Patent Application
20240271164
Integration of transgenes into a genome often leads to low levels of gene expression or gene inactivation. Such events limit genetic strategies. Optimal genome sites for expressing transgenes are important in, for example, insect gene-drive control strategies, insect sterile-release control programs, transgenic plants (e.g., designed to express genes for insect control), human cell and gene therapies, and for expression of proteins important for industry, nutrition, and medicine. However, current methods for finding optimal genome sites and for transgene integration have limitations. New strategies are needed.
Certain embodiments of the invention provide a synthetic genomic safe harbor (sGSH) as described herein (e.g., a cargo-loaded sGSH comprising a complementation gene and a transgene; or a minimal, receiving sGSH comprising a complementation gene and a landing sequence capable of receiving one or more transgene(s) to be inserted). Certain embodiments of the invention provide a synthetic genomic safe harbor (sGSH) in a genome, the synthetic GSH comprising an exogenous fusion sequence that is inserted at the locus of an endogenous target gene of the genome, wherein the fusion sequence comprises:
Certain embodiments of the invention provide a synthetic genomic safe harbor (sGSH) in a genome, the synthetic GSH comprising an exogenous fusion sequence that is inserted at the locus of an endogenous target gene of the genome, wherein the fusion sequence comprises:
Certain embodiments of the invention provide a method of making a synthetic genomic safe harbor (sGSH) as described herein (e.g., a single step method to arrive at a cargo-loaded sGSH directly, or a method of making a receiving sGSH first, and then inserting a transgene into the landing sequence of the receiving sGSH).
In certain embodiments, a synthetic genomic safe harbor described herein is capable of matching the developmental, tissue, and/or cellular expression specificity of a transgene with that of the endogenous target gene or its neighboring gene(s). For example, a synthetic GSH may comprise expression cassettes or promoters capable of matching (temporally and spatially) the developmental, tissue, and/or cellular expression specificity of the transgene with that of the endogenous target gene/the rescued target gene. In certain embodiments, the sGSH comprises two different promoters that are similarly regulated. In certain embodiments, the sGSH comprises two promoters having 100% sequence identity to each other. In certain embodiments, the sGSH comprises one or two promoters having 100% sequence identity to the native promoter sequence of the endogenous target gene.
Certain embodiments of the invention provide a method of making a synthetic GSH in a genome, the method comprising:
In certain embodiments, the endogenous target gene is not an essential gene (inactivation of which may lead to severe or lethal fitness cost, such as infertility, etc.). In certain embodiments, the endogenous target gene is a non-essential gene (inactivation of which may lead to small or mild fitness cost, such as eye color change, or impaired pair mating etc.). As used herein, an “essential gene” is a gene that inactivation of which (homozygous loss) will result in lethality or stop an individual subject's reproduction and propagation. As used herein, an “non-essential gene” is a gene that inactivation of which (homozygous loss) will not result in lethality or stop an individual subject's reproduction and propagation.
In certain embodiments, the endogenous target gene has a simple structure (e.g., no intron, or only has 1, 2, or 3 short intron(s) of length<1 kb) and a simple regulatory mechanism, e.g., primarily or only regulated by transcriptional control and no alternative splicing.
Certain embodiments of the invention provide a method of delivering a gene of interest (transgene sequence) to a cell comprising a sGSH described herein, comprising inserting a sequence comprising a transgene sequence encoding the transgene product into a landing pad of the sGSH, wherein the sGSH comprises an exogenous fusion sequence comprising: (a) a landing sequence described herein, and (b) a complementation sequence comprising a rescue gene sequence that encodes the target gene product as described herein.
Certain embodiments of the invention provide a polynucleotide as described herein (e.g., comprising an exogenous fusion sequence described herein).
Certain embodiments of the invention provide a method as described herein (e.g., a genome editing method), including a method of delivering a gene of interest to a cell, the method comprising contacting the cell with polynucleotide as described herein.
Certain embodiments of the invention provide a method of genome editing in a cell, comprising inserting an exogenous fusion sequence at the locus of an endogenous target gene, wherein the insertion of the fusion sequence inactivates the endogenous target gene, wherein the fusion sequence comprises (a) a transgene, and (b) a complementation sequence comprising a nucleic acid sequence of the target gene and a promoter sequence for the target gene.
Certain embodiments of the invention provide a method as described herein.
Certain embodiments of the invention provide a nucleic acid sequence described herein (e.g., comprising an exogenous fusion sequence described herein).
Certain embodiments of the invention provide a vector described herein (e.g., comprising an exogenous fusion sequence described herein).
A major problem in contemporary approaches to gene editing in the medical and agricultural fields relates to the challenges in finding sites into the target organism genome in which cassettes containing beneficial gene(s) can be accurately inserted with no side effects or fitness costs to the individual. Such sites are called genomic safe harbors (GSHs). Certain representative criteria have been proposed in the past to identify GSH computationally, in particular, these putative GSHs should: for example, (1) be >50 kb from a transcriptional start site, (2) not disrupt a transcriptional unit, (3) be >300 kb from miRNAs, and among other considerations. Thus, use of transcriptional unit and coding regions is effectively banned in such computational methods to identify GSHs for inserting a transgene. In model organisms, GSHs have remained difficult or elusive to find due to the immense cost and time needed to construct the genomic resources (e.g., annotated genome, chromosomal level genome assembly, transcriptomes, or knowledge of chromatin accessibility) to perform GSH identification bioinformatically and the absence of cell culture lines (for many organisms) to allow large-scale automated screens.
A simple approach that bypasses these strategies is described herein to create synthetic genomic safe harbors in selected target genes themselves. In this manner, synthetic genomic safe harbor (referred to as synthetic GSH, or sGSH) can be made to allow the insertion of a gene cassette having transgene into virtually any suitable target gene using the target-on-demand (ToD) strategy described herein. Thus, a target gene could be transformed into a synthetic genomic safe harbor. For example, the chosen endogenous target gene could express a single RNA and be surrounded by transcriptionally active genes. These are simple criteria, and the resources are often in place even in non-model organisms. By avoiding costly screening and the need for cell culture platforms or genetically tagged libraries, ToD is a fast and efficient GSH discovery/creation tool that could revolutionize gene-editing and transgenic strategies in all organisms, having especially high impact on non-model organisms and biotechnology.
Thus, the synthetic GSH as described herein comprises 1) a complementation gene (also referred to as a rescue gene); and 2) a transgene, and/or a landing sequence into which a transgene could be inserted. Thus, the synthetic GSH comprises exogenous, recombinant sequence introduced into the edited genome.
In certain embodiments, a synthetic GSH comprises 1) a complementation gene (also referred to as a rescue gene); and 2) a landing sequence. This synthetic GSH does not yet comprise an inserted transgene sequence that encodes a transgene product; such a synthetic GSH is termed a “minimal synthetic GSH” or “receiving synthetic GSH” that is capable of receiving a transgene sequence or for insertion of a transgene sequence.
In other embodiments, a synthetic GSH comprises 1) a complementation gene (also referred to as a rescue gene); and 2) a transgene. Such a synthetic GSH comprising a transgene sequence that encodes a transgene product is termed “cargo-loaded synthetic GSH”.
In certain embodiments, a “receiving synthetic GSH” is introduced into a genome first, and a transgene sequence is then inserted to arrive at a “cargo-loaded synthetic GSH” comprising a transgene sequence.
However, in certain embodiments, introduction of “receiving synthetic GSH” into a genome is not necessary and bypassed, namely, a “cargo-loaded synthetic GSH” comprising an exogenous fusion sequence that comprises a complementation sequence and a transgene sequence may be inserted into the genome directly.
For example, a targeted nuclease such as CRISPR-Cas9 could specifically home to and cut at the genomic locus of an endogenous target gene. A synthetic GSH sequence could be installed into the targeted genomic site via homology directed repair (HDR) or nonhomologous end joining (NHEJ). During this process, the original transcriptional unit of the target gene is disrupted so that functional product would not be expressed from the now disrupted original genomic sequence. However, the successfully installed synthetic GSH at the locus could complement (i.e., rescue) the loss of target gene function. For example, a cargo-loaded synthetic GSH could not only express the transgene but also express the otherwise inactivated target gene, because the synthetic GSH sequence comprises: (a) a transgene sequence encoding the transgene product and (b) a complementation sequence comprising a sequence encoding the target gene product, facilitating expression of the transgene product without fitness cost to host cell thanks to the expression of the rescued target gene product.
As long as the introduced synthetic GSH in the edited genome is capable of facilitating expression of the transgene product, and rescue gene product (which is identical to the endogenous target gene product), the fitness cost from inserting the synthetic GSH into the target gene locus could be minimized or prevented. A variety of synthetic GSH embodiments capable of achieving such functional outcome are described herein.
Briefly, the cargo-loaded synthetic GSH comprises at least two genes (transgene gene sequence and rescue gene sequence) sequences that encode two products (transgene product and target gene product). In its simplest form of execution (smaller synthetic GSH construct), the rescue gene could be placed upstream of the transgene. Alternatively, the transgene could be placed upstream of the rescue gene, which may require delivery of the entire target gene promoter and cDNA. The two products could be two separate and distinct products, or the two products may be a target gene-transgene fusion protein. It is to be understood that in certain embodiments, the target gene's promoter is not proposed to drive the transgene so the two genes should be expressed under two separate promoters respectively; however, it is also possible to express two genes under a single promoter using an IRES (internal ribosomal entry site) sequence, or 2A peptide (e.g., T2A) encoding sequence in between the two genes sequences that encode the products (e.g., two small gene products and/or to save from using a second promoter of great length).
Accordingly, certain embodiments of the invention provide a synthetic genomic safe harbor (GSH) in a genome, and a method of making a synthetic genomic safe harbor in a genome. In certain embodiments, the genome is a mammalian genome, a plant genome, an insect genome, a fungal genome, an oomycete genome, or a bacterium genome
In certain embodiments, the insect genome is from an insect Bemisia tabaci or Homalodisca vitripennis, but the technology can be applied to any insect species amenable to gene editing.
In certain embodiments, the insect genome is a genome of an insect in the order Diptera, Lepidoptera, Coleoptera, Hemiptera, or Orthoptera. In certain embodiments, the insect genome is a genome of an insect in the Aleyrodidae family. In certain embodiments, the insect genome is a genome of a psyllid, sharpshooter, leafhopper, planthopper, aphid, Bagruda bug, Lygus bug, box elder bug, chili thrip, crape myrtle bark scale, four-lined plant bug, pink hibiscus mealybug, scale insect, cycad aulacaspis scales, or wax scales on holly.
Certain embodiments of the invention provide a synthetic genomic safe harbor (GSH) in a genome, the synthetic GSH comprising an exogenous fusion sequence that is inserted at the locus of an endogenous target gene, wherein the exogenous fusion sequence comprises:
Certain embodiments of the invention provide a synthetic genomic safe harbor (e.g., a cargo-loaded sGSH) in a genome, the synthetic GSH comprising an exogenous fusion sequence that is inserted at the locus of an endogenous target gene, wherein the exogenous fusion sequence comprises:
Certain embodiments of the invention provide a synthetic genomic safe harbor (e.g., a receiving sGSH) in a genome, the synthetic GSH comprising an exogenous fusion sequence that is inserted at the locus of an endogenous target gene of the genome, wherein the fusion sequence comprises:
The term “cutting sequence” refers to a nucleic acid sequence capable of being cut by a targeted nuclease, such as a Cas nuclease, and the nucleic acid sequence is not naturally present at the locus of the endogenous target gene. In certain embodiments, the cutting sequence is not naturally present throughout the entire original genomic sequence of the genome (e.g., no off-target effect when the cutting sequence is cut by a targeted nuclease). In certain embodiments, the cutting sequence is a unique sequence, wherein the locus of the endogenous target gene or the entire original genomic sequence of the genome has no sequence having 100% sequence identity to the cutting sequence. In certain embodiments, the locus of the endogenous target gene or the original genomic sequence of the genome has no sequence having at least 99% (e.g., at least 97%, 95%, 90%, 85%, 80%, or 75%) sequence identity to the cutting sequence. In certain embodiments, the locus of the endogenous target gene or the original genomic sequence of the genome has no sequence having at least 70% (e.g., at least 65%, 60%, 55%, or 50%) sequence identity to the cutting sequence.
In certain embodiments, the cutting sequence comprises a protospacer adjacent motif (PAM) site sequence, and a gRNA related sequence (so that a Cas nuclease could cut the cutting sequence).
In certain embodiments, the cutting sequence comprises a PAM sequence, and a gRNA related sequence, wherein the gRNA related sequence has a length of about 18-25 nt, 19-23 nt, or 20-22 nt (e.g., about 20 nt). In certain embodiments, the gRNA related sequence's first 6-7 nt adjacent to the PAM sequence is a unique sequence, the locus of the endogenous target gene or the entire original genomic sequence of the genome has no sequence having 100% sequence identity to it.
In certain embodiments, the gRNA related sequence is a unique sequence, wherein the locus of the endogenous target gene or the entire original genomic sequence of the genome has no sequence having 100% sequence identity to the gRNA related sequence. In certain embodiments, the locus of the endogenous target gene or the original genomic sequence of the genome has no sequence having at least 99% (e.g., at least 97%, 95%, 90%, 85%, 80%, or 75%) sequence identity to the gRNA related sequence. In certain embodiments, the locus of the endogenous target gene or the original genomic sequence of the genome has no sequence having at least 70% (e.g., at least 65%, 60%, 55%, or 50%) sequence identity to the gRNA-related sequence.
In certain embodiments, the cutting sequence (e.g., comprising PAM site sequence and gRNA-related sequence) has a length of about 19-32 nt. In certain embodiments, the cutting sequence has a length of about 20-29 nt. In certain embodiments, the cutting sequence has a length of about 20-28nt. In certain embodiments, the cutting sequence has a length of about 20-26 nt. In certain embodiments, the cutting sequence has a length of about 20-24 nt.
In certain embodiments, the cutting sequence has a GC content of about 40-60%. In certain embodiments, the cutting sequence has a GC content of about 45-55%. In certain embodiments, the cutting sequence has a GC content of about 50%.
In certain embodiments, the landing sequence comprises two or more unique cutting sequences (e.g., each unique cutting sequence is separated by at least about 100 bp filler sequence). The nature of the filler sequence is not important so long as the filler sequence is different from all unique cutting sequences that the filler sequence will not be cut by a targeted nuclease that cut at a cutting sequence. In certain embodiments, the filler sequence has a length of about 100-500 nt, 100-400 nt, 100-300 nt, or 100-250 nt. In certain embodiments, the filler sequence is not homologous to sequence at the locus of the endogenous target gene. In certain embodiments, the filler sequence is homologous to sequence at the locus of the endogenous target gene.
In certain embodiments, the landing sequence comprises one cutting sequence and one or two filler sequence(s) that separate the cutting sequence from other sequences on the exogenous fusion sequence (e.g., such as the rescue gene sequence, certain regulatory sequences, and/or homology arm sequence).
In certain embodiments, the landing sequence has a length of about 200-600 nt. In certain embodiments, the landing sequence has a length of about 300-550 nt. In certain embodiments, the landing sequence has a length of about 400-500 nt.
As used herein, the term “landing sequence” or “landing pad” refers to a nucleic acid sequence wherein a transgene sequence could be inserted into, and the nucleic acid sequence is not naturally present at the locus of the endogenous target gene. In certain embodiments, the landing sequence is not naturally present throughout the entire original genomic sequence of the genome. In certain embodiments, the landing sequence is a unique sequence, wherein the locus of the endogenous target gene or the entire original genomic sequence of the genome has no sequence having 100% sequence identity to the landing sequence. In certain embodiments, the locus of the endogenous target gene or the original genomic sequence of the genome has no sequence having at least 99% (e.g., at least 97%, 95%, 90%, 85%, 80%, or 75%) sequence identity to the landing sequence. In certain embodiments, the locus of the endogenous target gene or the original genomic sequence of the genome has no sequence having at least 70% (e.g., at least 65%, 60%, 55%, or 50%) sequence identity to the landing sequence. In certain embodiments, the landing sequence comprises one cutting sequence. In certain embodiments, the landing sequence comprises one or more (e.g., two or more) cutting sequences, and one or more filler sequences.
As used herein, the term “the locus of an endogenous target gene” refers to the genomic locus of the single expression cassette of regulatory sequences and encoding sequence for the endogenous target gene (no other gene product, or expression cassette of other gene product is included in this specific locus of the endogenous target gene). However, this specific locus of the endogenous target gene could be located in a genomic region with actively transcribed, neighboring gene(s). As used herein, the term “encoding sequence”, “sequence that encodes a product”, or “sequence encoding a product” refers to the encoding nucleic acid sequence, such as exon(s) sequences (e.g., cDNA), or exon(s) and intron(s) sequence that could be transcribed and processed into an RNA (e.g., mRNA). In certain embodiments, the encoding sequence is a full-length encoding sequence that encodes the entire product, for example, a full-length cDNA sequence that encodes the entire product.
In certain embodiments, the rescue gene sequence (e.g., full-length cDNA sequence) encodes the entire target gene product.
In certain embodiments, the rescue gene sequence comprises partial cDNA sequence fused to exon(s)/intron(s) sequence for the endogenous target gene (e.g., partial downstream cDNA sequence is fused to upstream exon(s)/intron(s)), wherein the rescue gene sequence encodes the entire target gene product.
In certain embodiments, the rescue gene sequence comprises full-length cDNA that comprises native encoding sequence of the endogenous target gene (i.e., a full-length cDNA having 100% sequence identity to the native exon sequence(s) of the endogenous target gene).
In certain embodiments, the rescue gene sequence comprises full-length cDNA that does not comprise an altered codon(s) relative to the native encoding sequence (such as exon sequence(s), or in mRNA) of the endogenous target gene.
In certain embodiments, the rescue gene sequence comprises full-length cDNA sequence having at least 98%, 99%, or 100% sequence identity to the native encoding sequence of the target gene.
In certain embodiments, the complementation sequence further comprises a promoter sequence for the target gene (i.e., the rescue gene), therefore, the complementation sequence may comprise the rescue gene sequence encoding the target gene product, and a promoter sequence for the rescue gene. In certain embodiments, the complementation sequence further comprises 5′UTR sequence and/or 3′ UTR sequence.
In certain embodiments, the cDNA could be recoded to minimize nucleic acid sequence identity with the endogenous target gene. In these cases, the protein derived from the recoded cDNA region is identical to the endogenous target gene protein.
In certain embodiments, the rescue gene sequence has a length that is shorter than the native sequence of the endogenous target gene (e.g., the rescue gene sequence lacking one or more, or all intron sequences of the endogenous target gene). In certain embodiments, the rescue gene sequence comprises one or more introns of the endogenous target gene but not all intron sequences of the endogenous target gene. In certain embodiments, the rescue gene sequence is missing at least one intron of the endogenous target gene. In certain embodiments, the rescue gene sequence does not comprise intron(s) of the endogenous target gene. In certain embodiments, the rescue gene sequence comprises the cDNA sequence of the endogenous target gene.
In certain embodiments, the rescue gene sequence has a length that is the same as the length of the native sequence of the endogenous target gene (e.g., preserving all intron sequences of the endogenous target gene).
In certain embodiments, the endogenous target gene does not comprise intron(s). In these cases, the rescue gene sequence has the same length as that of the endogenous target gene. In these cases, alternative regulatory sequence (e.g., 3′UTRs) and/or use of alternate codons may be used to minimize gene encoding sequence identity between the endogenous target gene and the rescue gene.
In certain embodiments, the exogenous fusion sequence comprises a promoter sequence.
In certain embodiments, the exogenous fusion sequence comprises a promoter for the target gene (i.e., the rescue gene). In certain embodiments, the exogenous fusion sequence further comprises a promoter sequence for the transgene.
Thus, in certain embodiments, the exogenous fusion sequence comprises a first promoter sequence and a second promoter sequence. In certain embodiments, the two separate promoter sequences comprise different nucleic acid sequences. In certain embodiments, the two separate promoter sequences both comprise the same nucleic acid sequence.
In certain embodiments, the promoter sequence for the rescue gene comprises the native promoter sequence for the endogenous target gene. For example, as shown in
In certain embodiments, the promoter sequence for the rescue gene comprises a non-native promoter sequence for the target gene. In certain embodiments, the non-native promoter comprises a viral promoter sequence. In certain embodiments, the non-native promoter is a viral promoter suitable for insects (e.g., a baculovirus promoter such as OpIE2). In certain embodiments, the non-native promoter is a viral promoter suitable for mammalian cells (e.g., a CMV promoter). In certain embodiments, the non-native promoter is a viral promoter suitable for plants (e.g., a cauliflower mosaic virus (CaMV) promoter such as CaMV35S). In certain embodiments, the non-native promoter is a promoter suitable for bacteria. In certain embodiments, the non-native promoter is a bacteriophage promoter (e.g., a T7 promoter). In certain embodiments, the promoter for the transgene is a promoter suitable for fungi or oomycete. In certain embodiments, the non-native promoter is a non-viral promoter (e.g., a protomer derived from a mammalian genome, a plant genome, an insect genome, a fungal genome, an oomycete genome, or a bacteria genome).
In certain embodiments, the promoter for the rescue gene is a constitutive promoter In certain embodiments, the promoter for the rescue gene is an inducible promoter. In certain embodiments, the promoter for the rescue gene is a tissue-specific promoter. In certain embodiments, the exogenous fusion sequence (that comprises or does not comprise a landing sequence) further comprises a promoter sequence for the transgene.
In certain embodiments, the exogenous fusion sequence further comprises an optional promoter sequence (e.g., that is downstream of the complementation sequence, and upstream of the landing sequence). Such optional promoter sequence might be suitable for driving expression of a transgene encoding sequence once the transgene encoding sequence is inserted into the landing sequence.
In certain embodiments, the promoter for the transgene is a constitutive promoter. In certain embodiments, the promoter for the transgene is an inducible promoter. In certain embodiments, the promoter for the transgene is a tissue-specific promoter.
In certain embodiments, the promoter sequence for the transgene comprises a viral promoter sequence. In certain embodiments, the promoter for the transgene is a viral promoter suitable for insects (e.g., a baculovirus promoter such as OpIE2). In certain embodiments, the promoter for the transgene is a viral promoter suitable for mammalian cells (e.g., a CMV promoter). In certain embodiments, the promoter for the transgene is a viral promoter suitable for plants (e g., a cauliflower mosaic virus (CaMV) promoter such as CaMV35S) In certain embodiments, the promoter for the transgene is a promoter suitable for bacteria. In certain embodiments, the promoter for the transgene is a bacteriophage promoter (e.g., a T7 promoter). In certain embodiments, the promoter for the transgene is a promoter suitable for fungi or oomycete. In certain embodiments, the promoter for the transgene is a non-viral promoter (e.g., a protomer derived from a mammalian genome, a plant genome, an insect genome, a fungi genome, an oomycete genome, or a bacteria genome).
In certain embodiments, the exogenous fusion sequence comprises one promoter sequence. In certain embodiments, the exogenous fusion sequence could drive transcription of an RNA and co-expression of both rescue gene product and transgene product from the RNA. In certain embodiments, the fusion sequence comprises an internal ribosomal entry site (IRES) sequence, or a 2A peptide (also referred to as 2A self-cleaving peptide, e.g., T2A, P2A, E2A, or F2A) encoding sequence placed between the complementation sequence and the transgene sequence. For example, in certain embodiments, rescue gene (upstream) and transgene (downstream) could be expressed under one promoter for the rescue gene, and the transgene sequence does not have its own separate promoter sequence. In certain embodiments, transgene (upstream) and rescue gene (downstream) could be expressed under one promoter for the transgene, and the rescue gene sequence does not have its own separate promoter sequence. Thus, in certain embodiments, the exogenous fusion sequence comprises one expression cassette comprising one promoter, and an IRES sequence or 2A peptide encoding sequence between two genes sequences. In certain embodiments, exogenous fusion sequence comprises 3′-regulatory sequence (e.g., 3′-UTR sequence) in the expression cassette. In certain embodiments, exogenous fusion sequence comprises 5′-regulatory sequence and/or 3′-regulatory sequence in the expression cassette. In certain embodiments, exogenous fusion sequence comprises 5′-UTR sequence and/or 3′-UTR sequence in the expression cassette In certain embodiments, the exogenous fusion sequence comprises two expression cassettes (two separate promoters for each of the two genes respectively, thus, one expression cassette for rescue gene product and another expression cassette for transgene product). In certain embodiments, exogenous fusion sequence further comprises 3′-regulatory sequence (e.g., 3′-UTR sequence) in each expression cassette. In certain embodiments, exogenous fusion sequence comprises 5′-regulatory sequence and/or 3′-regulatory sequence in each expression cassette. In certain embodiments, exogenous fusion sequence comprises (i) 5′-UTR sequence and/or 3′-UTR sequence in a first expression cassette (e.g., for rescue gene or for transgene), and (ii) 5′-UTR sequence and/or 3′-UTR sequence in a second expression cassette (e.g., for transgene or for rescue gene).
In certain embodiments, the exogenous fusion sequence comprises a first expression cassette capable of expressing rescue gene product (i.e., target gene product), and a second expression cassette capable of expressing transgene product.
In certain embodiments, the exogenous fusion sequence comprises a first expression cassette capable of expressing rescue gene product (i.e., target gene product), a second expression cassette capable of expressing a first transgene product and a third expression cassette capable of expressing a second transgene product.
In certain embodiments, the exogenous fusion sequence comprises a complementation sequence as described herein, a first transgene sequence encoding a first transgene product (e.g., Cas nuclease, or gRNA), and a second transgene sequence encoding a second transgene product (e.g., gRNA, or Cas nuclease). In certain embodiments, a transgene product is an sgRNA gene (U6:sgRNA), or a Cas9, or Cas9-t2A-dsRed gene, or another value added transgene such as one that encodes an enzyme for production of the chemical or protein of interest). Any of these could be added via an sgRNA specific for the landing pad site (landing sequence) adjacent to the rescue gene.
In certain embodiments, the exogenous fusion sequence comprises only one transgene sequence.
In certain embodiments, the exogenous fusion sequence does not comprise a transgene sequence that encodes a fluorescent protein product. In certain embodiments, the exogenous fusion sequence does not comprise a transgene sequence that encodes a gRNA product. In certain embodiments, the exogenous fusion sequence does not comprise a transgene sequence that encodes a Cas nuclease product. In certain embodiments, the exogenous fusion sequence does not comprise a transgene sequence that encodes a product selected from the group consisting of a fluorescent protein, a Cas nuclease, and a gRNA.
In certain embodiments, the exogenous fusion sequence comprises a promoter sequence capable of driving expression in a germline cell (e.g., an insect germline cell). In certain embodiments, the exogenous fusion sequence comprises a first promoter sequence and a second promoter sequence, both of which are capable of driving expression in a germline cell (e g., an insect germline cell).
In certain embodiments, the insect cell is from Bemisia tabaci and Homalodisca vitripennis, but the technology can be applied to any insect species amenable to gene editing. In certain embodiments, the insect cell is from an insect in the order Diptera, Lepidoptera, Coleoptera, Hemiptera, or Orthoptera. In certain embodiments, the insect cell is from an insect in the Aleyrodidae family. In certain embodiments, the insect cell is a cell of psyllid, sharpshooter, leafhopper, planthopper, aphid, Bagruda bug, Lygus bug, box elder bug, chili thrip, crape myrtle bark scale, four-lined plant bug, pink hibiscus mealybug, scale insect, cycad aulacaspis scales, or wax scales on holly. In certain embodiments, the insect cell is not a mosquito cell.
In certain embodiments, the exogenous fusion sequence, from 5′ to 3′, comprises a) the complementation sequence and b) the landing sequence, or the transgene sequence (i.e., the landing sequence or the transgene sequence is downstream of the complementation sequence). For example, in certain embodiments, the exogenous fusion sequence, from 5′ to 3′, comprises:
In certain embodiments, the exogenous fusion sequence, from 5′ to 3′, comprises:
In certain embodiments, the exogenous fusion sequence, from 5′ to 3′, comprises:
In certain embodiments, the exogenous fusion sequence, from 5′ to 3′, comprises a) the landing sequence, or the transgene sequence, and b) the complementation sequence (i.e., the landing sequence, or the transgene sequence is upstream of the complementation sequence). For example, in certain embodiments, the exogenous fusion sequence, from 5′ to 3′, comprises:
In certain embodiments, the exogenous fusion sequence, from 5′ to 3′, comprises:
In certain embodiments, the exogenous fusion sequence, from 5′ to 3′, comprises:
In certain embodiments, the complementation sequence comprises a promoter sequence for the rescue gene, wherein the promoter sequence is homologous to, or is the native promoter sequence for the endogenous target gene. Accordingly, for example, if a targeted nuclease cuts the original genome near or at the junction between native promoter sequence and encoding sequence of the endogenous target gene, the promoter sequence comprised within the exogenous fusion sequence could serve as upstream homology arm to facilitate integration. Therefore, the exogenous fusion sequence may already comprise a homologous sequence (e.g., promoter sequence (or a portion thereof) as upstream homology arm, or as a non-limiting example, a promoter sequence (or a portion thereof) and exon sequence (or a portion thereof) could together serve as upstream homology arm) in the complementation sequence.
In certain embodiments, the exogenous fusion sequence further comprises one or two flanking sequence that is homologous to sequence at the locus of the endogenous target gene. In certain embodiments, the one or two flanking sequence is at least 95%, 96%, 97%, 98%, 99%, or 100% homologous to sequence at the locus of the endogenous target gene described herein. In certain embodiments, the exogenous fusion sequence further comprises only one flanking sequence (e.g., the exogenous fusion sequence only comprises one 3′ downstream flanking homology arm sequence and does not comprise any upstream flanking sequence because the complementation sequence of the exogenous fusion sequence already has a promoter sequence that could serve as upstream homology arm).
In certain embodiments, the exogenous fusion sequence, from 5′ to 3′, comprises:
In certain embodiments, the 3′ flanking sequence is homologous to the encoding sequence and/or 3′ regulatory sequence at the locus of the endogenous target gene on the unedited genome. In certain embodiments, the 3′-flanking sequence is about 500 to 1000 nt in length. In certain embodiments, the 3′-flanking sequence is homologous to a downstream region of the endogenous target gene. In certain embodiments, the 3′-flanking sequence is homologous to the last exon. In certain embodiments, the 3′-flanking sequence is homologous to sequence downstream of the last exon. In certain embodiments, the 3′-flanking sequence is homologous to the 3′-regulatory sequence of the endogenous target gene.
In certain embodiments, the 3′-flanking sequence is homologous to exon 1, intron 1, or exon 1 and intron 1 of the endogenous target gene.
In certain embodiments, the exogenous fusion sequence may comprise two flanking sequences (e.g., see
Accordingly, in certain embodiments, the exogenous fusion sequence further comprises one or two flanking sequences that are homologous to sequences at the locus of the endogenous target gene. In certain embodiments, each flanking sequence independently has a length of about 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 nt. In certain embodiments, one or both flanking sequences independently have a length of about 100-2000 nt, 100-350 nt, 100-300 nt, 100-200 nt, 300-1200 nt, 500-1600 nt, 500-1000 nt, or 100-2000 nt. In certain embodiments, one or both flanking sequences have a length of about 100-1500 nt, 500-1000 nt, or 600-1000 nt. In certain embodiments, one or both flanking sequences have a length of about 500 nt or 1000 nt.
In certain embodiments, one or both flanking sequences are homologous to a segment of the target gene sequence. As a non-limiting example for illustration purpose, if an exemplary endogenous target gene comprises exon 1, intron 1, exon 2, and the target gene is cut by a targeted nuclease in the middle of intron 1, to facilitate integration, the exogenous fusion sequence of the synthetic GSH may comprise a first flanking sequence that is homologous to the upstream segment of the severed intron 1, and a second flanking sequence that is homologous to the downstream segment of the severed intron 1.
In certain embodiments, the first flanking sequence is homologous to a sequence that is 800-1000 nt upstream of the cut site, and the second flanking sequence is homologous to a sequence that is 800-1000 nt downstream of the cut site.
As another non-limiting example for illustration purpose, if a targeted nuclease cuts the target genome at the junction between regulatory sequence (e.g., promoter sequence or 5′ untranslated region sequence) and exon 1, to facilitate integration, the exogenous fusion sequence of the synthetic GSH may comprise a first flanking sequence (i.e., upstream flanking homology arm) that is homologous to the regulatory sequence (e.g., promoter sequence, and/or 5′-untranslated region sequence), and a second flanking sequence (i.e., downstream flanking homology arm) that is homologous to exon 1 sequence.
As another non-limiting example for illustration purpose, if a targeted nuclease cuts the target genome at the regulatory sequence (e.g., promoter sequence or 5′ untranslated region sequence), to facilitate integration, the fusion sequence of the synthetic GSH may comprise a first flanking sequence that is homologous to upstream segment of the severed regulatory sequence (e.g., promoter sequence, or 5′ untranslated region sequence), and a second flanking sequence that is homologous to the downstream segment of the severed regulatory sequence (e.g., promoter sequence, or 5′ untranslated region sequence).
In certain embodiments, the exogenous fusion sequence, from 5′ to 3′, comprises:
In certain embodiments, the exogenous fusion sequence, from 5′ to 3′, comprises:
In certain embodiments, the exogenous fusion sequence, from 5′ to 3′, comprises:
In certain embodiments, the exogenous fusion sequence, from 5′ to 3′, comprises the transgene sequence and the complementation sequence (i.e., the transgene sequence is upstream of the complementation sequence). For example, in certain embodiments, the exogenous fusion sequence, from 5′ to 3′, comprises:
In certain embodiments, the exogenous fusion sequence, from 5′ to 3′, comprises:
In certain embodiments, the exogenous fusion sequence, from 5′ to 3′, comprises:
As used herein, the term “original genomic sequence” or “native genomic sequence” refers to the untouched genomic sequence that is not edited or engineered by insertion of a synthetic GSH as described herein.
As used herein, the term “target gene” refers to an endogenous target gene in a genome that is suitable for insertion of a synthetic GSH as described herein. For example, in certain embodiments, the target gene encodes a protein. In certain embodiments, the target gene encodes an RNA that does not have alternatively spliced RNA isoforms. For example, in certain embodiments, the target gene encodes a single protein that does not have other isoforms derived from alternative splicing events. In certain embodiments, the target gene is in a transcriptionally active region of the genome. In certain embodiments, the target gene is located at a DNase I hypersensitive site (DHS) and/or open chromatin such as unmethylated region of the genome. In certain embodiments, the target gene is in a transcriptionally active region that contains two or more genes, for example, the target gene and its adjacent gene(s) are all in a transcriptionally active status. In certain embodiments, the target gene is a single-copy gene in the genome. In certain embodiments, the target gene encodes a non-coding RNA (e.g., miRNA or lncRNA). In certain embodiments, the target gene encodes a microRNA (miRNA). In certain embodiments, the target gene encodes a long non-coding RNA (lncRNA).
In certain embodiments, the synthetic GSH described herein is located within a cluster of genes on the genome. For example, in certain embodiments, the synthetic GSH may be inserted at the locus of one endogenous target gene without disrupting neighboring gene(s). In certain embodiments, the cluster comprises two or more genes (e.g., 2, 3, 4, 5, 6, 7, 8 or more). In certain embodiments, the cluster is in a transcriptionally active region of the genome. In certain embodiments, the cluster is part of a DNase I hypersensitive site (DHS) and/or unmethylated region of the genome. Methods of assessing DHS of the genome are known in the art, for example, as described in Wenfei Jin et al., Nature, volume 528, pages 142-146 (2015), which is incorporated by reference herein.
Certain conventional GSH may be preferably located at a region (e.g., intergenic region) that does not disrupt a transcriptional unit of the original genomic sequence. However, the synthetic GSH described herein could disrupt a transcriptional unit of the original genomic sequence due to insertion, nonetheless the fitness cost is reduced or eliminated by the inserted synthetic GSH.
Certain conventional GSH may be preferably located at a distance of greater than 50 kb from a transcriptional start site. However, the synthetic GSH described herein is inserted at the locus of an endogenous target gene (e.g., within a transcriptionally active region of genes). In certain embodiments, the synthetic GSH described herein is located within a distance of 50 kb from one or more transcriptional start sites. Namely, if the edited genome sequence having the synthetic GSH is aligned or superimposed with the unedited original genomic sequence, the synthetic GSH described herein can be located within a distance of 50 kb from one or more transcriptional start sites of the original genomic sequence. In certain embodiments, the 5′ end of the synthetic GSH is located within a distance of 50 kb from one or more transcriptional start sites of the original genomic sequence. In certain embodiments, the 3′ end of the synthetic GSH is located within a distance of 50 kb from one or more transcriptional start sites of the original genomic sequence. In certain embodiments, the entire length of the synthetic GSH is located within a distance of 50 kb from one or more transcriptional start sites of the original genomic sequence.
Likewise, certain conventional GSH may be preferably located at a distance of greater than 300 kb from a miRNA gene or at a distance of greater than 100 kb from a lncRNA gene. However, the synthetic GSH described herein could be located close to miRNA or lncRNA gene(s). For example, in certain embodiments, the synthetic GSH described herein is located within a distance of 300 kb, 100 kb, or 50 kb from miRNA or lncRNA gene(s). Namely, if the edited genome sequence having the synthetic GSH is aligned or superimposed with the original genomic sequence, the synthetic GSH described herein can be located within a distance of 300 kb, 100 kb, or 50 kb from miRNA or lncRNA gene(s) of the original genomic sequence. In certain embodiments, the 5′ end of the synthetic GSH is located within a distance of 300 kb, 100 kb, or 50 kb from miRNA or lncRNA gene(s) of the original genomic sequence. In certain embodiments, the 3′ end of the synthetic GSH is located within a distance of 300 kb, 100 kb, or 50 kb from miRNA or lncRNA gene(s) of the original genomic sequence. In certain embodiments, the entire length of the synthetic GSH is located within a distance of 300 kb, 100 kb, or 50 kb from miRNA or lncRNA gene(s) of the original genomic sequence.
As used herein, the term “transgene” refers to a gene that is not natively present at the locus of the endogenous target gene. In certain embodiments, the transgene is an exogenous gene (i.e., a non-native gene that is not present in the genome of the cell) In certain embodiments, the transgene encodes an exogenous protein. In certain embodiments, the transgene is an endogenous gene that is separate and distinct from the target gene (i.e., not an allele of the target gene), thus, the transgene could be ectopically installed at the locus of the target gene as part of the cargo-loaded synthetic GSH, or in the landing pad site (landing sequence) of the receiving synthetic GSH In certain embodiments, the transgene encodes an endogenous protein (e.g., an endogenous wildtype protein). For example, if a host cell has a deficient or mutant gene X on chromosome 1, and the locus of the chosen target gene Y for synthetic GSH insertion is located at chromosome 2 of the host cell, then the synthetic GSH may comprise rescue gene Y and wildtype gene X (WT gene X is the “gene of interest”/transgene to confer benefits to the host cell).
After insertion of synthetic GSH at the locus of the endogenous target gene on the genome, the synthetic GSH could be surrounded by residual vestige sequences of the endogenous target gene that are now separated by the inserted synthetic GSH. In certain embodiments, the synthetic GSH is inserted at an exon sequence of the endogenous target gene of the original genomic sequence. In certain embodiments, the synthetic GSH is inserted at an intron sequence of the endogenous target gene of the original genomic sequence. In certain embodiments, the synthetic GSH is inserted at an exon-intron junction of the endogenous target gene of the original genomic sequence. In certain embodiments, the synthetic GSH is inserted at a regulatory sequence (e.g., promoter or 5′ untranslated region (5′UTR)) of the endogenous target gene of the original genomic sequence. In certain embodiments, the synthetic GSH is inserted at a junction between a regulatory sequence (e.g., promoter or 5′ untranslated region (5′UTR)) and the encoding sequence (e.g., exon 1 and/or intron 1) of the endogenous target gene of the original genomic sequence. In certain embodiments, the synthetic GSH exogenous fusion sequence may be inserted immediately downstream of target gene's promoter and/or 5′-UTR sequence of the endogenous target gene of the original genomic sequence.
In certain embodiments, the exogenous fusion sequence comprises a first flanking sequence that is homologous to an exon. In certain embodiments, the exogenous fusion sequence comprises a second flanking sequence that is homologous to an exon.
In certain embodiments, the exogenous fusion sequence comprises a first flanking sequence that is homologous to an intron. In certain embodiments, the exogenous fusion sequence comprises a second flanking sequence that is homologous to an intron.
In certain embodiments, the exogenous fusion sequence comprises a first flanking sequence that is homologous to an exon. In certain embodiments, the exogenous fusion sequence comprises a second flanking sequence that is homologous to an intron.
In certain embodiments, the exogenous fusion sequence comprises a first flanking sequence that is homologous to a regulatory sequence (e.g., promoter sequence, and/or 5′-UTR sequence). In certain embodiments, the exogenous fusion sequence comprises a second flanking sequence that is homologous to a regulatory sequence (e.g., promoter sequence, and/or 5′-UTR sequence).
In certain embodiments, the exogenous fusion sequence comprises a first flanking sequence that is homologous to a regulatory sequence (e.g., promoter sequence, and/or 5′-UTR sequence). In certain embodiments, the exogenous fusion sequence comprises a second flanking sequence that is homologous to an exon and/or intron sequence.
In certain embodiments, the synthetic GSH exogenous fusion sequence has a length of about 1000, 2000, 3000, 4000, 5000, 6000, 7000, or 8000 nt. In certain embodiments, the synthetic GSH exogenous fusion sequence has a length of about 1000-8000 nt, 2000-8000 nt, 3000-8000 nt, 4000-8000 nt, 5000-8000 nt, 6000-8000 nt, or 7000-8000 nt. In certain embodiments, the synthetic GSH exogenous fusion sequence has a length of about 2000-7000 nt, 3000-7000 nt, 4000-7000 nt, 5000-7000 nt, or 6000-7000 nt. In certain embodiments, the synthetic GSH exogenous fusion sequence has a length of about 2000-6000 nt, 3000-6000 nt, 4000-6000 nt, or 5000-6000 nt. In certain embodiments, the synthetic GSH exogenous fusion sequence has a length of about 1000-5000 nt, 2000-5000 nt, 3000-5000 nt, or 4000-5000 nt.
The size of the minimal, receiving synthetic GSH comprising landing site is dependent on the size of the cDNA (variable with the chosen target gene) and the size of landing sequence having one or more unique sgRNA site. In certain embodiments, two homology arms are included on both ends of the exogenous fusion sequence. For example, in certain embodiments, 5′ homology arm is a sequence having about 1 kb of promoter sequence and 3′ homology arm is a sequence having about 1 kb of an exon, intron, or exon/intron boundary.
A cargo-loaded synthetic GSH can also be assembled without the landing site so that the cargo-loaded GSH comprising rescue gene and transgene(s) can be inserted directly in the genome at the same time.
In certain embodiments, the synthetic GSH is inserted via HDR. In certain embodiments, the synthetic GSH is inserted via nonhomologous end joining (NHEJ).
In certain embodiments, the genome is an insect genome. In certain embodiments, the genome is a bacterial genome. In certain embodiments, the genome is a fungal or oomycete genome. In certain embodiments, the genome is a plant genome. In certain embodiments, the genome is a mammalian genome.
In certain embodiments, the genome is a chromosomal genome. In certain embodiments, the genome is a plasmid genome.
In certain embodiments, the synthetic GSH is inserted into a genome of a cell. In certain embodiments, the synthetic GSH is inserted into a genome of an insect cell. In certain embodiments, the synthetic GSH is inserted into a genome of a mammalian cell. In certain embodiments, the synthetic GSH is inserted into a genome of a bacterial cell. In certain embodiments, the synthetic GSH is inserted into a genome of a fungal or oomycete cell. In certain embodiments, the synthetic GSH is inserted into a genome of a plant cell.
Certain embodiments of the invention provide a method of delivering a gene of interest to a cell, or a method of genome editing in a cell, or a method of introducing a synthetic GSH to a cell, the method comprising contacting the cell with a polynucleotide as described herein (e g., an exogenous fusion sequence as described herein).
Certain embodiments of the invention provide a method of making a synthetic GSH (e.g., a minimal receiving sGSH having landing sequence, or a cargo-loaded sGSH having transgene sequence) in a genome of a cell, contacting the cell with a polynucleotide as described herein.
It is possible to make a receiving sGSH first and convert it to a cargo-loaded sGSH by inserting transgene sequence into the landing sequence of receiving sGSH; alternatively, a cargo-loaded sGSH having transgene sequence can be directly made in the genome without making a receiving sGSH first.
Certain embodiments of the invention provide a method of making a synthetic GSH in a genome of a cell, the method comprising:
Certain embodiments of the invention provide a method of making a synthetic GSH in a genome of a cell, the method comprising:
Certain embodiments of the invention provide a method of delivering a gene of interest (transgene) to a cell comprising a sGSH described herein, comprising inserting a sequence comprising a transgene sequence encoding the transgene product into a landing sequence of the sGSH, wherein the sGSH comprises an exogenous fusion sequence comprising: (a) a landing sequence described herein, and (b) a complementation sequence comprising a rescue gene sequence that encodes the target gene product as described herein.
Certain embodiments of the invention provide a method of making a synthetic GSH (e.g., a minimal, receiving sGSH) in a genome of a cell, the method comprising:
In certain embodiments, a method described herein comprises converting the minimal, receiving sGSH into a cargo-loaded sGSH. For example, the method comprises inserting a second exogenous fusion sequence at the landing sequence, wherein the second fusion sequence comprises a transgene sequence encoding the transgene product.
In certain embodiments, the second fusion sequence further comprises regulatory sequences (e.g., promoter, 5′-UTR, and/or 3′-UTR) as described herein. In certain embodiments, the second fusion sequence comprises a promoter sequence for the transgene as described herein. In certain embodiments, the second fusion sequence comprises 5′-UTR, and/or 3′-UTR sequence(s).
In certain embodiments, the second fusion sequence further comprises two flanking sequences (homology arms upstream and downstream of the transgene sequence).
In certain embodiments, the second fusion sequence comprises a 5′-flanking sequence that is homologous to sequence at the minimal, receiving sGSH. In certain embodiments, the 5′-flanking sequence is homologous to the landing sequence (landing sequence segment upstream of the cutting sequence). In certain embodiments, the 5′-flanking sequence is homologous to a complementation sequence described herein. In certain embodiments, the 5′-flanking sequence is homologous to rescue gene sequence (e.g., last exon). In certain embodiments, the 5′-flanking sequence is homologous to a regulatory sequence, such as a 3′-UTR sequence or a promoter sequence in the minimal, receiving sGSH (e.g., the receiving sGSH may comprise a promoter sequence upstream of the landing sequence and downstream of the complementation sequence).
In certain embodiments, the second fusion sequence comprises a 3′-flanking sequence that is homologous to sequence at the minimal, receiving sGSH. In certain embodiments, the 3′-flanking sequence is homologous to the landing sequence (landing sequence segment downstream of the cutting sequence). In certain embodiments, the 3′-flanking sequence is homologous to endogenous target gene sequence (e.g., downstream segment of the endogenous target gene sequence such as last exon). In certain embodiments, the 3′-flanking sequence is homologous to a regulatory sequence, such as a 3′-UTR sequence of the endogenous target gene sequence.
In certain embodiments, the second fusion sequence comprises two or more transgene sequences encoding two or more transgene products.
As used herein, the term “inactivation of endogenous target gene” refers to the disruption of the transcriptional unit of the endogenous target gene and no intact/functional target gene product could be expressed from the original genomic sequence that encodes the target gene.
In certain embodiments, the complementation sequence is a complementation sequence as described herein.
In certain embodiments, the complementation sequence further comprises a promoter sequence for the rescue gene sequence.
In certain embodiments, the complementation sequence is capable of rescuing the inactivated endogenous target gene. In certain embodiments, the inactivated target gene is rescued by the rescue gene sequence (e.g., comprising full-length cDNA) that encodes the entire target gene product.
In certain embodiments, the method comprises delivering site-specific genome editing enzyme(s) (also referred to as targeted nuclease) to the cell (e.g., delivering CRISPR-Cas enzyme and/or guide RNA to the cell).
Targeted nucleases, and methods of delivery, are known in the art and described herein. In certain embodiment the targeted nuclease is a CRISPR-Cas nuclease (also referred to as a Cas nuclease). In certain embodiments, the Cas nuclease is a CRISPR-Cas9 nuclease or a CRISPR-Cas12a nuclease. In certain embodiments, the Cas9 nuclease is derived from S. pneumoniae, S. pyogenes, S. thermophiles, F. novicida, S. aureus, N. meningitidis, or C. jejuni Cas9, and may include mutations as a Cas9 variant (e.g., Cas9 D10A nickase). In some embodiments, the Cas9 nuclease is SpCas9, SaCas9, StCas9, NmeCas9, or CjCas9. In some embodiments, the Cas12a nuclease is derived from L. bacterium or Acidaminococcus sp. and may include mutations as a Cas12a variant. In some embodiments, the Cas12a nuclease is LpCpf1 or AsCpf1. In certain embodiments, the Cas nuclease is derived from Streptococcus pyogenes Cas9 (e.g., see NCBI Accession NO: WP_010922251).
A guide RNA (e.g., a single guide RNA (sgRNA)) confers target sequence specificity/selectivity for Cas nuclease. Specifically, the guide RNA (gRNA), designed to guide Cas nuclease to cut specific sequence at the locus of the endogenous target gene, complexes with the Cas nuclease and directs cutting at the desired site. gRNA design techniques are described herein and known in the art (see, e.g., U.S. Pat. Nos. 9,790,490; 9,840,702; 9,981,020; 10,106,820 and 10,240,145, which are incorporated by reference herein).
In certain embodiments, the targeted nuclease cuts the original genomic sequence or the landing sequence with a double-stranded DNA break (including blunt end or sticky end).
In certain embodiments, the targeted nuclease cuts the original genomic sequence or the landing sequence with a single-stranded DNA break (e.g., using a nickase).
In certain embodiments, the targeted nuclease cuts the original genomic sequence within an exon sequence of the endogenous target gene. In certain embodiments, the targeted nuclease cuts the original genomic sequence within an intron sequence of the endogenous target gene. In certain embodiments, the targeted nuclease cuts the original genomic sequence at an exon-intron junction of the endogenous target gene. In certain embodiments, the targeted nuclease cuts the original genomic sequence at a regulatory sequence (e.g., promoter or 5′ untranslated region (5′UTR)) of the endogenous target gene. In certain embodiments, the targeted nuclease cuts the original genomic sequence at a junction between a regulatory sequence (e.g., promoter or 5′ untranslated region (5′UTR)) and encoding sequence of the target gene.
In certain embodiments, the method comprises delivering an exogenous fusion sequence described herein to a cell (e.g., a cell having unedited original genome, or a cell having a minimal, receiving sGSH).
In certain embodiments, the method comprises delivering a first exogenous fusion sequence described herein to a cell (e.g., a cell having unedited, original genome). In certain embodiments, the method comprises delivering an exogenous fusion sequence (e.g., a second exogenous fusion sequence) described herein to a cell (e.g., a cell having the receiving sGSH in the genome). In certain embodiments, an exogenous fusion sequence described herein is delivered as single-stranded DNA (ssDNA). In certain embodiments, an exogenous fusion sequence described herein is delivered as double-stranded DNA dsDNA. In certain embodiments, the method comprises delivering a vector (e.g., a plasmid) comprising an exogenous fusion sequence as described herein to the cell. In certain embodiments, the vector (e.g., a plasmid) comprising one or two gRNA sequence(s) that flank the synthetic GSH exogenous fusion sequence as described herein, so that targeted nuclease could cut the gRNA sequence(s) on the vector to release the synthetic GSH exogenous fusion sequence and/or to linearize the vector. In certain embodiments, the method comprises delivering a first vector described herein to a cell (e.g., a cell having unedited, original genome). In certain embodiments, the method comprises delivering a vector described herein (e.g., a second vector) to a cell (e.g., a cell having the receiving sGSH in the genome). In certain embodiments, the method comprises delivering a linearized vector described herein.
In certain embodiments, the method comprises delivering a first targeted nuclease (e.g., a first Cas nuclease/gRNA) described herein to a cell (e.g., a cell having unedited, original genome). In certain embodiments, the method comprises delivering a targeted nuclease (e.g., a second Cas nuclease/gRNA) described herein to a cell (e.g., a cell having the receiving sGSH in the genome).
In certain embodiments, the chosen endogenous target gene has a gRNA sequence that is absent on the synthetic GSH exogenous fusion sequence. For example, the chosen endogenous target gene may have a gRNA sequence at an intron, and the complementation sequence comprises a cDNA sequence for the target gene and therefore does not comprise the intronic sequence targeted by the gRNA/Cas nuclease.
Additionally, the chosen endogenous target gene may have a gRNA sequence at an exon, and the complementation sequence comprises a cDNA sequence comprising alternate codons for the target gene and does not comprise the original exon sequence targeted by the gRNA/Cas nuclease, as long as the complementation sequence comprise a sequence capable of encoding the same target gene product. For example, in certain embodiments, the complementation sequence comprises a rescue gene encoding sequence (e.g., exon(s) and intron(s)) having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the native encoding sequence for the endogenous target gene. In certain embodiments, the complementation sequence comprises a cDNA sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the native encoding sequence (such as exon sequence(s), or in mRNA) for the endogenous target gene.
Similarly, the chosen endogenous target gene may have a gRNA sequence at the regulatory sequence (e.g., promoter and/or 5′ UTR), and the complementation sequence may comprise a modified regulatory sequence (e.g., promoter and/or 5′ UTR) that lacks the gRNA sequence targeted by gRNA/Cas nuclease. For example, in certain embodiments, the complementation sequence comprises a promoter sequence (for the rescue gene) having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the native promoter sequence for the endogenous target gene. In certain embodiments, the complementation sequence comprises a 5′ UTR sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the native 5′ UTR sequence for the endogenous target gene.
Delivering targeted nuclease and delivering exogenous fusion sequence can be concurrent or sequential. In certain embodiments, delivering targeted nuclease is followed by delivering exogenous fusion sequence. In certain embodiments, delivering exogenous fusion sequence is followed by delivering targeted nuclease.
Deliveries of protein, nucleic acids, complex thereof, and/or vectors into cells are known in the art and are described herein. Targeted nucleases, gRNA, and/or exogenous fusion sequence can be introduced into a cell via lipid-mediated transfection (e.g., cationic lipid), polymer-mediated transfection (e.g., PEG), liposome, nanoparticle, electroporation, microinjection or any suitable methods such as deterministic mechanoporation (DMP) (Nano Lett. 2020 Feb 12; 20(2):860-867). Targeted nucleases can be delivered via intracellular delivery/expression of a vector comprising a nucleic acid encoding the targeted nuclease and/or gRNA. Alternatively, targeted nucleases can be delivered as a protein via intracellular or intranuclear delivery. In certain embodiments, targeted nucleases can be delivered as pre-assembled ribonucleoprotein particles (RNPs) into a cell. For example, Cas nuclease can be mixed with gRNA to form pre-assembled RNPs prior to delivery into a cell.
In certain embodiments, the synthetic GSH is inserted into the genome via homology directed repair (HDR). In certain embodiments, an unedited, original genome is edited into the genome comprising a sGSH by one HDR event with a delivered exogenous fusion sequence or vector described herein. In certain embodiments, a genome having a minimal, receiving sGSH is converted into a genome comprising a cargo-loaded sGSH by one HDR event with a delivered exogenous fusion sequence or vector described herein. In certain embodiments, the synthetic GSH is inserted into the genome via non-homologous end joining (NHEJ).
In certain embodiments, the first exogenous fusion sequence further comprises one or two flanking sequence(s) that are homologous to sequence(s) at the locus of the endogenous target gene. In certain embodiments, the first exogenous fusion sequence does not comprise flanking sequence that is homologous to sequences at the locus of the endogenous target gene.
In certain embodiments, the present invention provides a cell having a genome comprising the synthetic GSH as described herein (e.g., a receiving sGSH having landing sequence, or a cargo-loaded sGSH having transgene). In certain embodiments, the cell is a prokaryotic cell (e.g., a bacterial cell). In certain embodiments, the cell is a fungal or oomycete cell. In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the cell is a plant cell. In certain embodiments, the cell is an insect cell. In certain embodiments, the cell is a non-mammalian animal cell (e.g., a fish cell). In certain embodiments, the cell is a mammalian cell (e.g., a mouse, rat, dog, cat, monkey, rabbit, hamster, horse, cow, sheep, pig, goat, or camelids cell). In certain embodiments, the cell is a human cell.
In certain embodiments, the present invention provides a non-human organism having a genome comprising the synthetic GSH as described herein (e.g., a receiving sGSH having landing sequence, or a cargo-loaded sGSH having transgene).
In certain embodiments, the organism is a prokaryotic organism (e.g., a bacterium). In certain embodiments, the organism is a fungal or oomycete organism. In certain embodiments, the organism is a eukaryotic organism. In certain embodiments, the organism is a plant. In certain embodiments, the organism is an insect. In certain embodiments, the insect organism is Bemisia tabaci and Homalodisca vitripennis, but the technology can be applied to any insect species amenable to gene editing.
In certain embodiments, the insect organism is from the order Diptera, Lepidoptera, Coleoptera, Hemiptera, or Orthoptera. In certain embodiments, the insect organism is from the Aleyrodidae family. In certain embodiments, the insect organism is a psyllid, sharpshooter, leafhopper, planthopper, aphid, Bagruda bug, Lygus bug, box elder bug, chili thrip, crape myrtle bark scale, four-lined plant bug, pink hibiscus mealybug, scale insect, cycad aulacaspis scales, or wax scales on holly. In certain embodiments, the insect is not a mosquito.
In certain embodiments, the organism is a non-mammalian organism (e.g., a fish). In certain embodiments, the organism is a mammalian organism (e.g., a mouse, rat, dog, cat, monkey, rabbit, hamster, horse, cow, sheep, pig, goat, or camelids cell). In certain embodiments, the organism is a non-human organism.
The term “nucleic acid” and “polynucleotide” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base which is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues. A “nucleic acid fragment” is a fraction of a given nucleic acid molecule. Deoxyribonucleic acid (DNA) in the majority of organisms is the genetic material while ribonucleic acid (RNA) is involved in the transfer of information contained within DNA into proteins. The term “nucleotide sequence” refers to a polymer of DNA or RNA that can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers. The terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid fragment,” “nucleic acid sequence or segment,” or “polynucleotide” may also be used interchangeably with gene, cDNA, DNA and RNA encoded by a gene, e.g., genomic DNA, and even synthetic DNA sequences. The term also includes sequences that include any of the known base analogs of DNA and RNA.
“Naturally occurring” is used to describe an object that can be found in nature as distinct from being artificially produced. For example, a protein or nucleotide sequence present in an organism (including a virus), which can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory, is naturally occurring.
A “variant” of a molecule is a sequence that is substantially similar to the sequence of the native molecule.
“Recombinant nucleic acid molecule” is a combination of nucleic acid sequences that are joined together using recombinant nucleic acid technology and procedures used to join together nucleic acid sequences as described, for example, in Sambrook and Russell (2001), Gibson et al. Nature Methods. 6 (5): 343-345. (2009). As used herein, the term “recombinant nucleic acid,” e.g., “recombinant DNA sequence or segment” refers to a nucleic acid, e.g., to DNA, that has been derived or isolated from any appropriate cellular source, that may be subsequently chemically altered in vitro, so that its sequence is not naturally occurring, or corresponds to naturally occurring sequences that are not positioned as they would be positioned in a genome that has not been transformed with exogenous DNA. An example of preselected DNA “derived” from a source would be a DNA sequence that is identified as a useful fragment within a given organism, and which is then chemically synthesized in essentially pure form. An example of such DNA “isolated” from a source would be a useful DNA sequence that is excised or removed from said source by chemical means, e.g., by the use of restriction endonucleases or the polymerase chain reaction (PCR), so that it can be further manipulated, e.g., amplified, for use in the invention, by the methodology of genetic engineering.
Thus, recovery or isolation of a given fragment of DNA from a restriction digest can employ separation of the digest on polyacrylamide or agarose gel by electrophoresis, identification of the fragment of interest by comparison of its mobility versus that of marker DNA fragments of known molecular weight, removal of the gel section containing the desired fragment, and separation of the gel from DNA. Therefore, “recombinant DNA” includes completely synthetic DNA sequences, semi-synthetic DNA sequences, DNA sequences isolated from biological sources, and DNA sequences derived from RNA, as well as mixtures thereof.
The term “gene” is used broadly to refer to any segment of nucleic acid associated with a biological function. Thus, genes include coding sequences and/or the regulatory sequences required for their expression. For example, gene refers to a nucleic acid fragment that expresses mRNA, functional RNA, or specific protein, including regulatory sequences. Genes also include nonexpressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters. In addition, a “gene” or a “recombinant gene” refers to a nucleic acid molecule comprising an open reading frame and including at least about one exon and (optionally) an intron sequence.
A “vector” is defined to include, inter alia, any plasmid, cosmid, phage, or binary vector in double- or single-stranded linear or circular form which may or may not be self-transmissible or mobilizable, and which can transform a host cell either by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication).
“Expression cassette” as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a nontranslated RNA, in the sense or antisense direction. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least about one of its components is heterologous with respect to at least about one of its other components. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter, developmentally regulated, tissue or cell specific promoter, or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus.
The term “RNA transcript” or “transcript” refers to the product resulting from RNA polymerase catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript, or it may be an RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA. “Messenger RNA” (mRNA) refers to the RNA that is without introns and that can be translated into protein by the cell. “cDNA” refers to a single- or a double-stranded DNA that is complementary to and derived from mRNA.
“Regulatory sequences” are nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include enhancers, promoters, translation leader sequences, introns, and polyadenylation signal sequences. They include natural and synthetic sequences as well as sequences that may be a combination of synthetic and natural sequences. As is noted above, the term “suitable regulatory sequences” is not limited to promoters. However, some suitable regulatory sequences useful in the present invention will include, but are not limited to constitutive promoters, development-specific promoters, regulatable promoters, and viral promoters.
“5′-UTR (non-coding sequence)” or “5′-untranslated region” refers to a nucleotide sequence located 5′ (upstream) to the coding sequence. It is present in the fully processed mRNA upstream of the initiation codon and may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency.
“3′-UTR (non-coding sequence)” or “3′-untranslated region” refers to nucleotide sequences located 3′ (downstream) to a coding sequence and may include polyadenylation signal sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor.
“Promoter” refers to a nucleotide sequence, usually upstream (5′) to its coding sequence, which directs and/or controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. “Promoter” includes a minimal promoter that is a short DNA sequence comprised of a TATA-box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression. “Promoter” also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements that is capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an “enhancer” is a DNA sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. It is capable of operating in both orientations (normal or flipped), and is capable of functioning even when moved either upstream or downstream from the promoter. Both enhancers and other upstream promoter elements bind sequence-specific DNA-binding proteins that mediate their effects. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments. A promoter may also contain DNA sequences that are involved in the binding of protein factors that control the effectiveness of transcription initiation in response to physiological or developmental conditions.
“Expression” refers to the transcription and/or translation of an endogenous gene, heterologous gene or nucleic acid segment, or a transgene in cells. Expression may also refer to the production of protein.
“Coding sequence” refers to a DNA or RNA sequence that codes for a specific amino acid sequence and excludes the non-coding sequences. It may constitute an “uninterrupted coding sequence”, i.e., lacking an intron, such as in a cDNA or it may include one or more introns bounded by appropriate splice junctions. An “intron” is a sequence of RNA which is contained in the primary transcript but which is removed through cleavage and re-ligation of the RNA within the cell to create the mature mRNA that can be translated into a protein.
The terms “open reading frame” and “ORF” refer to the amino acid sequence encoded between translation initiation and termination codons of a coding sequence. The terms “initiation codon” and “termination codon” refer to a unit of three adjacent nucleotides (‘codon’) in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation).
As used herein, the term “operably linked” refers to a linkage of two elements in a functional relationship. For example, “operably linked” may refer to a linkage of polynucleotide elements or polypeptide elements in a functional relationship. A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, a regulatory DNA sequence is said to be “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation. “Operably-linked” also refers to the association two chemical moieties so that the function of one is affected by the other, e.g., an arrangement of elements wherein the components so described are configured so as to perform their usual function.
The term “amino acid” includes the residues of the natural amino acids (e.g., Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl, Hyp, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as well as unnatural amino acids (e.g., dehydroalanine, homoserine, phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, citruline, α-methyl-alanine, para-benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine). The term also comprises natural and unnatural amino acids bearing a conventional amino protecting group (e.g., acetyl or benzyloxycarbonyl), as well as natural and unnatural amino acids protected at the carboxy terminus (e.g., as a (C1-C6)alkyl, phenyl or benzyl ester or amide; or as an α-methylbenzyl amide). Other suitable amino and carboxy protecting groups are known to those skilled in the art (See for example, T. W. Greene, Protecting Groups In Organic Synthesis; Wiley: New York, 1981, and references cited therein) The term also comprises natural and unnatural amino acids bearing a cyclopropyl side chain or an ethyl side chain.
The terms “polypeptide” and “protein” are used interchangeably herein. A protein molecule may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell or bacteriophage. Fragments and variants of the disclosed proteins or partial-length proteins encoded thereby are also encompassed by the present invention. By “fragment” or “portion” is meant a full length or less than full length of the amino acid sequence of a protein.
By “portion” or “fragment,” as it relates to a nucleic acid molecule, sequence or segment of the invention, when it is linked to other sequences for expression, is meant a sequence having at least about 80 nucleotides, more preferably at least about 150 nucleotides, and still more preferably at least about 400 nucleotides. If not employed for expressing, a “portion” or “fragment” means at least about 9, preferably 12, more preferably 15, even more preferably at least about 20, consecutive nucleotides, e.g., probes and primers (oligonucleotides), corresponding to the nucleotide sequence of the nucleic acid molecules of the invention.
The invention encompasses isolated or substantially purified protein compositions. In the context of the present invention, an “isolated” or “purified” polypeptide is a polypeptide that exists apart from its native environment and is therefore not a product of nature. A polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell. For example, an “isolated” or “purified” protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. A protein that is substantially free of cellular material includes preparations of protein or polypeptide having less than about 30%, 20%, 10%, 5%, (by dry weight) of contaminating protein. When the protein of the invention, or biologically active portion thereof, is recombinantly produced, preferably culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein-of- interest chemicals. Fragments and variants of the disclosed proteins or partial-length proteins encoded thereby are also encompassed by the present invention. By “fragment” or “portion” is meant a full length or less than full length of the amino acid sequence of, a polypeptide or protein.
The terms “introduce to a cell” and “delivery to a cell” refers to contacting a cell with a composition described herein for intracellular delivery or administration of the composition. The delivered components can be provided as isolated or purified protein, nucleic acids (such as DNA or RNA), a vector, or any combination thereof. Thus, the methods of introduction or delivery can be a combination of delivery methods. For example, a polypeptide or an RNA can be introduced via intracellular delivery/expression of a vector comprising a nucleic acid encoding the recombinant polypeptide or the RNA. Non-limiting examples of vector delivery methods include transformation (e.g., transduction), viral and non-viral based delivery, nanoparticle delivery, liposomal delivery, etc. Alternatively, polypeptide(s) and nucleic acids can be introduced through the use of non-limiting examples of nanoparticles, liposomes, electroporation, microinjection, and gene gun, etc.
The term “transformation” refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. A “host cell” is a cell that has been transformed, or is capable of transformation, by an exogenous nucleic acid molecule. Host cells containing the transformed nucleic acid fragments are referred to as “transgenic” cells.
“Transformed,” “transduced,” “transgenic” and “recombinant” refer to a host cell into which a heterologous nucleic acid molecule has been introduced. The term “transformation” is used herein to refer to delivery of DNA into prokaryotic (e.g., E. coli) cells. The term “transduction” is used herein to refer to infecting cells with viral particles. The nucleic acid molecule can be stably integrated into the genome generally known in the art. Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like. For example, “transformed,” “transformant,” and “transgenic” cells have been through the transformation process and contain a foreign gene integrated into their chromosome. The term “untransformed” refers to normal cells that have not been through the transformation process.
“Genetically altered cells” denotes cells which have been modified by the introduction of recombinant or heterologous nucleic acids (e.g., one or more DNA constructs or their RNA counterparts) and further includes the progeny of such cells which retain part or all of such genetic modification.
“Homology” refers to the percent identity between two polynucleotides or two polypeptide sequences. Two DNA or polypeptide sequences are “homologous” to each other when the sequences exhibit at least about 75% to 85% (including 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, and 85%), at least about 90%, or at least about 95% to 99% (including 95%, 96%, 97%, 98%, 99%) contiguous sequence identity over a defined length of the sequences.
As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to a specified percentage of residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window, as measured by sequence comparison algorithms or by visual inspection. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well known to those of skill in the art. Typically, this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California).
As used herein, “comparison window” makes reference to a contiguous and specified segment of an amino acid or polynucleotide sequence, wherein the sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least about 20 contiguous amino acid residues or nucleotides in length, and optionally can be 30, 40, 50, 100, or longer.
As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polypeptide or polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, at least about 90%, 91%, 92%, 93%, or 94%, and at least about 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least about 70%, at least about 80%, 90%, or at least about 95%.
The term “substantial identity” in the context of a peptide indicates that a peptide comprises a sequence with at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, at least about 90%, 91%, 92%, 93%, or 94%, or 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window. An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution.
For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity or complementarity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
The invention will now be illustrated by the following non-limiting Examples.
Integration of transgenes into a genome often leads to low levels of gene expression or gene inactivation (
Genomic safe harbors (GSHs) are sites within an organism's genome, where transgenes can be inserted without negative fitness costs, promoting stable and high-level transgene expression, and having a low probability of silencing. The vast majority of GSH papers focus on the identification and use of GSHs for transgene expression for human cell and gene therapies and production of therapeutic proteins (Papapetrou and Schambach 2016a; Yamamoto and Gerbi 2018). GSHs are most often identified by large-scale screens using insertional mutagenesis of mammalian cells in culture or by T-DNA or transposon mutant collections of plants (Papapetrou and Schambach 2016a; Rozov et al. 2022; Dong et al. 2020). For agribusiness, hundreds of transgenic plants must be screened for the transgene in an insertion site that allows for optimal expression; this must be followed by rigorous biochemical characterization to assure that there are no off-target impacts or fitness costs.
While important for gene-drive strategies for insect control, the importance of the site for gene-drive cassette insertion is only now beginning to be acknowledged. In fact, there is only one report of an insect GSH. Cell cultures of the sleeping chironomid, Polypedilum vanderplanki (Diptera) were screened for high level expression of transgenes. (Miyata et al. 2022a). Four GSHs were identified; three were in intergenic regions and one was within an intron. The larger genomic context (i.e., proximity to transposons, other repetitive elements or chromatin status) was not reported.
Computational approaches have also been used to identify potential GSHs (Autio et al. 2021; Furukawa et al. 2022; Aznauryan et al. 2022). Certain criteria for putative GSHs have been set for human gene therapies and some of these criteria may be useful for the identification of potential GSHs in insects. These putative GSHs should: (1) be >50 kb from a transcriptional start site, (2) not disrupt a transcriptional unit, (3) be >300 kb from miRNAs or >100 kb from IncRNAs, (4) be located outside of DNase I hypersensitivity clusters, which are likely enriched for binding sites for regulatory factors, and (5) be outside of ultra-conserved regions of the genome (Papapetrou and Schambach 2016a). Finally, GSHs should promote stable gene expression of transgenes in all tissue types across multiple generations.
For non-model organisms, the genomics resources (i.e., multiple annotated genomes, a chromosomal level genome assembly, large numbers of transcriptomes from different organs or populations, a knowledge of chromatin accessibility, and location of miRNAs and IncRNAs) needed for computational identification of GSHs are largely lacking. Arras et al., (2015) working with the yeast Cryptococcus neoformans identified two criteria for GSHs: that they be flanked by convergently transcribed genes and that they be in one of the larger intergenic regions. C. neoformans has a very compact genome and so the lengths of intergenic regions very small relative to those of insects. Furthermore, most non-model insects do not have insertional mutant collections or cell culture lines that enable high-throughput screens for GSH identification. At least for this reason, identifying GSHs in non-model insects is challenging, but remains critical for the successful deployment of sustainable gene-drive strategies. For example, at present, there are only two reports of gene drive in an insect species outside the Order Diptera, and both are in the diamondback moth, Plutella xylostella, a major pest of international agriculture (Asad et al. 2022; Xu et al. 2022). Gene drive was weak in the study by Asad et al (2022) and not observed in the study of Xu et al (2022). Xu et al. (2022) suggested that safe harbors for gene-drive cassette insertion should be sought. GSHs are also important for our proposed methods of insect control that rely on transgene expression of double-stranded RNAs or Cas9 and sgRNAs in plants. For this reason, we propose a novel method for making synthetic GSHs, we call Target-on-Demand (ToD). We discuss the ToD concept in the context of insect gene drive. However, it could have wide ranging impact on mammalian, plant and insect biotechnology.
Our approach is based on gene complementation. The ability of a wild-type cDNA to substitute for the mutated gene and, simultaneously, tailor transcription of the transgene to the desired tissues and levels.
The site of integration of transgene cassettes influences the sustainability and effectiveness of a gene drive and its level of expression. Surprisingly, while acknowledged as an important feature for successful transgenesis and gene drive, GSHs have received relatively little attention in the insect gene-drive literature or in the insect community overall. This is due to the fact that identification of GSHs in non-model organisms has few precedents. Described herein are exemplary methods to create a synthetic GSH that can transform “any” gene into a GSH and so increases dramatically the number of sites that can be identified and tested (a targets on demand, ToD).
To date, genome insertion site targets for gene-drive cassettes have been genes with an easily identified morphological phenotypes, such as eye pigmentation or body color. When used as target genes in mosquitoes, eye-color genes w and cn exhibit different gene drive efficiencies, 59% and 38%, respectively. Moreover, in our hands, w and cn mutations have a mild fitness cost impacting the success of Glassy-winged sharpshooter (GWSS) paired matings (but, luckily, not pool matings). Therefore, these genes are not GSHs in GWSS. In whitefly, w mutations are lethal. It is clear new gene-drive insertion sites are needed.
Therefore, to enable a robust and sustainable GWSS or whitefly gene drive, we need to identify GSH loci as a landing and launching pads for gene drive in these insects. Optimal target sites are also needed for the insertion of genes for sterile insect control programs.
For non-model organisms, a simple and yet widely applicable method for creating a GSH would revolutionize our ability to express gene products and develop durable gene drives. Described herein is an exemplary method to custom design a synthetic GSH—a target on demand (
The ToD scheme is illustrated with an experimental design using the GWSS cn gene (
Four possible G0 phenotypic classes could be generated: mosaic cn− eyes, mosaic cn− eyes and dsRED fluorescence, wild-type eyes, wild-type eyes and dsRED fluorescence. Insects in each class are pooled and virgin adults from this pool are pair mated. G0 insects that have wild-type eyes (cn+) and are dsRed+ (phenotype indicative of success) should be the result of complementation. Paired matings of these insects should have rates of egg hatch similar to wild-type insects, further indicating the success of complementation using the ToD strategy. Whereas cn−/dsRed+ insects should yield no progeny from pair matings; they would represent a failure of complementation.
Integration of transgenes into a genome often leads to low levels of gene expression or gene inactivation (
Genomic safe harbors (GSHs) are sites within an organism's genome, where transgenes can be inserted without negative fitness costs, promoting stable and high-level transgene expression, and having a low probability of silencing. The vast majority of GSH papers focus on the identification and use of GSHs for transgene expression for human cell and gene therapies and production of therapeutic proteins (Papapetrou and Schambach 2016a; Yamamoto and Gerbi 2018). GSHs are most often identified by large-scale screens using insertional mutagenesis of mammalian cells in culture or by neutron particle, T-DNA or transposon mutant collections of plants (Papapetrou and Schambach 2016a; Rozov et al. 2022; Dong et al. 2020). For agribusiness, hundreds of transgenic plants must be screened for the transgene in an insertion site that allows for optimal expression; this must be followed by rigorous biochemical characterization to assure that there are no off-target impacts or fitness costs.
While important for gene-drive strategies for insect control, the importance of the site for gene-drive cassette insertion is only now beginning to be acknowledged (Xu et al, 2012). In fact, there is only one report of an insect GSH. Cell cultures of the sleeping chironomid, Polypedilum vanderplanki (Diptera) were screened for high level expression of transgenes. (Miyata et al. 2022a). Four GSHs were identified; three were in intergenic regions and one was within an intron. The larger genomic context (i.e., proximity to transposons, other repetitive elements or chromatin status) was not reported.
To date, there are relatively few strategies that have been used to identify GSHs and all are labor-intensive. These strategies have relied on: (1) large screens of transgene expression cells in culture (in mammals and insects) (
In mammals and insects, cell cultures are used to identify GSHs. Transgenic cells are sorted to identify cells expressing a fluorescent reporter gene at high levels inferring a GSH (
An alternative strategy was used to identify GSHs in rice. In this case, morphological records and the whole-genome sequencing data of a fast-neutron rice mutant collection was surveyed and five mutant loci were identified with no apparent fitness costs (Li et al. 2017; Jung et al. 2008). These loci were tested for use as GSHs and one allowed stable expression of a 5.2-kb transgene cassette that promoted carotenoid production (Dong et al. 2020).
Computational approaches have also been used to identify potential GSHs (Autio et al. 2021; Furukawa et al. 2022; Aznauryan et al. 2022; Arras et al. 2015; Balmas et al. 2023; Dabiri et al. 2023; Ittiprasert et al. 2023). In these studies, the foremost concerns are to assure that GSHs will promote stable gene expression of transgenes (e.g., in all tissue types) across multiple generations and transgenes will not directly or indirectly impact potential cancer-inducing genes (Dabiri et al. 2023). About eight criteria have been iterated for bioinformatic identification of putative GSHs (Papapetrou et al. 2011; Chekulaeva and Filipowicz 2009; Van Meter et al. 2020; Dabiri et al. 2023; Papapetrou and Schambach 2016b; Odak et al. 2020). To assure that the transgene does not inactivate a critical gene or regulatory element (e.g., small RNAs) and is not influenced by regional enhancers, silencers or insulators, people have proposed that a GSH should be: >50 kb from a transcriptional start site (1st criterion); not disrupt a transcriptional unit (2nd criterion); be >300 kb from miRNAs (3rd criterion); be >300 kb from known cancer-associated genes (4th criterion); >100 kb from non-coding RNAs (eg., lncRNAs) (5th criterion); and be outside of ultra-conserved regions of the genome (Papapetrou and Schambach 2016a), which may harbor essential genes or structural elements (6th criterion). Additionally, to assure that a transgene is expressed at desired levels and is not silenced in subsequent generations, GSHs should be located in open chromatin domains to allow transgene expression (7th criterion) and easy access of DNA-cutting enzymes critical for gene insertion (8th criterion).
For non-model organisms, the genomics resources (i.e., multiple annotated genomes, a chromosomal level genome assembly, large numbers of transcriptomes from different organs or populations, a knowledge of chromatin accessibility, and location of miRNAs and IncRNAs) needed for computational identification of GSHs are largely lacking. Collecting these deep genomic resources is costly and time consuming and not feasible for many non-model organisms. Most non-model organisms do not have cell cultures that allow for large scale screens to identify GSHs (
Therefore, alternative criteria have been used in some non-model organisms. For example, two criteria for GSHs were used for identifying GSHs in the yeast Cryptococcus neoformans, which has a compact genome and short intergenic regions (Arras et al. 2015) relative to those of insects. GSHs are flanked by convergently transcribed genes (criterion 1) and in a large intergenic region (criterion 2). Other non-model organisms have also stressed the need for GSHs. Approaches have included: (1) testing GSH regions identified in other organisms (i.e, ROSA26, AAVS1, H11 and COLIA1) in chickens (Ma et al. 2022), (2) combining chromatin accessibility (epigenome) and genome resources in blood flukes (Ittiprasert et al. 2023), (3) using epi/genome resources and a large scale screen of Cas9 mutational hotspots in microalgae (Jeong et al. 2023), and (4) leveraging the serendipitous discovery of a TGFβ receptor 2-like gene in Xenopus as a safe harbor (Shibata et al. 2023; Shibata et al. 2022).
Overall, identifying GSHs in model and non-model insects is challenging, but remains important for the successful deployment of sustainable gene-drive strategies. At present, there are only two reports of gene drive in an insect species outside the Order Diptera, and both are in the diamondback moth, Plutella xylostella, a major pest of international agriculture (Asad et al. 2022; Xu et al. 2022). Gene drive was weak in the study by Asad et al (2022) and not observed in the study of Xu et al (2022). Xu et al. (2022) suggested that genomic safe harbors (GSHs) for gene-drive cassette insertion should be sought. GSHs are also important for our proposed methods of insect control that rely on transgene expression of double-stranded RNAs or Cas9 and sgRNAs in plants.
The site of integration of transgene cassettes influences the sustainability and effectiveness of a gene drive and its level of expression. Surprisingly, while acknowledged as an important feature for successful transgenesis and gene drive, GSHs have received relatively little attention in the insect gene-drive literature or in the insect community overall. This is due to the fact that identification of GSHs in non-model organisms has few precedents. For this reason, we propose a novel method for making synthetic GSHs, we call Target-on-Demand (ToD). A competitive matrix of the current and our proposed method for GSH discovery is provided in
The ToD technology creates a synthetic GSH that could transform “any” gene into a GSH. Our strategy is simple. Since insertion of a cassette into a target gene causes loss of function, it often has a fitness cost (
The ToD technology breaks from the current dogma for GSH identification, which deliberately avoids insertional inactivation of a target gene due to potential fitness costs to an organism. Several other important features speak to the novelty of the ToD technology. While some genomics resources would be useful for the deployment of ToD technology in an organism, they are not essential. The ToD technology is not dependent on numerous deep and costly epi/genomics resources, the ability to propagate a species' cells in culture, access to large collections of insertional mutants, or large foot-print screens of mature transgenic organisms (
The ToD strategy uses transcriptional units as the target sites for transgene integration. The minimal ToD gene cassette has a rescue gene and a landing site for the integration of one or more transgenes. Alternatively, a cargo-carrying ToD gene cassette includes a rescue gene and a transgene that encodes a value-added product. We restore function of the inactivated target gene by the integration of a ‘rescue’ gene that provides the target gene's product (
Target genes (potential GSHs) can be identified by one of many strategies. Knowledge about orthologous genes in other species may help identify a target gene in a non-model organism. Alternatively, if RNA-seq data and a genome sequence (even at the scaffold level) are available, predicted expression of a target gene and its neighboring genes can be deduced to enable optimal target genes for the ToD strategy. As the ToD strategy is not based on robust genomics resources, testing a few (e.g., 5-6) target genes for their efficacy in a ToD strategy may assure that one or more GSHs are identified. It is noteworthy that even with robust genomics resources, multiple putative GSHs have been tested in most studies published to date.
In this Example, the deployment and development of the ToD technology in insects are further discussed, as GSHs are important for the successful deployment of sustainable gene drives. To date, genome insertion site targets for gene-drive cassettes have been genes with an easily identified morphological phenotypes, such as eye pigmentation or body color; in addition, genes critical for sex determination (e.g, doublesex) have been used in gene drive strategies in Anopheles gambiae (Kyrou et al. 2018) and Drosophila suzukii (Yadav et al. 2023). When used as target genes in mosquitoes, eye-color genes white (w) and cinnabar (cn) exhibit different gene drive efficiencies, 59% and 38%, respectively. Moreover, in our hands, w and cn mutations have a mild to severe fitness costs in Homalodisca vitripennis (glassy-winged sharp shooter, GWSS) and Bemisia tabaci (whitefly), respectively. For GWSS, disruption of cn interferes with paired matings but, luckily, not pool matings. GWSS w mutants have poor eclosion and slowed development. Despite these fitness costs, we have been able to maintain GWSS w and cn mutant colonies using pool matings for over 11 generations. Therefore, while cn is currently being used as a target gene for transgene insertion, cn is not optimal GSH for GWSS. In whitefly, w mutations are lethal. It is clear new target gene sites are needed. The ToD technology can solve these mild to severe fitness costs and provide optimal integration sites for transgene expression.
The ToD technology should enable robust and sustainable gene-drive strategies in insects as GSH loci that serve as optimal landing and launching pads for gene drive in insects are needed. Optimal target sites are also needed for the insertion of genes for sterile insect control programs and in transgenic strategies that would block pathogen transmission. There is substantial information indicating that the chromosomal integration sites for Cas9 and sgRNAs which are critical for many contemporary gene drives influences the success of gene drive strategy (López Del Amo et al. 2020).
Several simple criteria can be used to select a putative GSH (target gene) in a non-model organism with limited genomics resources. For example, a non-limiting, exemplary target gene may:
The transgene may confer a value-added trait to the organism. In testing the ToD technology, we will use a fluorescent reporter/marker gene to follow gene insertion events; this is important for organisms where CRISPR-mediate gene insertion occurs at low frequency. In using synthetic GSHs for interrogating biological processes or for biotechnology, the value-added trait includes traits beneficial to the organism or traits useful for pest insect control or traits useful for making product having industrial or therapeutic applications (e.g., product can be isolated or purified further). The transgene can use any native, alien or synthetic promoter, coding sequence, and 3′-flanking region. It would be advantageous to select a promoter to drive the transgene that is expressed in a similar manner to the target gene; but the target gene's promoter is not proposed to drive the transgene in certain embodiments, although it is possible one promoter could drive two genes with an IRES sequence or 2A peptide encoding sequence in between the two genes.
The rescue gene is constructed using knowledge of the target gene (the potential GSH). The rescue gene may utilize the target gene's promoter and 3′-flanking sequences to direct the expression of the target gene's protein in the correct cell types and tissue. The rescue gene's coding region could be the target gene's cDNA. If intronic sequences are important in modulating the expression level of the target gene, the rescue gene could include one or more introns that are known to be essential for driving native gene expression. However, this level of knowledge is not known for most genes in model or non-model organisms. For this reason, we focus on genes with simple structures. In addition, since complementation is achieved by using a single cDNA, it is important that alternative splicing of the target gene (if any) is not critical for its function.
Two types of ToD constructs can be made. The minimal, receiving ToD cassette that harbors the rescue gene and landing pad (
We illustrate the ToD scheme in
Once a minimal, receiving synthetic GSH is identified, we can extend this technology for easy integration of other transgenes. For this application, a minimal, receiving ToD cassette is used (
We test this ToD strategy using eye color genes in Homalodisca vitripennis (GWSS) and Bemisia tabaci (whitefly) due to our success with editing of these genes in these insects (de Souza Pacheco et al. 2022; Pacheco et al. 2022)(Atkinson and Walling, unpublished results). White (w) and cinnabar (cn) are used in GWSS and w and vermilion (v) are used with whiteflies. We know that the GWSS cn is not a GSH, as cn mutants have mild fitness costs that interfere with pair matings. We have shown that we can integrate genes into the GWSS cn with high efficiency using HDR and NHEJ technologies and we establish and maintain lines with pool matings (which bypasses the need for pair matings). Using the ToD strategy, four possible GO phenotypic classes could be generated: mosaic cn− eyes, mosaic cn− eyes and dsRED fluorescence, wild-type eyes, wild-type eyes and dsRED fluorescence (
Impact: virtually “any” gene can be designed to be a synthetic GSH. The ToD strategy may challenge the dogma of avoiding insertion into transcriptionally active genes.
Further testing of optimal target genes for testing the synthetic GSH strategy will occur. To assure that the ToD strategy is easy to execute, the target genes could express a single RNA and be surrounded by transcriptionally active genes; these are simple criteria and the resources (even in non-model organisms) are often in place. We will have transcriptome data from seven GWSS organs that should allow selection of optimal target genes. A small number of target genes may need to be tested in each organism to provide the GSH site that promotes accurate and developmentally correct expression. This fast and efficient ToD method for GSH discovery could revolutionize gene-drive strategies in all organisms, having especially high impact on non-model organisms. If successful, this technology could potentially revolutionize biotechnology initiatives to express transgenes and gene drives in plants, animals and microbes.
We will make the simpler minimal ToD cassette that will insert the rescue gene and a landing pad into the GWSS cn gene, or another target gene (an optimal target gene for sGSH). The landing pad will have one or multiple unique sgRNA cutting sites for insertion of transgene(s). Due to the high efficiency of HDR gene insertion in GWSS, we can avoid the use of a reporter gene in this construct and directly screen for gene insertion events by PCR.
Optimal synthetic GSHs will have a simple gene structure with few or no introns, be surrounded by actively transcribed genes in the genome, and be constitutively expressed. With the limited genomics resources at hand for GWSS, we will identify such candidate genes.
The methods being used are similar to the GWSS cn gene. The w gene cargo-loaded ToD construct with be the 2nd proof-of-concept experiment due to the ease of GWSS editing. The w ToD construct will use the w promoter, w cDNA and w homology arm. The reporter gene and its promoter will be the OpIE2:dsRed construct. A minimal ToD cassette will also be assembled and tested for use for integrating transgenes as described for the cn minimal ToD cassette.
The methods being used to construct the vermilion (v) and w ToD constructs will be similar to the GWSS cn gene ToD. The two B. tabaci genes will be the 3rd and 4th proof-of-concept experiments for the ToD technology. The w ToD construct comprising w rescue gene will include the w promoter, w cDNA and a w homology arm. The v ToD construct comprising w rescue gene will include the v promoter, v cDNA and v homology arm. The transgene (reporter gene and its promoter) will be the OpIE2:dsRed construct. The rescue gene and transgene will be assembled to form the cargo-loaded ToD cassette. The methods for introducing Cas9, sgRNAs, and plasmids into B. tabaci embryos are described in US patent application publication No. US 20210105986 (Atkinson and Walling 2018), which is incorporated by reference herein.
Whiteflies will be assessed for phenotypes (eye-color, mortality, dsRed fluorescence) to assess the utility of the rescue genes in this insect. Minimal ToD cassettes will be assembled and tested as described for GWSS.
There is comparatively limited organ-specific transcriptome data for B. tabaci. We have salivary gland and abdomen, as well as whole insect and virus-infected transcriptomes to use for identification of transgenes that are constitutively expressed. The steps for identifying and testing candidate target genes as synthetic GSHs will follow the protocols described above.
The ToD technology would have a large impact on crop biotechnology and plant cell cultures used for bioreactor production of macromolecules, as well as the study of model plants such as Arabidopsis thaliana. The criteria for a GSH for transgene expression in intact plant vs plants cells grown in bioreactors may be different. Many genes essential for plant development and growth are not needed in plant cell culture; there are marked distinctions in intact plants versus immortalized plant cell culture transcriptomes (Tanurdzic et al. 2008; Iwase et al. 2005). The GSHs for transgenes used in plant cell culture-based biotechnologies would emphasize high transgene expression with high yields of recombinant proteins or metabolites (Rozov et al. 2022). Rozov et al (2022) inserted a modified human interferon gene into a transcriptionally active region upstream of a Histone3 gene that is expressed constitutively during prophase. Protein yields were 2-5 fold more than random transgenic insertion events. In addition, large gene cassettes have also been inserted into a region adjacent to a constitutively expressed ubiquitin gene by Cre-lox technologies and both regulated and constitutive promoters were accurately used (Pathak and Srivastava 2020).
The proof-of-concept experiments are proposed for rice. CRISPR/Cas-mediated integration of a 5.2-kb carotenoid biosynthesis construct into two GSHs of rice has been successful (Dong et al. 2020).
Detailed surgical procedures required for vasectomies, removing embryos from pregnant, euthanized mice, microinjection of embryos in vitro, incubation of embryos in vitro, and subsequent insertion of these embryos into the ampulla of the oviduct via the infundibulum in anesthetized females can be found in Bunting et al. (2022). The outcome of the experiments described in this paper are mice that have been gene edited, these being confirmed both by phenotype and by DNA sequencing of PCR products generated from the target site of the gene editing. To test our ToD approach in these mice, additions or modifications to this protocol are as follows:
All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.
This application claims priority to U.S. Provisional Application No. 63/413,572 filed on 5 Oct. 2022. The entire content of the application referenced above is hereby incorporated by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| 63413572 | Oct 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2023/034566 | Oct 2023 | WO |
| Child | 18634406 | US |
