
		QGNFKLLD
		MLGLSESRQVTVAKTLS
		TDALISSVDIVHMFASK
		ARKMNLVTV
Danio	R2Dr	MESTAKGKSYWMARRPV	33489	AATCCCCCCT	33499	AAATCCCAGC	33511	AB097126.1
rerio		EGATEGSLGRVPFVTRD		ACCCAATCCC		GGGATACAGC
		PKRKPEA		CCCGTCGTGA		AAGAAGGTAT
		KRTLTHGLGLRECSVVL		CCTCCAGGCC		CGGATCTAAT
		TRLIEGRRGRDHTPSGW		AGGAATCACG		AAGGTTGAGC
		NAQRGMP		AGCGTACGAC		GAGGAGAGG
		NDESSVEEPNGPIPSNP		AGTGGCCATC		GTGGAGATCC
		IPTGTQALPEPMADGEQ		CGGCAATGAC		TTTGGGGGGG
		GEHPGVVV		AATAGCGTGA		GTCGGGCTAA
		TLPLRDLNCPLCGGSAS		CTAACGACAA		GTTCCCCTCT
		TAVKVQRHLAFRHGTVP		TGAGTCAGAT		CGGGTCCTCC
		VRFSCESC		CCATGACCCT		CACGGTGACG
		GKTSPGCHSVLCHIPKC		TGGAGTGGGT		CTCTACCCCT
		RGPTGEPPEKVVKCEGC		TAACCTCCGC		CCCTCCTCGC
		SRTFGTRR		CTCTTTAAAAA		TCGTAGAACC
		ACSIHEMHVHSEIRNRK		C		CAACGGTGAA
		RIAQDRQEKGTSTDGEG				CACGGTTGGC
		RAGVERAD				AGGATGAAGT
		AGEGPSGEGIPPKRPRR				GACGTGAGGG
		ARTPREPSEPPANPPIL				GTAAGACATG
		SPQPDLPP				CGTACGTGAG
		GGLRDLLREVASGWVRA				CGCGCATTTT
		ARDGGTVIDSVLAAWLD				TGCTGTTCTC
		GNDRLPE				TGGACTGGGT
		LVDAATQRTLQGLPAGR				TTCGTCCCCC
		LARRPATFVAPNRRRGR				TCACAACCAT
		WGRRLKL				CACTTACACT
		LAKRRAYHDCQIRFRKD				ATAGGGGCAC
		PARLAANILDGKSETSC				AGCGGCTCCT
		PINEQAIHEH				ACCTCCCTCC
		FRNKWANPSPFGGLGRF				CTATGACCCC
		GTENRANNAHLLGPISK				CCCTTCCCAT
		SEVQTSLR				ACCGATCCAT
		NASNASTPGPDGVGKRD				GGCTGTTCTA
		ISNWDPECETLTQLFNM				GTCTGGACCG
		WWFTGVI				AGGGTCGGAC
		PSRLKKSRTVLLPKSSD				GGGGCATTTG
		PGAEMEIGNWRPITIGS				AAGGTAGCTG
		MVLRLFTRVI				GAATCCTCCG
		NTRLTEACPLHPRQRGF				CTGCTGCGAG
		RRSPGCSENLEVLECLL				CCTGAGGTCG
		RHSKEKRS				ATGGTTAGAG
		QLAVVFVDFAQAFDTVS				GTGAAATACT
		HEHMLSVLEQMNVDPHM				TGGGAGGAGA
		VNLIREIYT				CACAGCCTCC
		NSCTSVELGRKEGPDIP				GGAGAGCCCC
		VRVGVKQGDPLSPLLEN				TCCCGGGTGG
		LALDPLIQS				TCATCATGGC
		LERTGKGCEAEGHKVTA				AACCGGGTGA
		LAFADDLALVAGSWEGM				AACCTTACGG
		AHNLALV				TTTCACTTACG
		DEFCLTTGLTVQPKKCH				AAACAGCACC
		SFMVRPCRGAFTVNDCP				ATAACAGCGC
		PWVLGGK				CGTAATAGCG
		ALQLTNIENSIKYLGVK				CACCGGTGTG
		VNPWAGIEKPDLTVALD				ACTACTGTCC
		RWCKRIGKSL				AGTGCTGATA
		LKPSQKVYILNQFAIPR				TTCTCATCTG
		LFYLADHGGAGDVMLQN				GAGAATACAA
		LDGTIRKAV				CACGGGTAAT
		KKWLHLPPSTCNGLLYA				GGCAGAGTAT
		RNCNGGLGICKLTRHIP				TCAAAACCCA
		SMQARRMF				AATGTTTACG
		RLANSSDPLMKAMMRGS				ATCGACCAAC
		RVEQKFKKAWMRAGGEE				GGAGTCGTTC
		SALPRV				CCTTGCATCT
		FGANQYQEGEEVANDLV				AGGCCGGACC
		PRCPMPSDWRLEEFQHW				CGAAACTGCC
		MGLPIQ				GTAATTGCCC
		GVGIAGFFRNRVANGWL				GTCCCCAAGG
		RKPAGFKERHYIAALQL				TAGCCTCTTA
		RACVYPTL				GAAAACCGAA
		EFQQRGRSKAGAACRRC				GCCCGGTCG
		SSRLESSSHILGKCPAV				GGGCGGTGG
		QGARIRRH				TTGCGGCGGC
		NKICDLLKAEAETRGWE				GCTGCGGGG
		VRREWAFRTPAGELRRL				GCCTGCTGCT
		DLVLILGDE				CGGGCGGCG
		ALVIDVTVRYEFAPDTL				TCGGTGTGCC
		QNAGKDKVSYYGPHKEA				GCGGTGGTTG
		IARELGVRR				CGGTGGTGCG
		VDIHGFPLGARGLWLAS				GCGGGGATCT
		NSKVLELMGLSRERVKV				CGGTCCTTGC
		FSRLLSRR				GGTGCCGCTG
		VLLYSIDIMRTFYATLQ				TGCCGCCGCG
						GTCGCGTCGG
						TGGCGCTGGG
						GTGGTGGCCC
						GAGTGGCGTC
						GGCGTGCCAC
						TGCCCATAGT
						CGCCCGCGG
						ATCTGGAGGG
						GGGCGACCG
						ATCTGGAGGG
						GCGAGGGGG
						CTCGCGGGAC
						TTTAACGAGA
						AACGGAACGC
						AACTTCTCGC
						ATCGCTCCCG
						GGACTTTCCC
						CCCTCGTTCA
						GCCGAGGGAT
						GCCAAAAGGC
						ATGAAAGGTA
						AGTACCATAC
						CGGTCCGCAA
						AACTCTCTTCT
						GACTCGGTTC
						TCTGTTGGTTT
						TCTAGAGTAA
						CAACGAGGTG
						GAGGAGAGG
						GACATGGCAG
						GGACTCCCAT
						TCGTGCCAGC
						GGGTGGGGA
						CAGATCGAAG
						GAACGGTTCG
						AGGGCGTAAC
						AGACGAGAGG
						GAATCCGGTC
						ACACATTGAT
						GCCATGCCTA
						AATAGGCGAG
						GTTTGTATTTC
						TACTTTGTGG
						GTTCAGTATA
						GTCGGAGCAT
						ATGGTCGGTT
						GTCCCGTTGT
						TTTCACGGCG
						GGCAAGCGAC
						TATCATGATAA
						AGTAGAATGG
						GAGACGGGCT
						CCCTGACAAA
						CCCGGAAAGG
						CGCCCCCCCG
						TGGTTCGTAG
						CAGCTGACGG
						ATCACGCTCG
						AAGAAAAATG
						AGTGAGAGGG
						GACGCCGCAA
						CCAC
Oryzias	R2Ol	MGTDTVYVGQDYPSGLS	33490	CGCACAGGG	33500	GGGGGACAG	33512	LC349444.1
latipes		KRVPARLVAGPMLRERS		GACACAGAGC		CTGGGAGTCT
		CHAHVFR		CTGCCCAAGT		CGGCATGATT
		AGHMWNWRTSLPSGRWD		ACCGCTCCCG		ACAAATCTTG
		QPALEKSRVLTRSVATA		AGGGAGCGG		CGCTGCACTC
		TDPEITS		GAAACGGGG		GGATGTCGTC
		YPGKSVSTSTQVQEEDW		GGGTGACTAT		CCCGTGACGG
		CSRESGWISPGLAPEEP		CCCCTGGGGT		ACACATTAATC
		SVVSEITA		CCGGCGAGA		CGGAAAGCGA
		SMVATMRVATEEVVLEP		GCGCTGGTCT		GTGGTGACTC
		QPEQVVTILPEHGRNVP		ACGGACCAGG		GCCTCAAG
		PGLAEQDT		GGTGGCTGTG
		ASPIEVSVLLPDLAENC		GGCAGGCTGC
		PLCGVPSGGLRLLGKHF		TCCTCAGGCC
		AVRHAGVPV		AGTTGATTAG
		TYECRKCAWRSPNSHSI		TTACGCATGG
		SCHVPKCRGRARMPSGD		GCTGTACCTC
		PGIACDL		CACGTGGTCC
		CEARFATEVGVAQHKRH		CGCTGGTAAC
		VHPVEWNKVRLERRGAR		GACTTGTCGG
		GGGIKAT		CTAAATCAGC
		KLWSVAEVETLIRLIRE		CCGCCCACCA
		HGDSGATYQLIADELGR		TCTGGGATAT
		GKTAEQVRS		GGTTGACCGT
		KKRLLRIDTASNSPDDA		CTAACCCCAG
		EVEEERLESLAVRSSSR		TACTCAGGTC
		SPPSLVATR		ACAAACAAA
		VREAVARGESEGGEEIR
		AIAALIRDVDQNPCLIE
		TSASDIISKLG
		RRVDGPKRPRPVVREQT
		QEKGWVRRLARRKREYR
		EAQYLYS
		RDQARLAAQILDGAASQ
		ECALPVDQVYGAFREKW
		ETVGQFH
		GLGEFRTGARADNWEFY
		SPILAAEVKENLMRMAN
		GTAPGPD
		RISKKALLDWDPRGEQL
		ARLYTTWLIGGVIPRVE
		KECRTKLLP
		KSSDPVELQDIGGWRPV
		TIGSMVTRLFSRILTMR
		LTRACPINP
		RQRGFLASSSGCAENLL
		IFDEIVRRSRRDGGPLA
		VVFVDFARA
		FDSISHEHILCVLEEGG
		LDRHVIGLIRNSYVDCV
		TRVGCVEGM
		TPPIQMKVGVKQGDPMS
		PLLFNLAMDPLIHKLET
		AGTGLKWG
		DLSIATLAFADDLVLVS
		DSEEGMGRSLGILEKFC
		QLTGLRVQP
		RKCHGFFMDKGVVNGCG
		TWEICGSPIHMIPPGES
		VRYLGVQV
		GPGRGVMEPDLIPTVHT
		WIERISEAPLKPSQRMR
		VLNSFALPR
		IIYQADLGKVTVTKLAQ
		IDGIVRKAVKKWLHLSP
		STCNGLLYSR
		NRDGGLGLLKLERLIPS
		VRTKRIYRMSRSPDIWT
		RRMTSHSVS
		KSDWEMLWVQAGGERGS
		APVMGAVEAAPTDVERS
		PDYPDW
		RREENLAWSALRVQGVG
		ADQFRGDRTSSSWIAEP
		ASVGFAQ
		RHWLAALALRAGVYPTR
		EFLARGKEKSGAACRRC
		PARLESCS
		HILGQCPFVQANRIARH
		NKVCVLLATEAERFGWT
		VIREFRLED
		AAGGLKIPDLVCKKADT
		VLIVDVTVRYEMDGETL
		KRAASEKVK
		HYLPVGQQITDKVGGRC
		FKVMGFPVGARGKWPAS
		NNTVLAE
		LGVPAGRMRTFARLVSR
		RTLLYSLDILRDEMREP
		AGRGTRVA
		LIPAATGAAN

Example 8. Examination of R2Tg Activity

As R2Tg had the highest insertion activity, we continued to explore the programmability of this R2 system. The characterization of R2Tg enzymatic activities and payload flexibility at the 28S locus and a reprogrammed target in human cells were assessed (FIG. 30A-C, 32, 54A-C, 55A-C). We tested R2Tg for heterologous activity at the endogenous 28S locus by designing an EGFP payload flanked by the cognate R2Tg 5′ and 3′ UTRs and 28S homology arms. Co-transfection of this engineered payload together with wild-type R2Tg resulted in EGFP insertion into endogenous 28S loci, as determined by left and right PCR junctional analysis (FIG. 30A). To verify dependence on the retrotransposition mechanism, we introduced inactivating mutations in the RLE endonuclease domain (R2TgD1274A) and ZF domain (R2Tg_ZF2mut), and found these mutations ablated insertion activity (FIG. 30B). Alternatively, mutations introduced at catalytic residues in the RT domain (R2Tg_{D877A,D878A,D884A}) significantly reduced, but did not eliminate, insertion activity (FIG. 30B), which we confirmed by quantifying insertion events using next generation sequencing of targeting amplicons (NGS) (FIG. 30C). We validated these findings on inactivating mutations with our plasmid reporter assay by both luciferase production and editing by NGS (FIG. 30D and FIG. 31A-B). Based on NGS reads and gel electrophoresis readouts, all R2Tg insertions were found to be full length (FIG. 30), in contrast to observed partial insertions with R201 (Su et al., 2019 RNA. 25, 1432-1438). However, R2Tg integration was accompanied by indels at the 28S target, consistent with the previously observed non-templated addition of deoxycytidines by the RT domain (Bibillo, et al., 2004. Journal of Biological Chemistry. 279, pp. 14945-14953.) (FIG. 31C). To finely profile boundaries of functional domains, we tested N- and C-terminal truncations, finding that no C-terminal truncations were tolerated, likely due to loss of the RLE domain, whereas N-terminal truncations were tolerated up to the ZF motifs (FIG. 32). These results show the necessity of the ZF and RLE domains for activity and demonstrate that the N-terminal domain upstream of the ZF motifs does not critically contribute to the insertion process.

Having determined that the R2TgZF2mut mutant ablated integration, we speculated that supplementing additional DNA binding or nicking activity could rescue R2 integration activity at the 28S target site. We mutated Cas9 from Streptococcus pyogenes to generate either nickase (SpCas9^H840A) or dead (SpCas9D10A,H840A) variants and fused these Cas9 variants via an XTEN linker to the N-terminus of R2TgΔ1-184,ZF2mut, which contains both a truncation that retains activity (Δ1-184) and the inactivating ZF2 domain mutation (ZF2mut). We then designed Cas9 guides against the 28S target region and coupled these with the R2Tg variants (FIG. 33A). We found that both single guides and paired guides around the target site were able to recruit with SpCas9^H840A-R2TgZF2mut and restore integration at the locus, up to 72% of that of WT R2Tg (FIG. 33B-C). However, fusions of SpCas9D10A,H840A with the ZF2 mutant failed to restore activity, implying that the ZF2 binding was necessary for successful nicking (FIG. 34A). We also observed that SpCas9^H840A fusion to the RLE deficient mutant R2TgD1274A failed to rescue insertion activity, suggesting involvement of the native nicking of the RLE domain in insertion process, perhaps for second strand nicking or initiation of second strand synthesis (FIG. 34B). These observations held over a larger panel of mutations in the RT and RLE domains as well (FIG. 34C). Therefore, the insertion process involving the RLE and RT domains can be rescued when combined with SpCas9^H840A nickase, suggesting a possible route for evolutionary retargeting dependent on ZF evolution or reengineering via Cas9 supplementation.

Example 9. Identification of Factors Affecting Integration Dynamics

Given that the homology of the RNA template is a strong determinant of the target site, we probed the necessary homology for integration. We tested iterative truncations of either the 5′ or 3′ homology regions (FIG. 35A), finding that, while the 5′ region was sensitive to truncation and required a minimum homology between 20 and 40 nt, the 3′ region was robust to truncations, allowing efficient integration even in the absence of homology (FIG. 35B). This leniency may be due in part to promiscuous priming of the R2 systems, suggesting an avenue for evolving preferences to new loci via asymmetric acquisition of homology that would be compatible with Class 1 insertions observed in our computational exploration.

The malleable constraints of RNA cargo homology, especially at the 3′ end, prompted us to test cargo components. We next tested whether priming could occur internally to cargo, which would allow for successful integration after swapping the UTR and homology regions. Successful insertion from internal homology allows for scarless integration, with significant gene editing applications (FIG. 35C). We evaluated a panel of cargo permutations (FIG. 36A), swapping or duplicating homology elements to investigate whether internal homology could allow for template insertion. Moving homology internal to the UTR resulted in successful scarless insertions (FIG. 36C and FIG. 36B), as confirmed by sanger sequencing, suggesting flexible template priming. Traces of payloads with homology external to the 5′ UTR, had a loss of phasing at the 5′ junction due to multiple populations, and next generation sequencing confirmed these were due to non-templated addition of nucleotides, similar to what we observed at the wildtype 28S locus (FIG. 37A-B). Analysis of cross-junctional PCR products of insertion products with cargos having internal homology showed a reduction of size corresponding to a complete absence of the UTR region (FIG. 37C, FIG. 38A-D, FIG. 60, and FIG. 83A), confirming scarless insertion, the flexibility of the retrotransposon cargo architecture, and the efficiency of scarless integration especially with cargo 6 (FIG. 38C-D). Targeted integration with internal homology also required homology to the 28S region, as alternative homology to the A/AVS1 locus failed to show detectable insertion (FIG. 83B). These results highlight that priming off the template is very flexible, implying a very direct path for an expressed retrotransposon to acquire new priming sequences by landing in new areas via promiscuous priming of novel target sites or acquiring nearby targets and supporting both the Class 1 and Class 2 insertion mechanism.

While permutations of cargo components and complete removal of the 3′ UTR were tolerated, deletion of the 5′ UTR region resulted in significantly lower integration rates (FIG. 38A); as integration was not completely eliminated, this suggests that some element of the 28S homology region is still recognized by R2Tg for integration. All tested payloads also had some amount of residual indel formation (FIG. 37B, FIG. 38A-B). Overall, moving homology regions internal to the UTRs could produce scarless insertion at both 5′ and 3′ junctions while still maintaining efficient integration, especially with cargo 9 (FIG. 38A). These modifications also show that scar formation of R2Tg due to external homology is not a necessary component for the system to function and suggested the feasibility of acquiring new sites via similarity to internal regions.

Example 10. Programming of R2Tg for Integration at Specific Loci

We next programmed the R2Tg system to integrate at different loci by swapping target homologies (FIG. 39A). We designed scarless insertion payloads with homology arms to either A/AVS1 or NOLC1 loci and co-transfected these payloads with R2Tg in HEK293FT cells, finding targeted insertion at NOLC1 at ˜0.5%, which depended on the RLE catalytic residues of R2Tg, and no detectable insertion at A/AVS1 (FIG. 39B). To improve efficiencies of retargeting R2Tg at both loci, we tested whether SpCas9^H840A could improve insertion through additional nicking activity. We designed a pair of guide RNAs to introduce nicks on the bottom and top strands of the NOLC1 locus or a single guide RNA to introduce a nick at the A/AVS1 locus. We co-delivered these along with a cargo carrying transgene payloads, 5′ and 3′ R2Tg UTRs, and homology arms directed around the nicking site of 100 or 50 nt for A/AVS1 and NOLC1 respectively. We found that SpCas9^H840A-R2Tg fusion had increased efficiency at both NOLC1 (˜3%) and A/AVS1 (˜0.5%) (FIG. 39B), showing that SpCas9^H840A could significantly improve R2Tg insertion efficiency.

To find optimal payloads for efficient insertion at new loci, we designed a panel of payloads following integration guidelines that were effective at the 28S locus (FIG. 39C). Across designs, including internally located homology arms, deletion of the 3′ UTR, and truncation of the 5′ UTR, we progressively observed increased integration at the A/AVS1 locus up to 6% (FIG. 39D). Importantly, addition of the 5′ 28S homology arm upstream of the 5′ UTR in payloads 2, 6, and 7 substantially improved integration activity. Payloads 4-7 with homology arms directly flanking the insert had scarless insertion at the A/AVS1 locus, with minimal indel formation and perfect insertions representing more than 99% of the editing activity. The most truncated 5′ UTR sequence had the highest editing, suggesting that R2Tg recognition of the payload only required a small sequence region. As was expected from 28S payload characterization, the 3′ UTR was also dispensable for A/AVS1 insertion. We also examined whether insertion activity at the endogenous A/AVS1 locus could be optimized via better SpCas9^H840A fusions to different R2Tg truncations, again finding that C-terminal truncations were not tolerated, whereas the 1-184 residue truncation of R2Tg had maximum activity compared to other truncations while offering a more compact version of the SpCas9^H840A-R2Tg fusion (FIG. 40A-B). To test the dependence of these SpCas9^H840A-R2Tg fusions on the binding and nicking activities of SpCas9^H840A independently, we compared the SpCas9^H840A-R2Tg fusion to the dead SpCas9D10A,H840A-R2Tg fusion, as well as SpCas9^H840A and R2Tg delivered in trans as separate proteins. We found that, while the SpCas9D10A,H840A-R2Tg had no insertion activity, co-delivery of SpCas9^H840A and R2Tg was sufficient to efficiently insert the payload at both NOLC1 and A/AVS1 loci, showing that the nicking activity, but not binding activity, of SpCas9^H840A was enhancing insertion efficiency (FIG. 40C).

As some R2 retrotransposons have been proposed to function as a homodimer upon binding their cognate RNA templates (Yang et al., 1998. Mol. Cell. Biol. 18, 3455-3465), we were motivated to explore whether dual guides on opposing DNA strands might emulate dual nicking and recruitment of R2Tg and stimulate more efficient integration. Comparing single and dual guides, we found that certain paired guides achieved up to 15% integration with minimal indels generated and near perfect integration >99% using payloads with 100 nt of homology (FIG. 41A). Specific combinations of paired guides had low levels of integration with SpCas9^H840A alone, indicating some contribution from HDR mediated insertion of the payload off the DNA vector, and this effect was less prominent with single guides. Interestingly, top strand nicking guides, such as guide A4, could promote insertion, suggesting that the RLE domain of the R2Tg protein could initiate bottom strand nicking at the A/AVS1_target (FIG. 41A). To reduce HDR background, we tested payloads with homology arms reduced from 100 nt to 50 nt and found that these designs maintained insertion while blunting HDR byproducts (FIG. 41B). Surprisingly, while integration with SpCas9^H840A-R2Tg had minimal indel formation, SpCas9^H840A alone generated substantially more indels at the WT locus, indicating competition between complete integration and continued nicking and indel formation (FIG. 41A).

Example 11. Further Development and Integration of Re-Targeted Retrotransposons

We next determined whether diverse non-LTR retrotransposons could be repurposed for integration in cells despite failing at the 28S locus. To compensate for potentially ineffective binding or cleavage at the 28S locus in mammalian cells, we fused a panel of 11 additional retrotransposon candidates to SpCas9^H840A and tested them for additional guided insertion improvements at the 28S locus. We found that several of the retrotransposons, including many without activity at 28S target, had significant increases in 28S insertion when paired with targeting guides (FIG. 42A), including R2Bm, R2Ci, HeroDr, R10Mbr, R2Oi, and R2Tsp, whereas R2Tg and R2Mes did not have further increases, likely due to their already high natural integration.

We modified corresponding payloads for scarless insertion by rearranging homology regions internal to UTRs, and reprogrammed homology regions and SpCas9 guides to target payloads to the A/AVS1 locus (FIG. 58B). We found that our framework for retrotransposon retargeting generalized, with 6 out of 12 orthologs tested efficiently reprogrammed (FIG. 42B-D) showing efficient reprogramming, with minimal indel formation accompanying the insertion activity (FIGS. 58B and 68). R2Toc had a high efficiency of reprogramming with strongly reduced background 28S insertion (FIG. 29A).

Example 12. Site-specific Target-primed Insertion via Targeted CRISPR Homing of Retroelements (STITCHR)

After developing our SpCas9^H840A-R2Toc-based insertion system, which we refer to as Site-specific Target-primed Insertion via Targeted CRISPR Homing of Retroelements (STITCHR), we explored multiple applications for STITCHR-based programmable gene insertion in mammalian cells.

We first demonstrated STITCHR biochemically by purifying recombinant R2Toc protein and co-incubating it with SpCas9^H840A to reprogram RNA templated insertion at an A/AVS1 target. When R2Toc was assisted by SpCas9^H840A nicking using two sgRNAs, it had 9% insertion at the target site, compared to 1.5% insertion without SpCas9^H840A (FIG. 92A). As with R2Tg, R2Toc alone generated a unique cleavage pattern on the A/AVS1 target, indicating it also could be reprogrammed for cleavage and TPRT via its payload RNA. When STITCHR was examined in human cells, it could insert with 7% and 8% efficiencies at the NOLC1 and A/AVS1 targets, respectively. RLE and RT mutants eliminated insertion activity (FIG. 92B). As with R2Tg, the RT and RLE mutants complemented each other, as co-delivery of both mutants in trans rescued insertion activity (FIG. 92B-C). To ensure expression level differences of mutants and wild-type R2Tg and R2Toc proteins are not causing differences in integration efficiency, we measured the expression of each ortholog's wild-type and mutant versions, finding fairly uniform expression levels (FIG. 92D).

To generalize STITCHR reprogramming to other loci beyond A/AVS1, we targeted the NOLC1 and SERPINA1 loci with panels of single and dual guides, finding that dual guides integration efficiencies up to 13% and 10% insertion at NOLC1 and SERPINA1, respectively (FIG. 43A-D). While single guides had measurable integration activity, dual guides tended to better promote integration. Assaying the fidelity of insertion, we found low indel formation at these sites, indicating that integration was with high fidelity and that integration by SpCas9^H840A-R2Toc outcompeted indel formation, as indels were higher with SpCas9^H840A alone (FIG. 43A, 43D). Moreover, integration was scarless, as would be expected from our payloads with internal homology regions. While many dual guide combinations and single guides enabled STITCHR gene insertion, minimal editing was observed with SpCas9^H840A alone. Interestingly, single top strand nicking guides, in addition to bottom strand guides, could stimulate STITCHR insertion, suggesting R2Toc RLE domain participation in bottom strand nicking (FIG. 43A, 43C). Furthermore, we found that elimination of payload homology at NOLC1 or substitution for non-homologous sequences ablated editing (FIG. 44A). Conversely, increasing payload homology did not substantially increase integration efficiency, but led to higher background due to HDR (FIG. 44B). We also found that for high background loci, reducing payload homology could support gene integration with reduced HDR background, as observed at the A/AVS1 and SERPINA1 loci with SpCas9^H840A-R2Toc where we could achieve 8% gene integration with no background from HDR (FIG. 44C-D). Interestingly, we observed that R2Toc, like R2Tg, was also capable of programmable insertion without the assistance of Cas9 via the payload homology as the non-targeting guide conditions had 2% NOLC1, 1.3% A/AVS1, and 0.35% SERPINA1 insertion in HEK293FT cells (FIG. 43A-B, FIG. 44C, FIG. 71A) and up to 5% in HepG2 cells (FIG. 71A-C).

To take advantage of scarless genome insertion with R2Toc, we investigated whether we could place an EGFP tag in-frame to a protein target. We chose NOLC1 due to its distinct nuclear organization and designed our template in the reverse direction to prevent constitutive expression of the EGFP off the template cargo (FIG. 45A). We found that STITCHR-mediated GFP insertion led to NOLC1 tagging, as verified by confocal imaging and corresponding colocalization with immunofluorescence staining (FIG. 45B). We then explored additional payload flexibility of the STITCHR system at the A/AVS1 locus, using a panel of cargo sequences of different lengths. Evaluating various therapeutically relevant genes, including BTK, CEP290, HBB, HEXA, OTC, and PAH, we found insertion efficiencies of 10-20% at the A/AVS1 locus with minimal insertion using SpCas9^H840A alone (FIG. 45C-D, FIG. 74). These payloads varied in size between 0.7-7.7 kb, showing that STITCHR mediated insertion can insert a wide range of insert sizes.

Multiple types of genomic edits by STITCHR, including single base edits, small insertions, and a range of large payload insertions are enabled by the flexible nature of the retrotransposon insertion pathway (FIG. 59A-B, FIG. 61, FIG. 69A-C, FIG. 70A-C). We tested a panel of edits at the NOLC1 locus, spanning single base changes (transitions and transversions), small insertions (1-10 bp), therapeutic cargos, and large gene insertions. STITCHR could effectively install these diverse edit types (FIG. 59C), including therapeutically relevant genes of different sizes, such as BTK, CEP290, HBB, HEXA, OTC, and PAH, and synthetic sequences up to 10.9 kb. STITCHR also inserted these therapeutic genes at A/AVS1 (FIG. 45D). For cargos carrying small edits, we found both that extending the transcript beyond the homology arms further improved editing, presumably due to stabilization of the RNA transcript or better expression from the Pol II promoter (FIG. 59C), and U6 promoters could effectively produce smaller templates and genomic edits (FIG. 72). To combine STITCHR-mediated insertion with deletion, we tested simultaneous replacement of genomic regions by separating the homology target sites by 50-150 bp (FIG. 60A), which we refer to as STITCHR-replace. We found that an EGFP payload could be simultaneously inserted while replacing 50-150 bp of genomic sequence with 6-7% integration efficiency (FIG. 60B and FIG. 73).

Testing STITCHR in other cell types, we observed 4-10% integration at the NOLC1 and EMX1 loci in HepG2 cells and 12-30% gene integration at the A/AVS1, NOLC1, and EMX1 loci in Huh-7 cells (FIG. 71A-C).

To test STITCHR activity in a non-dividing context, we inhibited HDR using the cell cycling inhibitor aphidicolin, which traps cells at the G1/S phase transition and inhibits HDR activity. We found that STITCHR integration of an EGFP cargo at the NOLC1 locus was not inhibited by increasing concentrations of aphidicolin and led to increases in efficiency at intermediate aphidicolin concentrations (FIG. 46A). In contrast, HDR integration by SpCas9 nuclease at the EMX1 locus was inhibited by up to 94% by aphidicolin (FIG. 46B), and residual NOLC1 HDR insertion observed with SpCas9^H840A alone was eliminated at all tested aphidicolin concentrations, further demonstrating a retrotransposition and HDR-independent based mechanism for insertion (FIG. 46B). HDR insertion at NOLC1 of the GFP STITCHR template with 50 bp homology arms was inhibited up to 80% by aphidicolin (FIG. 46C). We also found that other cell cycle inhibitors, mirin and double thymidine similarly inhibited HDR but still maintained efficient editing with STITCHR at the A/AVS1 locus (FIG. 74).

To further apply STITCHR in non-dividing cells, an adenoviral delivery of the STITCHR machinery was designed and tested. Using one adenoviral vector carrying the STITCHR protein vector and a second vector expressing guides and template, STITCHR could achieve 6% integration in HEK293FT cells (FIG. 97A) and, in quiescent primary human hepatocytes, 0.5% gene integration was achieved (FIG. 97B). To further improve STITCHR efficiency, we eliminated potential re-nicking of the integrated sequence by removal of the PAM in the payload homology, resulting in a substantial increase in integration rate (FIG. 97C). To confirm that guide orientation was critical, we explored whether nicking the opposite strands using the D10A version of the SpCas9 nickase would still promote TPRT by STITCHR, finding that this substantially reduced integration efficiencies (FIG. 97D).

To extend STITCHR to multiplexed editing without reliance on cell division, we investigated whether STITCHR could mediate multiplexed integration at two different sites in the genome. We simultaneously delivered guide RNAs and cargos targeting the A/AVS1 and NOLC1 loci for Gluc and EGFP insertion, respectively, finding that multiplexed insertion was possible with 12% and 6% integration at the A/AVS1 and NOLC1 loci, respectively (FIG. 47A-B).

We also examined STITCHR in the context of a concurrent insertion/deletion approach. We compared a SpCas9^H840A-R2Toc using a single fixed guide RNA (N4, see table 7) to target the NOLC1 locus to that of the non-targeting, SpCas9^H840A alone. An EGFP insert was used as a payload. When homology arms on the payload template were separated by 0 bp, 50 bp, 100 bp, or 150 bp, we were successfully able to delete the genomic target while concurrently inserting the EGFP payload into the NOLC1 locus (FIG. 49). Exemplary sequences for the 50 bp deletion, 100 bp deletion, and 150 bp deletion, as well as the Guide sequence used for this experiment are found in Table 7.

TABLE 7
EGFP Cargo and Guide Sequences for Concurrent Insertion/Deletion
		SEQ ID	Guide	SEQ
Cargo	Sequence	NO:	Sequence	ID NO:
−50 deletion	atccttaatattcaatgaagcgcgggtaaacggcgggagtaactatgactc	33513	GACGCGTAT	33425
	tcttaaggtctagttacaactggTCCTGAGTCGTGCTGCGTCGAC		TGCCTGGAG
	AACGGTAGTGACGCGTATTGCCTGGAGGCCGCCACCATG		GA
	CCCGCCATGAAGATCGAGTGCCGCATCACCGGCACCCTG
	AACGGCGTGGAGTTCGAGCTGGTGGGCGGCGGAGAGG
	GCACCCCCGAGCAGGGCCGCATGACCAACAAGATGAAG
	AGCACCAAAGGCGCCCTGACCTTCAGCCCCTACCTGCTGA
	GCCACGTGATGGGCTACGGCTTCTACCACTTCGGCACCTA
	CCCCAGCGGCTACGAGAACCCCTTCCTGCACGCCATCAAC
	AACGGCGGCTACACCAACACCCGCATCGAGAAGTACGAG
	GACGGCGGCGTGCTGCACGTGAGCTTCAGCTACCGCTAC
	GAGGCCGGCCGCGTGATCGGCGACTTCAAGGTGGTGGG
	CACCGGCTTCCCCGAGGACAGCGTGATCTTCACCGACAA
	GATCATCCGCAGCAACGCCACCGTGGAGCACCTGCACCC
	CATGGGCGATAACGTGCTGGTGGGCAGCTTCGCCCGCAC
	CTTCAGCCTGCGCGACGGCGGCTACTACAGCTTCGTGGT
	GGACAGCCACATGCACTTCAAGAGCGCCATCCACCCCAG
	CATCCTGCAGAACGGGGGCCCCATGTTCGCCTTCCGCCG
	CGTGGAGGAGCTGCACAGCAACACCGAGCTGGGCATCG
	TGGAGTACCAGCACGCCTTCAAGACCCCCATCGCCTTCGC
	CAGATCTCGAGCTCGAGCTCGGCTTCCTGCGCGATAACC
	AACTCTCAGAGGTGGCCAATAAGTTCG
	atccttaatattcaatgaagcgcgggtaaacggcgggagt
−100	aactatgactctcttaaggtctagttacaactggTCCTGAGTCGTGCT	33514	GACGCGTAT	33425
deletion	GCGTCGACAACGGTAGTGACGCGTATTGCCTGGAGGCCG		TGCCTGGAG
	CCACCATGCCCGCCATGAAGATCGAGTGCCGCATCACCG		GA
	GCACCCTGAACGGCGTGGAGTTCGAGCTGGTGGGCGGC
	GGAGAGGGCACCCCCGAGCAGGGCCGCATGACCAACAA
	GATGAAGAGCACCAAAGGCGCCCTGACCTTCAGCCCCTA
	CCTGCTGAGCCACGTGATGGGCTACGGCTTCTACCACTTC
	GGCACCTACCCCAGCGGCTACGAGAACCCCTTCCTGCAC
	GCCATCAACAACGGCGGCTACACCAACACCCGCATCGAG
	AAGTACGAGGACGGCGGCGTGCTGCACGTGAGCTTCAG
	CTACCGCTACGAGGCCGGCCGCGTGATCGGCGACTTCAA
	GGTGGTGGGCACCGGCTTCCCCGAGGACAGCGTGATCTT
	CACCGACAAGATCATCCGCAGCAACGCCACCGTGGAGCA
	CCTGCACCCCATGGGCGATAACGTGCTGGTGGGCAGCTT
	CGCCCGCACCTTCAGCCTGCGCGACGGCGGCTACTACAG
	CTTCGTGGTGGACAGCCACATGCACTTCAAGAGCGCCAT
	CCACCCCAGCATCCTGCAGAACGGGGGCCCCATGTTCGC
	CTTCCGCCGCGTGGAGGAGCTGCACAGCAACACCGAGCT
	GGGCATCGTGGAGTACCAGCACGCCTTCAAGACCCCCAT
	CGCCTTCGCCAGATCTCGAGCTCGACCAAAGCGACAGGA
	GCTGTGAGTTCCGGGCTTGGGGCGGGGACCGGGCTG
−150	atccttaatattcaatgaagcgcgggtaaacggcgggagtaactatgactc	33515	GACGCGTAT	33425
deletion	tcttaaggtctagttacaactggTCCTGAGTCGTGCTGCGTCGAC		TGCCTGGAG
	AACGGTAGTGACGCGTATTGCCTGGAGGCCGCCACCATG		GA
	CCCGCCATGAAGATCGAGTGCCGCATCACCGGCACCCTG
	AACGGCGTGGAGTTCGAGCTGGTGGGCGGCGGAGAGG
	GCACCCCCGAGCAGGGCCGCATGACCAACAAGATGAAG
	AGCACCAAAGGCGCCCTGACCTTCAGCCCCTACCTGCTGA
	GCCACGTGATGGGCTACGGCTTCTACCACTTCGGCACCTA
	CCCCAGCGGCTACGAGAACCCCTTCCTGCACGCCATCAAC
	AACGGCGGCTACACCAACACCCGCATCGAGAAGTACGAG
	GACGGCGGCGTGCTGCACGTGAGCTTCAGCTACCGCTAC
	GAGGCCGGCCGCGTGATCGGCGACTTCAAGGTGGTGGG
	CACCGGCTTCCCCGAGGACAGCGTGATCTTCACCGACAA
	GATCATCCGCAGCAACGCCACCGTGGAGCACCTGCACCC
	CATGGGCGATAACGTGCTGGTGGGCAGCTTCGCCCGCAC
	CTTCAGCCTGCGCGACGGCGGCTACTACAGCTTCGTGGT
	GGACAGCCACATGCACTTCAAGAGCGCCATCCACCCCAG
	CATCCTGCAGAACGGGGGCCCCATGTTCGCCTTCCGCCG
	CGTGGAGGAGCTGCACAGCAACACCGAGCTGGGCATCG
	TGGAGTACCAGCACGCCTTCAAGACCCCCATCGCCTTCGC
	CAGATCTCGAGCTCGAAGATGACCACAAGGCTTCAGGCC
	CTGACGTGCTTAGGTTTCCAGGTGGGG

We further examined the possibility of using STITCHR to create single nucleotide edits and small nucleotide insertions. SpCas9^H840A-R2Toc was used with dual guides N4 and N8 (N8 Sequence: GGGA/ACCACGCGGCGA/ATGC (SEQ ID NO: 33429)) with a payload of either a GFP insert (FIG. 50A, columns 1-2,) a payload with a 1 bp mismatch to the NOLC1 locus (FIG. 50A, columns 3-8), or a payload with a small nucleotide insert (FIG. 50A, columns 9-14). When compared to the non-targeting SpCas9^H840A, the SpCas9^H840A-R2Toc system was able to make single base pair edits, as well as small nucleotide inserts (1-50 bp). We also examined the effect of the promoter in driving the STITCHR cargo (FIG. 50B). Use of the CAG promoter to express the cargo resulted in slightly higher editing levels, potentially due to higher expression of the template RNA sequence. Sequences used in these experiments are found at table 8.

The fidelity of STITCHR insert sequence was also assessed. We amplified the EGFP insert at the NOLC1 locus and performed long-read sequencing. Only a single amplicon species was detected. This amplicon aligned completely with a single, scarless TPRT insertion of EGFP at NOLC 1 (FIG. 78A). We also examined potential off-target insertions by linear amplification PCR and found that the majority of inserts occurred at the on-target site. The majority of off-target inserts occurred oat one of four sites on other chromosomes. (FIG. 78B).

TABLE 8
Cargo and Guide Sequences for Concurrent Single Nucleotide Edits and Small
Nucleotide Inserts
Cargo           Sequence
1 bp mismatch A attcaatgaagcgcgggtaaacggcgggagtaactatgactctcttaaggtctagttacaactggTCCTGAGTCG
                TGCTGCGTCGACAACGGTAGTGACGCGTATTGCCTGGAGGACGGACGCCGGCATTCGCC
                GCGTGGTTCCCAGCGACCTGTATCCCCTCGT (SEQ ID NO: 33516)
1 bp mismatch C attcaatgaagcgcgggtaaacgggggagtaactatgactctcttaaggtctagttacaactggTCCTGAGTCG
                TGCTGCGTCGACAACGGTAGTGACGCGTATTGCCTGGAGGCCGGACGCCGGCATTCGCC
                GCGTGGTTCCCAGCGACCTGTATCCCCTCGT (SEQ ID NO: 33517)
1 bp mismatch T attcaatgaagcgcgggtaaacggcgggagtaactatgactctcttaaggtctagttacaactggTCCTGAGTCG
                TGCTGCGTCGACAACGGTAGTGACGCGTATTGCCTGGAGGTCGGACGCCGGCATTCGCC
                GCGTGGTTCCCAGCGACCTGTATCCCCTCGT (SEQ ID NO: 33518)
1 bp insert     attcaatgaagcgcgggtaaacgggggagtaactatgactctcttaaggtctagttacaactggTCCTGAGTCG
                TGCTGCGTCGACAACGGTAGTGACGCGTATTGCCTGGAGGAGCGGACGCCGGCATTCGC
                CGCGTGGTTCCCAGCGACCTGTATCCCCTCGT (SEQ ID NO: 33519)
10 bp insert    attcaatgaagcgcgggtaaacggcgggagtaactatgactctcttaaggtctagttacaactggTCCTGAGTCG
                TGCTGCGTCGACAACGGTAGTGACGCGTATTGCCTGGAGGATGCCCGCCAGCGGACGCC
                GGCATTCGCCGCGTGGTTCCCAGCGACCTGTATCCCCTCGT (SEQ ID NO: 33520)
50 bp insert    attcaatgaagcgcgggtaaacggcgggagtaactatgactctcttaaggtctagttacaactggTCCTGAGTCG
                TGCTGCGTCGACAACGGTAGTGACGCGTATTGCCTGGAGGATGCCCGCCATGAAGATCG
                AGTGCCGCATCACCGGCACCCTGAACGGCGTGCGGACGCCGGCATTCGCCGCGTGGTTC
                CCAGCGACCTGTATCCCCTCGT (SEQ ID NO: 33521)
80 bp           attcaatgaagcgcgggtaaacggcgggagtaactatgactctcttaaggtctagttacaactggTCCTGAGTCG
                TGCTGCGTCGACAACGGTAGTGACGCGTATTGCCTGGAGGATGCCCGCCATGAAGATCG
                AGTGCCGCATCACCGGCACCCTGAACGGCGTGGAGTTCGAGCTGGTGGGCGGCGGAGA
                GGGGCGGACGCCGGCATTCGCCGCGTGGT (SEQ ID NO: 33522)

Example 13. Trans Delivery of Nuclease Activity

We next tested whether the nuclease activity of the genome editing system had to be provided in cis or if it could be provided in trans. An EGFP payload (with 50 nt homology arms) was used in conjunction with a SpCas9^H840A-R2Toc, in which Cas9 is fused to the R2Toc element, targeting the NOLC1 locus with dual N4 and N8 guides, and was compared to the non-targeting SpCas9^H840A. In addition, the nuclease activity conferred by SpCas9^H840A was also examined with separate (trans) expression of R2Toc (FIG. 51, columns 5-6). This non-linked SpCas9^H840A and R2Toc exhibited a payload insertion level similar to that of the fused system, SpCas9^H840A-R2Toc. When the nuclease activity was not supplemented with the non-LTR site specific retrotransposon element, little payload insertion was observed.

Example 14. Methods of the Examples

Mammalian Cell Culture

HEK293FT cells (ATCC) were cultured in Dulbecco's Modified Eagle Medium with 4.5 g/l glucose, sodium pyruvate, GlutaMAX (Thermo Fisher Scientific) and supplemented with 10% (v/v) fetal bovine serum (FBS) and 1× penicillin-streptomycin (Thermo Fisher Scientific). Cells were maintained below confluency at 37° C. and 5% CO₂

Cell Transfection, Genomic DNA Extraction and Purification

Cells were transfected in 96 well poly-D-Lysine plates (Corning) 16-24 h after plating at a confluency of 70% using Lipofectamine 3000 according to the manufacturer's protocol. In brief, 50 ng R2-expressing plasmid, 50 ng cargo plasmid, 50 ng reporter plasmid (optional) and 30 ng of sgRNA-expressing plasmids were transfected. 72 h post transfection, genomic DNA was isolated by removing media and adding 50 μl QuickExtract (Lucigen) per well. After a 5 min incubation at room temperature, the lysate was transferred to a 96 well PCR plate and incubated at 65° C. for 15 min, 68° C. for 15 min, and 98° C. for 10 min and used as input for targeted deep sequencing. Lysates were further purified using AMPure magnetic beads (Beckman Coulter) according to the manufacturer's protocol and eluted in 25 μL water, if used as input for ddPCR or NGS-based assays.

Editing Quantification by Next Generation Sequencing or ddCPR

Insertion efficiencies into plasmid and genomic DNA were quantified using a 3-primer assay. Here, a forward primer was combined with two reverse primers, one of which binds in the uninserted DNA and the other in inserted DNA. The forward and two reverse primers in a 2:1:1 ratio were added at a total combined concentration of 0.5 μM for a first round PCR counting 20 cycles. A second round PCR with 12 cycles added barcoded primers for Illumina NGS. The 28S, A/AVS1, and SERPINA1 experiments were quantified by 3 primer NGS for total integration and indel rates. For NOLC1, the 3-primer assay was used for analyzing indels associated with integration events and the WT locus. NOLC1 total integration was assayed by digital droplet PCR (ddPCR) as described below.

To quantify NOLC1 integration efficiency by digital droplet PCR, 24 μL solutions were prepared in a 96-well plate containing 1) 12 μL 2× ddPCR Supermix for Probes (Bio-Rad) 2) primers for amplification of the integration junction at 250 nM-900 nM, 3) FAM probe for detection of the integration junction amplicon at 250 nM 4) 1.44 μL RPP30 HEX reference mix (Bio-Rad) 5) 0.12 μL FastDigest restriction enzyme for degradation of primer off-targets (Thermo Fisher) and 6) Sample DNA at 1-10 ng/μL. The 20 μL of reaction mix was transferred to a Dg8 Cartridge (Bio-Rad) and loaded into a QX2000 droplet generator (Bio-Rad). 40 μL droplets suspended in ddPCR droplet reader oil were transferred to a new 96-well plate and thermocycled according to manufacturer's specifications. Lastly, the 96-well plate was transferred to a QX200 droplet reader (Bio-Rad) and the generated data were analyzed using Quantasoft Analysis Pro to quantify DNA editing.

Example 15. Cas9-Assisted Retrotransposon Insertion

We next tested whether R2Tg could be reprogrammed to target a nicked substrate, such as one cut with a Cas9 nickase, allowing TPRT off a template with corresponding homology (FIG. 56). To simulate R2Tg-based insertion into a “pre-nicked” substrate, we tested primer extension of an EGFP-coding payload RNA with homology to the human NOLC1 locus instead of the 28S target. Hybridization of either of two complementary primers to this NOLC1 payload resulted in primer extension when incubated with the R2Tg protein (FIG. 84A). While a universal primer, but not a NOLC1-specific primer, enabled extension of a 28S template, no extension was observed in the absence of an RNA template (FIG. 84A). These results suggest that the RT domain of the R2Tg protein is flexible and can extend RNA templates beyond its endogenous 28S sequence.

SpCas9^H840A has the potential to improve insertion through recruitment and supplementation of nicking activity (FIG. 56A). We next probed NOLC1 TPRT product formation. SpCas9^H840A was co-incubated in combination with R2Tg, an EGFP payload RNA with NOLC1 homology arms and 5′ and 3′ UTRs, dNTPs, a dsDNA NOLC1 target, and either single or paired guides. TPRT was more active with paired Cas9 guides and was dependent on magnesium, dNTP and an RNA template (FIG. 84B-C). Surprisingly, some TPRT products could be observed in the absence of Cas9 suggesting R2Tg to be capable of reprogrammed TPRT directed by the payload homology arms (FIG. 84B-C). Moreover, in the R2Tg only condition with SpCas9^H840A, a unique cleavage pattern could be observed, further confirming that the R2Tg protein can be reprogrammed for cleavage and TPRT (FIG. 84B).

To determine if modifying homology sites to a new locus could direct insertion to a different target in cells, we designed scarless insertion payloads with internal homology to the NOLC1 locus and co-transfected plasmids expressing these payloads with R2Tg into HEK293FT cells. Reprogrammed insertion was achieved at NOLC1 at ˜0.1% that was dependent on the RLE and RT domains (FIG. 85), demonstrating homology-guided retargeting of non-LTR retrotransposons.

To determine if SpCas9^H840A could further improve insertion through supplementation of nicking activity, a pair of guide RNAs was designed to introduce nicks on the bottom and top strands of NOLC1 and co-delivered these guides with a cargo carrying transgene payloads, a 5′ R2Tg UTR, and internal 50 nt homology arms placed around the nicking site at the NOLC1 locus. SpCas9^H840A-R2Tg fusion was found to have increased efficiency at NOLC1 (˜0.6%) (FIG. 56B) in a guide and RLE-dependent fashion, demonstrating that SpCas9^H840A can significantly improve R2Tg insertion efficiency.

A panel of payloads was designed to optimize payload design for efficient insertion at retargeted loci. The panel was designed to target the NOLC1 locus to expand upon our initial findings from R2Tg natural insertion at the 28S locus (FIG. 57). Payloads were designed with varying 5′ UTR sequences by panning 65 nt windows of the annotated 5′ UTR, including regions upstream containing the 5′ 28S homology region to navigate around a potentially relevant HDV-like cleavage site occurring in said region in R2Bm and R2Tg 5,29. Windows overlapping the distal 5′ UTR region and 28S homology region upstream of a 5′ target homology and payload sequence, either with or without a 3′ UTR region, were found to be necessary and sufficient for reprogrammed insertion at NOLC1 (FIG. 57). A truncated 28S-5′ UTR sequence improved insertion efficiency over the complete 5′ UTR and retained significant secondary structure, indicative of potential conserved function (FIG. 66A). Retargeting at the A/AVS1 locus followed similar rules, requiring the upstream 28S and a minimal 15 nt 5′ UTR for efficient gene integration quantified using a validated sequencing assay (FIG. 66B-C, FIG. 75). Efficient integration was observed at A/AVS1, with 15% efficiency using the Cas9-assisted nicking approach and 4% efficiency using the R2Tg protein alone (FIG. 86A-B). Similarly, NOLC1 biochemical integration was also improved using this updated payload design, with 6-8% integration efficiency (FIG. 86C) and insertion was homology specific with non-homologous templates showing no insertion activity (FIG. 86C). Integration efficiency was dependent on target concentration (FIG. 87A). Small RNA templates carrying a single base mismatch or insertion were still functional for TPRT, yielding efficiency in the 1-2% range on the NOLC1_target (FIG. 87B). Interestingly, on the A/AVS1 target, as observed with NOLC1 (FIG. 84), a unique cleavage pattern was observed in the R2Tg only condition, suggesting that R2Tg could be reprogrammed for cleavage and TPRT in absence of SpCas9^H840A. SpCas9^H840A stimulated a higher rate of TPRT than with R2Tg alone (FIG. 86). These results shows that the 28S sequence together with a truncated 5′ UTR sequence, can function as a bona fide 5′ UTR, and that the entire R2Tg 3′UTR can be dispensable for retargeted insertion.

Insertion activity was tested at the endogenous A/AVS1 locus using additSpCas9^H840A-R2Tg fusion proteins with different R2Tg protein truncations. C-terminal truncations were found to be not tolerated, whereas the 1-184 residue N-terminal truncation of R2Tg retained activity while offering a more compact version of the SpCas9^H840A-R2Tg fusion (FIG. 67A-B). To probe the mechanism of retargeted insertion at the NOLC1 and A/AVS1 loci by the SpCas9^H840A-R2Tg truncation fusion, insertion with the mutagenized variation of the RT domain was tested, confirming that integration was dependent on the RT activities of the retrotransposon and consistent with a TPRT based mechanism for programmable integration (FIG. 58A and FIG. 67C).

To probe the mechanism of retargeted insertion by the SpCas9^H840A-R2Tg^Δ184 fusion in cells, we tested insertion with the mutagenized variation of the RT domain and confirmed that integration was RT-dependent, consistent with a TPRT-based mechanism for programmable integration (FIG. 88A-C). We tested whether co-incubation of R2Tg RLE and RT mutants in trans would rescue TPRT. While protein mutants with either RLE or RT domain inactivated were TPRT-deficient, in combination the two mutants partly restored insertion activity at both the NOLC1 and A/AVS1 loci (FIG. 88A). We also found that the R2Tg^RTmut in trans with SpCas9^H840A is inactive, but that wild-type R2Tg in trans with SpCas9^H840A is active, allowing for insertion at the NOLC1 and A/AVS1 loci (FIG. 89A-B). This is consistent with TPRT as the mechanism of payload insertion by R2Tg.

Example 16: Target Primed Reverse Transcription (TPRT) with R2Tg

We examined the biochemical mechanism of retrotransposon activity by testing to analyze TPRT. Unless otherwise noted, in vitro TPRT reactions were performed in a total volume of 10 μL, containing 165 nM purified recombinant R2 protein, 90 ng DNA target, 300 ng RNA template, murine RNase inhibitor (1×, NEB) in a buffer containing 50 mM Tris-HCl (pH 8), 200 mM NaCl, 2 mM MgCl2, 5 mM DTT and 500 μM of each dNTP. For Cas9-nicking assisted reprogramming of TPRT to new sites, reactions were further supplemented with 400 nM final concentration SpCas9^H840A nickase (IDT) and 200 nM final concentration of each synthetic nicking guide RNA (Synthego). TPRT reactions were incubated at 37° C. for 1 hour, followed by 80° C. for 15 minutes. 1 ul (7U) of RNase A (Qiagen) was then added to each reaction and incubated for a further 15 minutes at 50° C. The reactions were purified on MinElute columns (Qiagen), separated by electrophoresis on Novex 6% TBE Gels (Invitrogen) and visualized by SYBR Gold staining (Invitrogen). Using a purified R2Tg protein and an in vitro transcribed RNA payload containing the Gluc coding sequence, 100 nt 5′ and 3′ arms homologous to the 28S DNA target and either with or without 5′ and 3′ R2Tg UTRs, we observed magnesium- and dNTP dependent TPRT (FIG. 79). Insertion occurred with DNA double strand cleavage of the 28S target (FIG. 79A). TPRT was sensitive to RNA payload design: in the absence of UTRs, insertion was significantly diminished but there was no decrease in target cleavage (FIG. 79B). Target cleavage was influenced by the complete absence of the payload RNA, which substantially reduced cleavage (FIG. 79A, B). TPRT efficiency was also highest with 100 nt homology arms on the payload and progressively decreased with 60 and 30 nt arms, whereas 0 nt arms completely eliminated TPRT (FIG. 80A). R2Tg was most active on RNA payloads containing a 5′ cap modification and TPRT was substantially lower on templates containing poly-A tails (FIG. 80B-D). TPRT product formation was also dependent on R2Tg concentration and accumulated over time (FIG. 81 A-C).

We also examined the dependence of first and second strand synthesis on TPRT. To accomplish this, the top and bottom strands of the DNA target were labeled with different fluorophores. Cleavage of both strands of the DNA target was observed (FIG. 82A), confirming that the larger bands observed in FIG. 79 were indeed motility-shifted target-primed products containing a large insertion. Cleavage of only the bottom strand was TPRT dependent.

R2Tg was mutated to determine the catalytic effects on TPRT. Both RLE (D1274A) and RT (A875-878, FADD) R2Tg mutants eliminated TPRT (FIG. 82B). To verify that the RLE R2Tg mutant was capable of isolated reverse transcription activity, we performed a primer extension assay consisting of the RNA payload pre-primed with a DNA primer. Comparable primer extension efficiency was observed between the wild-type and RLE mutant R2Tg proteins (FIG. 82C). Co-incubation with both the RT and RLE mutant proteins rescues TPRT activity (FIG. 82B), further indicating that the nuclease and reverse transcriptase activities of the R2Tg protein can function in trans.

TPRT activity was also examined in mammalian cells. We tested R2Tg for heterologous activity at the endogenous 28S locus by designing an EGFP payload flanked by the cognate R2Tg 5′ and 3′ UTRs and 28S homology arms. We found that co-transfection of this engineered payload together with wild-type R2Tg resulted in EGFP insertion into endogenous 28S loci, as determined by left and right PCR junctional analysis (FIG. 90A). These R2Tg insertions were primarily full length, in contrast to observed partial insertions with R20119. To verify dependence on the retrotransposition mechanism, we introduced inactivating mutations in the RLE endonuclease domain (R2TgD1274A, hereafter referred to as R2TgRLEmut), RT domain (R2TgF875A, A876L, D877A,D878A, L879A, V880A, L881A, hereafter referred to as R2TgRTmut), and ZF domain (R2TgZF2mut). R2TgRTmut residues for mutagenesis were selected on the basis of the widely conserved YXDD RT motif, consisting of YXDD residues and three additional conserved hydrophobic residues essential for catalytic activity of the RT26. Each of the three mutants were incapable of insertion activity alone (FIG. 90A), suggesting that each domain has a role in the observed TPRT activity. To further confirm that insertion was RT dependent, we engineered additional RT modifications, including deleting all 7 residues of the YXDD box (R2Tg^Δ(874-884) hereafter referred to as R2TgΔRTmut) and mutagenesis of the two aspartates in the FADD motif to positively charged residues, which has been shown to inactivate retroviral RTs27. We similarly observed that all these mutants significantly inhibited insertion activity of the R2Tg system, concordant with an RT-dependent insertion mechanism via TPRT (FIG. 90B-C).

Example 17: Mechanisms of Payload Insertion

We tested mammalian insertion with in vitro transcribed RNAs, as additional evidence that insertion is occurring via reverse transcription from an RNA template. We synthesized mRNA templates with a 5′ cap and variable length poly A tail modifications and delivered them to cells along with a plasmid coding for R2Tg and a plasmid reporter target; to account for transcriptional lag, mRNA was transfected one day after plasmid transfection. Analysis of both the 28S plasmid reporter target and the endogenous genomic 28S locus through junction-spanning PCR revealed bands corresponding to the correct insertion product in an RT- and RLE-dependent fashion (FIG. 91A). Accordingly, luciferase expression off the reporter target plasmid was observed (FIG. 91B). Taken together, this evidence shows that R2Tg is capable of inserting payloads by reverse transcription of RNA by its RT domain and nicking of target DNA by its RLE domain.

Example 18: STITCHR Templates

To better understand the RNA templated mechanism, we explored STITCHR's ability to use small synthetic RNA templates in cells extended off the SpCas9 sgRNA scaffold instead of a 5′ R2 UTR sequence, allowing for template recruitment via SpCas9. We first tested reconstitution of a 20 bp deletion in a luciferase reporter to restore luciferase activity. When delivering SpCas9^H840A-R2Toc or SpCas9^H840A-R2Tg as plasmid constructs a day before, a synthetic RNA template extended off a SpCas9 sgRNA scaffold sequence generated 3-5% editing, compared with 7% with the same payload expressed off of a plasmid (FIG. 93A-D). Editing efficiencies corresponded to measurable levels of protein production as measured by luciferase activity (FIG. 93B-D). We next explored targeting an endogenous site with a similar synthetic RNA template extended off a SpCas9 sgRNA scaffold and lacking a 5′ UTR sequence. Using this approach, we detected significant installation of a 38 bp sequence (FIG. 94A). We next tested an in vitro transcribed mRNA template containing an optimal 5′ UTR sequence for insertion of a ˜700 bp GFP ORF at the NOLC1 locus and found 0.5-2% insertion efficiency using the R2Toc STITCHR system in both HEK293FT and HUH-7 cells (FIG. 94B-D), with detectable PCR junction bands corresponding to correct insertion events (FIG. 95A). R2Tg-based STITCHR demonstrated similar 1-1.5% integration efficiency using an in vitro transcribed RNA templated delivered to cells (FIG. 95B).

Example 19: Insertion and Integration Via STITCHR

To corroborate our findings that STITCHR utilizes an RNA templated integration mechanism, we designed a GFP-containing mRNA template carrying an intron and quantified insertion of spliced payloads by sequencing. Using this readout, we quantified 0.02-0.1% integration of spliced templates from both R2Tg and R2Toc based STITCHR systems (FIG. 95C). These data suggest that, in line with previous findings, STITCHR is utilizing an RNA templated mechanism of insertion.

Next, we explored multiple applications for STITCHR-enabled programmable gene insertion in mammalian cells. We generalized STITCHR reprogramming to other loci, targeting A/AVS1, NOLC1, LMNB1, and EMX1 loci with panels of single and dual guides, which produced integration efficiencies between 5% to 11% (FIG. 96A, FIG. 59A). Insertion was RT and RLE domain dependent, with minimal integration from the RT mutant (SpCas9^H840A-R2TocRTmut), RLE mutant (SpCas9^H840A-R2TocD1210A) or SpCas9^H840A alone (FIG. 69, FIG. 70, FIG. 96), supporting a retrotransposon mediated insertion mechanism. The SpCas9^H840A-R2TocRTmut reduced integration activity by 75-97% across all the guides and genes tested, indicating the importance of the RT domain for TPRT and integration. A/AVS1 insertion was quantified by both next-generation sequencing and digital-droplet PCR, finding similar levels of integration efficiency (FIG. 96A-B; FIG. 59A). Indel formation was low across all sites, suggesting relatively high product purity of SpCas9^H840A-R2Toc-mediated insertions (FIG. 96A-B; FIG. 59A). In contrast, SpCas9-stimulated HDR using the same insertion template designs had reduced efficiency of integration relative to STITCHR and resulted in indel formation from 12%-30% (FIG. 96A; FIG. 59A).

While SpCas9^H840A controls showed that insertion with STITCHR required retrotransposon activity, an alternative mechanism for insertion could rely on reverse-transcription mediated ssDNA generation stimulating HDR, as has been demonstrated with retron-based systems in mammalian cells (Kong, X. et al. Protein Cell 12, 899-902 (2021); Zhao et al. CRISPR j. 5, 31-39 (2022)). To determine contributions from HDR, we evaluated the cell-cycle dependence of STITCHR, using the cell cycling inhibitor aphidicolin, which traps cells at the G1/S phase transition (Borel, F. et al. J. Cell Sci. 115, 2829-2838 (2002). We found that STITCHR integration of an EGFP cargo at the NOLC1 locus was not inhibited by increasing concentrations of aphidicolin, and instead increased efficiency at intermediate aphidicolin concentrations (FIG. 46A). In contrast, SpCas9-stimulated HDR at the EMX1 locus of a small insert was inhibited by up to 94% by aphidicolin (FIG. 46B) and HDR insertion at NOLC1 of the GFP STITCHR template with 50 bp homology arms was inhibited up to 80% by aphidicolin (FIG. 46C). We also found that other cell cycle inhibitors, mirin and double thymidine similarly inhibited HDR but still maintained efficient editing with STITCHR at the A/AVS1 locus (FIG. 74A-B).

Example 20: Cas9 Independent R2Tg Retargeting

To test retargeting of R2 substrate nicking via reprogramming of homology arms independent of Cas9, we incubated R2Tg with a novel target and corresponding homology-containing payloads in vitro and assayed for both cleavage and TPRT Unless otherwise noted, in vitro TPRT reactions were performed in a total volume of 10 μL, containing 165 nM purified recombinant R2 protein, 90 ng DNA target, 300 ng RNA template, murine RNase inhibitor (1×, NEB) in a buffer containing 50 mM Tris-HCl (pH 8), 200 mM NaCl, 2 mM MgCl2, 5 mM DTT and 500 μM of each dNTP. TPRT reactions were incubated at 37° C. for 1 hour, followed by 80° C. for 15 minutes. 1 ul (7U) of RNase A (Qiagen) was then added to each reaction and incubated for a further 15 minutes at 50° C. The reactions were purified on MinElute columns (Qiagen), separated by electrophoresis on Novex 6% TBE Gels (Invitrogen) and visualized by SYBR Gold staining (Invitrogen). We designed an A/AVS-targeting RNA payload with 100 nt homology arms and incubated it with a PCR target generated from the A/AVS1 human locus. We found that in the presence of the RNA payload, R2Tg, and dNTPs, TPRT could be observed, with weak cleavage bands corresponding to cleaved, but unintegrated DNA products. Removal of dNTPs eliminated TPRT and correspondingly increased cleavage products. In absence of R2Tg, the RNA payload, or Mg2+, we did not observe either cleavage or TPRT products (FIG. 98A New). To map the cleavage fragments, we ligated adaptors to free DNA ends and amplified ligated products with an adapter specific primer and a primer targeting either the 5′ or 3′ regions of the target. These products, which reveal 3′ and 5′ cleavage regions, respectively, were profiled with next-generation sequencing (FIG. 98B New). Comparing the cleavage locations between the no dNTP reaction and no RNA payload reaction, we found a majority of cleavage ends flanked homology arm positions, suggesting direct retargeting of R2Tg nicking by the RNA homology (FIG. 98C New). In the no RNA payload conditions, no appreciable cleavage could be observed with the majority reads corresponding to the 5′ or 3′ ends of the uncleaved target (FIG. 98C New). We also evaluated reprogramming R2Tg towards a NOLC1 target, finding that the R2Tg protein was capable of RNA-dependent cleavage and TPRT off of this novel DNA sequence and that the cleavage sites could similarly be mapped using a ligation based sequencing assay, with some of the cleavage sites originating around the homology arms (FIG. 99A-C).

Example 21: Delivery of R2 Element and Payload as RNA

This example will describe the results of experiments in which an R2 element is delivered as part of an RNA delivery system. The example further describes the results of experiments in which the genome editing system as a whole is delivered as RNA. Both modified R2 elements and unmodified R2 elements will be used. The modified R2 elements will be directly delivered as RNA or delivered as part of a delivery system designed to improve administration of the RNA. The integration efficiency of these R2 element enzymes delivered as RNA will be assessed as in previous examples.

Claims

1. A genome editing system comprising: i) an R2 element enzyme; andii) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5′ homology region, a 3′ homology region, and a protein binding element,wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome, andwherein the R2 element enzyme comprises a reverse transcriptase domain, and a nickase domain.

2. The genome editing system of claim 1, wherein the R2 element enzyme further comprises a targeting domain.

3. (canceled)

4. The genome editing system of claim 1, wherein the nucleic acid insertion into the genome is a DNA or RNA insertion template.

5. The genome editing system of claim 1, wherein the R2 element enzyme is a modified R2 element enzyme.

6. (canceled)

7. The genome editing system of claim 5, wherein the modified R2 element enzyme is modified by an N-terminal or C-terminal truncation of the R2 element enzyme sequence.

8. The genome editing system of claim 5, wherein the modified R2 element enzyme comprises a linker.

9. (canceled)

10. The genome editing system of claim 1, wherein the genome editing system targets a genomic locus.

11. The genome editing system of claim 1, wherein the genome editing system targets a genomic locus other than the 28S rRNA locus.

12. The genome editing system of claim 11, wherein an N-terminal zinc finger domain of the R2 element enzyme is modified to target a genomic locus other than the 28S rRNA locus.

13. The genome editing system of claim 11, wherein a non-naturally occurring targeting region is fused to the N-terminus of the R2 element enzyme or inserted into the R2 element enzyme.

14. The genome editing system of claim 5, wherein the modified R2 element enzyme is a fusion protein.

15. (canceled)

16. The genome editing system of claim 5, wherein the modified R2 element is fused to a Cas12 protein that is fully active, catalytically dead, or functioning as a nickase.

17. (canceled)

18. The genome editing system of claim 5, wherein the modified R2 element is fused to a TALEN protein, zinc finger protein, argonaute, or meganuclease protein.

19. (canceled)

20.-28. (canceled)

29. The genome editing system of claim 1, wherein the payload RNA further comprises a 5′ untranslated region (UTR), a 3′ UTR, or both a 5′ and a 3′ UTR, wherein the UTRs are truncated.

30. (cancel)

31. (cancel)

32. The genome editing system of claim 1, wherein the payload RNA further comprises a nuclear retention element.

33. The genome editing system of claim 1, wherein the payload RNA further comprises a Cas9 or Cas12 guide RNA, and wherein the Cas9 or Cas12 guide RNA comprises an extension with a 5′ homology sequence, a 3′ homology sequence, a 5′ untranslated region (UTR), a 3′ UTR, an insertion template, or any combination thereof.

34. (canceled)

35. The genome editing system of claim 1, wherein the R2 element enzyme comprises a nuclear localization signal (NLS).

36. (canceled)

37. A method of inserting a large nucleic acid into a genome within a cell using a Cas9 or Cas12 fusion protein, wherein the method comprises supplying a Cas9 or Cas12 fusion protein to a cell, wherein the Cas9 or Cas12 fusion protein is supplied with a payload RNA template, wherein the RNA template is reverse transcribed by the Cas9 or Cas12 fusion protein prior to being inserted into the genome of the cell; and wherein the large nucleic acid is inserted into the genome of the cell.

38.-46. (canceled)

47. A genome editing system comprising: i) a payload RNA, wherein the payload RNA comprises an insertion template and optionally one or more of a 5′ homology region, a 3′ homology region, and a protein binding element,wherein the insertion template comprises a sequence for a nucleic acid insertion into the genome;ii) a non-LTR site specific retrotransposon element enzyme; wherein the non-LTR site specific retrotransposon element enzyme comprises a reverse transcriptase domain and, optionally, a nuclease or nickase domain, andwherein if the non-LTR-site specific retrotransposon element enzyme does not comprise the optional nuclease or nickase domain, the genome editing system further comprisesiii) a nuclease or nickase enzyme.

48.-116. (canceled)