Multiple gene editing
阅读说明：本技术 多重基因编辑 (Multiple gene editing ) 是由 S.C.法伦克鲁格 D.F.卡尔森 于 2015-04-28 设计创作，主要内容包括：呈现了用于在细胞中做出多重基因编辑的材料和方法。其它方法包括动物和生成动物的方法。(Materials and methods for making multiplex gene edits in cells are presented. Other methods include animals and methods of generating animals.)
1. A method for breeding an animal with a genetic edit that causes a growth incompetent phenotype, comprising obtaining a host blastocyst, embryo, morula, or zygote from a host animal that does not comprise the genetic edit that causes a growth incompetent phenotype, and introducing a donor cell from a donor animal that comprises the genetic edit that causes a growth incompetent phenotype into the host blastocyst, embryo, morula, or zygote to produce a chimeric blastocyst, embryo, morula, or zygote.
2. The method of claim 1, wherein the host animal comprises a Spermatogonial Stem Cell (SSC) knockout mutation and the donor animal does not comprise the SSC knockout mutation.
3. The method of claim 2, further comprising implanting said chimeric blastocyst, embryo, morula, or zygote into a surrogate mother to produce a chimeric animal derived from said chimeric blastocyst, embryo, morula, or zygote.
4. The method of claim 3, wherein said chimeric animal derived from a chimeric blastocyst, embryo, morula, or zygote produces a gamete comprising said genetic edit causing a growth failure phenotype but not said SSC knockout mutation.
5. The method of claim 3, wherein said chimeric animal is bred by sexual reproduction to produce an animal with said genetic edit that causes failure to thrive.
6. The method of claim 2, wherein said host animal comprising said SSC knockout mutation is incapable of producing a genetically functional gamete comprising said host animal.
7. The method of claim 2, wherein the SSC knockout mutation comprises a deletion in sperm-free (DAZL) knockout mutation.
8. The method of claim 1, wherein the genetic edit that causes the failure to thrive phenotype is generated using a genome editing tool selected from the group consisting of: transcription activator-like effector nucleases (TALENs), zinc finger nucleases, and RNA-guided endonucleases (RGENs).
9. The method of claim 8, wherein said RGEN comprises the CRISPR/cas9 system.
10. The method of claim 1, wherein the failure to thrive phenotype comprises a reduced ability to produce offspring that live to sexual maturity relative to an animal without the genetic edit that causes the failure to thrive phenotype.
11. The method of claim 1, wherein the failure to thrive phenotype comprises an immunodeficiency.
12. The method of claim 1, wherein the genetic edit that causes the failure-to-thrive phenotype comprises a genetic edit in a gene comprising interleukin 2 receptor subunit gamma (IL2Rg) or recombinant activation 2(RAG 2).
13. The method of claim 12, wherein the genetic edits causing the failure to thrive phenotype comprise genetic edits in the genes interleukin 2 receptor subunit gamma (IL2Rg) and recombination activation 2(RAG 2).
14. The method of claim 3, wherein said host animal, said donor animal, or an animal derived from a chimeric blastocyst, embryo, morula, or zygote is selected from the group consisting of: livestock, apes, dogs, cats, birds, fish, rabbits, pigs, cattle, buffalo, goats, sheep, and artiodactyls.
15. The method of claim 14, wherein the donor animal and the host animal are of the same species.
16. The method of claim 14, wherein said donor animal and said host animal are of the same breed.
17. The method of claim 14, wherein said donor animal and said host animal are of different breeds.
18. An animal produced from the chimeric blastocyst, embryo, morula, or zygote of claim 1.
19. Progeny of the animal of claim 18.
Technical Field
Background
Genetic modifications to cells, and animals made from such cells, can be used to alter gene expression. The field of genetic engineering is very active.
Disclosure of Invention
It would be very useful to make large vertebrates that have multiple variations in their genetic code in a single generation. As disclosed herein, this can be achieved by editing multiple genes simultaneously in a cell or embryo. Multiple genes can be targeted for editing in vertebrate cells or embryos using targeted nucleases and Homology Directed Repair (HDR) templates. These cells or embryos can be used to study or prepare whole animals. Multiple edits may be made in a single generation that would otherwise not be possible, such as through breeding or genetic engineering changes that are made one by one.
Drawings
Figure 1A depicts a method for making an animal homozygous for two knockouts using a single edit.
Fig. 1B depicts a hypothetical method of preparing an animal with multiple edits by making a single edit at a time.
FIG. 2 depicts the multiple gene editing used to establish founders at generation F0.
FIG. 3 multiplex Gene editing of porcine RAG2 and IL2R γ. Panel a) Surveyor and RFLP analysis to determine the efficiency of non-homologous end joining (NHEJ) and homology-dependent repair HDR on
FIG. 4 multiplex Gene editing of porcine APC and p 53. Panel a) Surveyor and RFLP analysis to determine the efficiency of non-homologous end joining (NHEJ) and homology-dependent repair HDR on
Fig. 5. Effect of oligonucleotide HDR template concentration on five-gene multiplex HDR efficiency. Indicated amounts of TALEN mRNA for porcine RAG2, IL2Rg, p53, APC and LDLR were co-transfected into porcine fibroblasts with either 2uM (panel a) or 1uM (panel b) of each cognate HDR template. The percentages NHEJ and HDR were measured by surfyor and RFLP assays.
FIG. 6 is a five gene multiplex dataset showing experimental data plots of the effect of oligonucleotide HDR template concentration on 5-gene multiplex HDR efficiency. Indicated amounts of TALEN mRNA for porcine RAG2, IL2Rg, p53, APC and LDLR were co-transfected into porcine fibroblasts with each of the associated HDR templates of 2uM (panel a) or 1uM (panel b). The percentages NHEJ and HDR were measured by surfyor and RFLP assays. Colony genotypes from 5-gene multiplex HDR: colony genotypes were assessed by RFLP analysis. Panel a) each line represents the genotype of one colony at each designated locus. Three genotypes can be identified; those with expected RFLP genotypes of heterozygous or homozygous HDR as well as those with RFLP-positive fragments, plus a second allele with a visible change (shift) in size indicating an insertion or deletion (indel) allele. The percentage of colonies with edits at the specified loci is indicated below each column. Panel b) counts of the number of colonies edited at 0-5 loci.
Figure 7 is another five-gene multiplex dataset showing a plot of experimental data for a second experiment relating to the effect of oligonucleotide HDR template concentration on five-gene multiplex HDR efficiency. The second colony genotype for the 5-gene multiplex assay. Panel a) each line represents the genotype of one colony at each designated locus. Three genotypes can be identified; those with expected RFLP genotypes of heterozygous or homozygous HDR as well as those with RFLP-positive fragments, plus a second allele with visible changes in size indicating an insertion or deletion (indel) allele. The percentage of colonies with edits at the specified loci is indicated below each column. Panel b) counts of the number of colonies edited at 0-5 loci.
FIG. 8 is another five-gene multiplex assay dataset showing colony genotypes. Panel a) each line represents the genotype of one colony at each designated locus. Three genotypes can be identified; those with expected RFLP genotypes of heterozygous or homozygous HDR as well as those with RFLP-positive fragments, plus a second allele with visible changes in size indicative of an insertion or deletion (indel) allele. The percentage of colonies with edits at the specified loci is indicated below each column. Panel b) counts of the number of colonies edited at 0-5 loci.
Fig. 9 depicts a method of making an F0 generation chimera with a targeted nuclease that produces a desired gene knockout or allele selection.
FIG. 10 depicts the establishment of F0 generation animals with normal phenotype and offspring with Failure To Thrive (FTT) phenotype and genotype.
FIG. 11 depicts a method of making a chimeric animal with gametes inherited from a donor embryo.
FIG. 12 depicts multiple edits at three target loci of NKX-2, GATA4 and
Figure 13 depicts multiplexed gene editing using a combination of TALENs and RGENs; assays of transfected cells assessed by RFLP revealed HDR at both sites.
Detailed Description
Methods of multiplex gene editing are described. Various genes can be modified in cells or embryos that can be used to study or prepare whole animals. Other embodiments relate to the supplementation of cell or organ loss by selective elimination (depopulation) of host niches (hostniches). These inventions provide for the rapid creation of animals to serve as models, food products, and as a source of cellular and cell-free products for industry and medicine.
Fig. 1A has a timeline illustrating why it takes several years to prepare livestock with only two edited alleles using a single edit, which is about six years for cattle. In this context, editing means selecting a gene and changing it. First, the gene of interest must be edited (e.g., Knocked Out (KO)) in cultured somatic cells that are cloned to create heterozygous calves with targeted KO. The heterozygotes were bred to maturity for breeding, about 2 years for cattle, to produce first generation (F1) male and female heterozygous calves, which will breed with each other to produce homozygous knockout calves (F2). It would be impractical to generate homozygotes for multiple targeted mutations in cattle using conventional methods. Depending on the particular protocol used, the number of years required and the number of animals required to make further edits increases in an approximately exponential manner, as shown in fig. 1B. Among vertebrates, even those having a large number of offspring per generation and a shorter gestation time than cattle require an excessively long time to achieve multiple edits. For example, pigs are bred with a large number of offspring each time, the gestation time is about half that of cattle, but the time to do multiple edits can take many years. Furthermore, a scheme that minimizes time by aggressive close-up propagation may not be reasonably possible for multiple edits. Furthermore, serial cloning is undesirable from a process and result standpoint, particularly if the animal is used as a livestock or laboratory model.
The opportunity presented by the present invention is shown in fig. 2, which shows multiple edits made in the first generation animal (F0). Embryos were prepared directly or by cloning with two or more edits independently selected as either heterozygous or homozygous and placed in surrogate females (offspring). The resulting animals were founders of the F0 generation. Multiple embryos can be prepared and placed in one or more surrogate to produce offspring of both sexes, or multiple cloned embryos can be prepared using well-known embryo division techniques. Livestock (e.g., swine) can be crossed and bred that typically produce litters of both sexes.
Targeted endonucleases and Homology Directed Repair (HDR) can be used to disrupt or otherwise edit multiple alleles in a cell or embryo, as described herein. One embodiment is a method of genetically modifying at a plurality of target chromosomal DNA sites in a vertebrate cell or embryo, comprising introducing into the vertebrate cell or embryo: a first targeting endonuclease directed to a first target chromosomal DNA site and a first Homology Directed Repair (HDR) template homologous to the first target site sequence; and a second targeting endonuclease directed to a second target chromosomal DNA site and a second HDR template homologous to the second target site sequence, wherein the first HDR template sequence replaces the native chromosomal DNA sequence at the first target site and the second HDR template sequence replaces the native chromosomal DNA sequence at the second target site sequence.
It is learned that, with unexpected and surprising and unpredictable results, multiple edits, such as knockouts or replacements, can be obtained. One theoretical mechanism is that there are few cells that can accept multiple edits because they are at a particular stage in the cell cycle. They respond easily when exposed to endonucleases and HDR templates. A relevant theory of operation is that the HDR templating (templating) process itself leads to multiple substitutions, as activation of the cellular repair mechanism for one targeted site also facilitates repair or HDR templating at other sites. HDR has historically been an inefficient process and thus it is clear that multiple HDR edits are not attempted, observed, or approved.
The results herein indicate that too much or too little endonuclease and/or HDR template can have negative effects, which may confound prior studies in this area. In fact, the inventors have observed that targeted endonucleases can be designed and prepared correctly, but fail because they are too efficient. Furthermore, the population of successfully modified cells generally does not improve over time. Cell-modifying technicians often look for the longevity and modification of cells as an indicator of stability and health for successful cloning or other uses. However, this desire is generally not helpful in the multiplexing (multiplexing) process herein. Furthermore, the inventors have observed that the efficiency of Homologous Recombination (HR) introgression is variable in a multiplex approach compared to single locus introgression. Some loci are very sensitive, but others are greatly reduced in efficiency. There is significant interference between endonucleases, but the net effect cannot be explained simply, for example by assuming that endonucleases compete for a common resource.
There are various well-known techniques for inserting many genes randomly or inaccurately into multiple locations in chromosomal DNA, or for performing many random edits that disrupt multiple genes. Clearly, a random or imprecise process would not help scientists who need to edit multiple specific targeted genes to achieve an effect. Thus, the HDR methods taught herein can be readily distinguished by edits made only at the intended target site and the resulting organism. One difference is that the inventive HDR editing embodiments can be performed without inserting additional gene copies and/or without destroying genes other than the gene targeted by the endonuclease. And a specific edit is made at one position because the HDR template sequence is not copied into a site with no proper homology. Embodiments include organisms and methods in which an exogenous allele replicates into chromosomal DNA only at the site of its cognate allele.
An advantage of HDR-based editing is that editing can be selected. In contrast, other attempts through the non-homologous end joining (NHEJ) process may generate indels at multiple positions such that the indels cancel each other without frameshifting. This problem becomes important when multiplexing is involved. The successful use of HDR provides that editing can be done to ensure that the target gene has the expected frameshift when desired. Furthermore, allele replacement requires HDR and cannot be accomplished by NHEJ, vector-driven nucleic acid insertion, transposon insertion, and the like. Furthermore, selecting organisms without unwanted editing further increases the difficulty.
However, it is generally believed that multiple modifications as described herein have not previously been achieved at targeted sites in cells or animals associated with livestock or large vertebrates. It is well known that animals with so many genetic lesions are created from high passage cell cloned animals that they cannot be used as F0 creators of laboratory models or livestock.
Moreover, gene editing is a random process; thus, the art has traditionally emphasized various screening techniques to identify a small percentage of cells that have been successfully edited. Since it is a random process, the technician can expect the difficulty of making multiple edits to increase exponentially as the number of intended edits increases.
One embodiment of the present invention provides a method of creating multiple targeted gene knockouts or other edits in a single cell or embryo, referred to herein as a method of multiple gene knockouts or edits. The term targeted gene refers to a site of chromosomal DNA selected for endonuclease attack by designing an endonuclease system (e.g., TALEN or CRISPR). The terms knock-out, inactivation, and disruption are used interchangeably herein to refer to altering the targeted site such that the gene expression product is eliminated or substantially reduced such that expression of the gene no longer has a significant effect on the entire animal. These terms are sometimes used elsewhere to refer to reducing the effect of a gene in an observable manner without substantially eliminating its effect.
Gene editing as the term is used herein refers to selecting a gene and altering it. Random insertion, gene trapping, etc. are not gene editing. Examples of gene editing are gene knock-out at a targeted site, addition of nucleic acids, removal of nucleic acids, elimination of all functions, allelic introgression, polymorphic changes, sub-allelic changes (hypomorphic alteration) and substitution of one or more alleles.
Replacement of an allele refers to a non-meiotic process in which an exogenous allele is replicated on an endogenous allele. The term replacement of an allele refers to a change from a native allele to an exogenous allele without indels or other changes, except in some cases degenerate substitutions. The term degenerate substitution refers to a change of a base in a codon to another base without changing the encoded amino acid. Degenerate substitutions may be selected to be in exons or introns. One use of degenerate substitutions is to create restriction sites for easy testing of the presence of introgression sequences. Endogenous alleles are also referred to herein as native alleles. The term gene is broad and refers to chromosomal DNA that is expressed to produce a functional product. The gene has an allele. A genotype is homozygous if there are two identical alleles at a particular locus, and heterozygous if the two alleles are different. An allele is an alternative form (one member of a pair) of a gene located at a particular position on a particular chromosome. Alleles determine different traits. Alleles have base pair (bp) differences that give rise to different traits and distinguish them from each other at specific positions in their DNA sequence (distinguishing positions or bp) that serve as allele markers. Alleles are generally described and described herein as being identical if they have the same base at a distinct position; animals naturally have some variation at other bp in other positions. When comparing alleles, the skilled artisan routinely modulates natural variation. The term identical is used herein to mean that there is absolutely no bp difference or indels in the DNA alignment.
A similar test for allele identity is to align chromosomal DNA in the altered organism with that of the exogenous allele, as it is recognized in nature. An exogenous allele will have one or more allelic markers. The DNA alignments upstream and downstream of the marker will be the same distance apart. This distance may be, for example, 10 to 4000bp, depending on the desired test. While HDR templates can be expected to be created with identical sequences, the bases on either side of the template region will of course have some natural variation. The skilled artisan routinely distinguishes alleles despite natural variation. The skilled artisan will immediately recognize that all ranges and values between the explicitly stated limits are contemplated, and any of the following distances may be used as an upper or lower limit: 15, 25, 50, 100, 200, 300, 400, 500, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000, 4000.
The skilled artisan is also able to distinguish gene editing from alleles that are the result of gene editing rather than sexual reproduction. This is trivial when the allele is from another species that cannot sexually reproduce to mix the alleles. Also, many edits are not found at all in nature. Even when a substitution is made that completely repeats the allele found naturally in another variety, the edits can be readily distinguished when the allele migrates from one variety to the next. Alleles are stably located on DNA most of the time. However, meiosis during gametogenesis results in the occasional exchange of male and female DNA for alleles, an event known as crossover (crossover). Cross-over frequency and genetic maps have been extensively studied and developed. In the case of livestock, the pedigree of the animal can be followed in great detail for many generations. In genetics, centimorgans (cM, also known as picture distance units (m.u.)) is a unit that measures genetic linkage. It is defined as the distance between chromosomal locations (loci or locus markers) for which the expected average number of intervening chromosomal crossings in a single generation is 0.01. Genes that are close to each other have a lower chance of crossing than genes that are far from each other on the chromosome. Crossover is a very rare event when two genes are adjacent to each other on a chromosome. The crossing of a single allele with respect to its two adjacent alleles is not possible, so that such events must be the product of genetic engineering. Even where animals of the same breed are involved, natural versus engineered allele substitutions can be readily determined when the parents are known. Also, by genotyping potential parents, the parents can be determined with high accuracy. Parental assays are routine in herds and humans.
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