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Lethal gene deletion in mouse zygotes via targeting the selected allele in F 1 hybrid mice using in vivo electroporation. (a) Schematic of Rad51 gene deletion in F 1 hybrid mice, which was generated in only the C57BL/6NCrSlc allele. (b) Alignment of sequences corresponding to the Rad51 intron 1 and intron 2 genomic breakpoint junctions. (c) Summary of the experimental efficiency of chromosomal deletion via in vivo electroporation.
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The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has facilitated dramatic progress in the field of genome engineering. Whilst microinjection of the Cas9 protein and a single guide RNA (sgRNA) into mouse zygotes is a widespread method for producing genetically engineered mice, in vitro and in vivo electroporation (w...
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... the normal chromosome showed discrete green and red signals from the Adamts20 and K18N loci, respectively, overlapping signals indicative of the inverted Adamts20-K18N locus were observed in the chromosome of line #9, suggesting that line #9 contained the 7.67 Mb inversion (Fig. 1d). Previous studies have demonstrated that recombination between the wild-type and chromosomal inversion line does not occur within these inversion events [17][18][19] (Supplementary Fig. 4a). To determine whether crossovers were suppressed in mouse line #9, heterozygous #9 (C57BL/6JJmsSlc background) www.nature.com/scientificreports ...
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... the 18 mice harbouring heterozygous inversions that were examined, there was no recombination detected within this inversion. In contrast, the mice with homozygous wild-type chromosomes did not show these suppressive effects, supporting the idea that inversion-suppressed recombination was present in line #9 ( Supplementary Fig. 4b,c). These results demonstrate that our electroporation method introduced a 7.67 Mb chromosomal inversion in the mouse zygotes, which is 1.5 times longer than the inversion produced in a previous study using microinjection-based methods 8 . ...
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... recessive lethal deletion in F 1 hybrid mice via in vivo electroporation. Next, we examined whether this strategy could detect a recessive lethal deletion (Fig. 4a). We attempted to delete an essential gene, Rad51, the loss of which in both alleles seemed to produce a lethal embryonic phenotype 22,23 . We www.nature.com/scientificreports www.nature.com/scientificreports/ electroporated the genome editing CRISPR/Cas9 mixture into the oviduct of C57BL/6NCrSlc females that had been mated to ...
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... genome editing CRISPR/Cas9 mixture into the oviduct of C57BL/6NCrSlc females that had been mated to C3H/HeJYokSlc males, and we used C57BL/6NCrSlc females mated to C57BL/6NCrSlc males as a control. In the C57BL/6NCrSlc/C3H/HeJYokSlc F 1 hybrid strains, we obtained two F 0 pups and found that one of them had a large deletion in the target locus (Fig. 4b,c). It should be noted that the control C57BL/6NCrSlc/ C57BL/6NCrSlc strain failed to deliver their pups, suggesting that these embryos died because of the deletion of both Rad51 gene copies (Fig. 4c). In fact, the deletion line #20 generated exhibited a recessive lethal embryonic phenotype ( Supplementary Fig. 6). Together, our results ...
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... In the C57BL/6NCrSlc/C3H/HeJYokSlc F 1 hybrid strains, we obtained two F 0 pups and found that one of them had a large deletion in the target locus (Fig. 4b,c). It should be noted that the control C57BL/6NCrSlc/ C57BL/6NCrSlc strain failed to deliver their pups, suggesting that these embryos died because of the deletion of both Rad51 gene copies (Fig. 4c). In fact, the deletion line #20 generated exhibited a recessive lethal embryonic phenotype ( Supplementary Fig. 6). Together, our results suggest that this unique method may be successfully applied for the generation of target-specific chromosomal rearrangements, such as large-scale inversions and recessive lethal ...
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Exploiting bacterial DNA-acting enzymes and expanding their repertoire for genome engineering have been major technological and conceptual advances in molecular biology over the last decade. The CRISPR-Cas9 system offers many attractive superiorities, such as multiplexing, high precision, low cost, and simplicity compared to other strategies/system...
Citations
... Since the report of GONAD/i-GONAD, many researchers have reproduced this technology using mice, rats, and hamsters, and some modifications have been made to these technologies [12]. For examples, these include production of KO or KI mice using ssODN as a donor [6,7,, large deletion (LD) of a target site in chromosomes [7,22,37], maintenance of lethal mutant mice using an inversion balancer identified from the C3H/HeJJcl strain [38], production of rodents with floxed alleles [20,22,31] or long DNA fragments at a target locus [7], epitope-tagging at a target gene [32] or in situ somatic gene replacement in oviductal epithelium [39]. Furthermore, these techniques have recently been applied to carcinogenic studies, where oviduct-targeted gene delivery of oncogenes can cause the generation of malignant tumors of oviduct origin [40,41]. ...
Gene-engineered animals created using gene-targeting technology have long been recognized as beneficial, valid, and valuable tools for exploring the function of a gene of interest, at least in early 2013. This approach, however, suffers from laborious and time-consuming tasks, such as the production of successfully targeted embryonic stem (ES) cells, their characterization, production of chimeric blastocysts carrying these gene-modified ES cells, and transplantation of those manipulated blastocysts to the recipient (pseudopregnant) females to deliver chimeric mice. Since the appearance of genome editing technology, which is now exemplified by the CRISPR/ Cas9 system, in late 2013, significant advances have been made in the generation of genome-edited animals through pronuclear microinjection (MI) of genome-editing components into fertilized eggs (zygotes) or electroporation (EP) of zygotes in the presence of these reagents. However, these procedures require the transfer of genome-edited embryos into the reproductive tracts of recipient females for further development. G enome editing via o viductal n ucleic a cids d elivery (GONAD) and its modified version, called “improved GONAD ( i -GONAD),” were developed as an alternative to the MI- or EP-based genome-edited animal production and now recognized to be very convenient and straightforward as genome editing can only be performed in vivo (within the oviductal lumen where fertilized embryos exist). This system also enables the simultaneous transfection of epithelial cells lining the oviductal lumen . In this review, we summarize the recent advances in GONAD/ i -GONAD and their derivatives and discuss the potential of these technologies to study various biological systems related to female reproduction.
... To circumvent these limitations, direct in vivo CRISPR editing approaches to produce genetically manipulated animals have been developed (7)(8)(9)(10)(11)(12). These approaches do not require development of stem cell lines or in vitro fertilization. ...
... Over the last few years, multiple groups have reported successfully delivering Cas9 ribonucleoprotein (RNP) complexes into the oviducts of recently mated mice to edit fertilized eggs in vivo (7)(8)(9)(10)(11)(12). Despite not having previous experience in these types of approaches, we wanted to determine if we could adapt the approach for use in our own laboratory and on the mouse C57BL/6J genetic background to be compatible with existing genetically modified mouse lines. ...
Although genetically modified mouse models have long been a powerful tool for microbiology research, the manipulation of the mouse genome is expensive, time consuming, and has historically remained the domain of dedicated animal facilities. The recent use of in vivo clustered regularly interspaced short palindromic repeats (CRISPR)-based editing technology has been reported to reduce the expertise, cost, and time required to generate novel mouse lines; it has remained unclear, however, if this new technology could meaningfully alter experimental timelines. Here, we report the optimization of an in oviduct murine genetic manipulation technique for use by microbiologists. We use this approach to generate a series of knockout mice and detail a protocol using an influenza A virus infection model to test the preliminary importance of a host factor in as short as 11 weeks (with a fully backcrossed knockout line in ~22 weeks) from initiation of the study. Broader use of this approach by the microbiology community will allow for more efficient, and rapid, definition of novel pathogenic mechanisms in vivo .
IMPORTANCE
Clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies have already begun to revolutionize biomedical science. An emerging application of this technology is in the development of genetically modified model organisms to study the mechanisms underlying infectious disease. Here, we describe a protocol using an in vivo CRISPR-based approach that can be used to test the importance of a candidate host factor for microbial pathogenesis in less than 3 months and before complete establishment of a new mouse line. Adoption of this approach by the broader microbiology community will help to decrease the resources and time required to understand how pathogens cause disease which will ultimately speed up the development of new clinical interventions and therapies.
... C57BL/6NCrSlc female mice (8-12 weeks old) in oestrus were mated with males (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24) weeks old) at 15:00-18:00 h. The presence of copulation plugs was confirmed the next morning via visual inspection, and plug-positive mice were subjected to i-GONAD experiments, as previously described 7 . ...
... Asymmetric disjunction results in a wide range of highly imbalanced gametes, many of which do not survive until the end of spermatogenesis 17 . Breakpoints in intergenic regions may disturb the interactions between the promoter and transcriptional units with its cis-acting regulators through positional effects, thus severely affecting gene expression 18 .We recently succeeded in inducing a 7.67 Mb inversion in WT mice19 ...
Complex chromosomal rearrangements (CCRs) are often observed in clinical samples from patients with cancer and congenital diseases but are difficult to induce experimentally. For generating animal models, these CCRs must be induced as desired, because they can generate profound genome instability and/or result in cell death. This is the first study to present the establishment of an animal model for CCRs. The disruption of Recql5 , which degrades RAD51 during DNA repair, successfully induces CRISPR/Cas9-mediated CCRs, establishing a mouse model containing triple fusion genes and megabase-sized inversions. Notably, some of these structural features of individual chromosome rearrangements use template switching and microhomology-mediated break-induced replication mechanisms and are reminiscent of the newly described phenomenon chromoanasynthesis. Whole-genome sequencing (WGS) analysis revealed that the structural variants in these mice caused only target-specific rearrangements but not target-independent complex ones. Thus, these data demonstrate that CCRs can be efficiently induced by manipulating DNA repair pathways.
... Iwata et al. [93] i-GONAD (protein) CRISPR/Cas9 (indels) NEPA21 ...
... As shown in Section 7-1, CRISPR/Cas9-mediated genome editing using 2 types of gRNAs results in frequent generation of LD in the sequence flanked by the two sites recognized by these gRNAs. Iwata et al. [93] applied this technique to introduce chromosomal inversions of several megabases (Mb) in mice. When mouse zygotes were subjected to in vitro EP, a 7.67 Mb inversion was successfully introduced, which is longer than any previously reported inversion produced using MI-based methods. ...
CRISPR-based genome engineering has been widely used for producing gene-modified animals such as mice and rats, to explore the function of a gene of interest and to create disease models. However, it always requires the ex vivo handling of preimplantation embryos, as exemplified by the microinjection of genome editing components into zygotes or in vitro electroporation of zygotes in the presence of genome editing components, and subsequent cultivation of the treated embryos prior to egg transfer to the recipient females. To avoid this ex vivo process, we have developed a novel method called genome-editing via oviductal nucleic acids delivery (GONAD) or improved GONAD (i-GONAD), which enables in situ genome editing of zygotes present in the oviductal lumen of a pregnant female. This technology does not require any ex vivo handling of preimplantation embryos or preparation of recipient females and vasectomized males, all of which are often laborious and time-consuming. In this chapter, recent advances in the development of GONAD/i-GONAD will be described.
... In plants, C. elegans, and mouse and human cells, inversions have been generated using CRISPR-Cas9 mutagenesis to induce simultaneously 2 double-strand breaks, which occasionally resolve as an inversion (Li et al. 2015;Iwata et al. 2016Iwata et al. , 2019Zhang et al. 2017;Korablev et al. 2017;Dejima et al. 2018;Schmidt et al. 2019). Similar efficient approaches have not been reported in Drosophila, and I have failed to recover inversions in D. melanogaster using this kind of approach to generate inversions on the order of 10 kb-1 Mbp. ...
Perhaps the most valuable single set of resources for genetic studies of Drosophila melanogaster is the collection of multiply-inverted chromosomes commonly known as balancer chromosomes. Balancers prevent the recovery of recombination exchange products within genomic regions included in inversions and allow perpetual maintenance of deleterious alleles in living stocks and the execution of complex genetic crosses. Balancer chromosomes have been generated traditionally by exposing animals to ionizing radiation and screening for altered chromosome structure or for unusual marker segregation patterns. These approaches are tedious and unpredictable, and have failed to produce the desired products in some species. Here I describe transgenic tools that allow targeted chromosome rearrangements in Drosophila species. The key new resources are engineered reporter genes containing introns with yeast recombination sites and enhancers that drive fluorescent reporter genes in multiple body regions. These tools were used to generate a doubly-inverted chromosome 3R in D. simulans that serves as an effective balancer chromosome.
... Multiple studies in numerous species have used electroporation to deliver CRISPR Cas9 genome-editing reagents into zygotes to generate knockout embryos and animals. Non-mosaic knockouts have been most efficiently produced in rats and mice (Hashimoto et al., 2016;Chen et al., 2019) targeting a wide range of genes, including LIF (Kim et al., 2020), Rad51 (Iwata et al., 2019), and Rosa26 (Troder et al., 2018). ...
The introduction of genome editing reagents into mammalian zygotes has traditionally been accomplished by cytoplasmic or pronuclear microinjection. This time-consuming procedure requires expensive equipment and a high level of skill. Electroporation of zygotes offers a simplified and more streamlined approach to transfect mammalian zygotes. There are a number of studies examining the parameters used in electroporation of mouse and rat zygotes. Here, we review the electroporation conditions, timing, and success rates that have been reported for mice and rats, in addition to the few reports about livestock zygotes, specifically pigs and cattle. The introduction of editing reagents at, or soon after, fertilization can help reduce the rate of mosaicism, the presence of two of more genotypes in the cells of an individual; as can the introduction of nuclease proteins rather than mRNA encoding nucleases. Mosaicism is particularly problematic in large livestock species with long generation intervals as it can take years to obtain non-mosaic, homozygous offspring through breeding. Gene knockouts accomplished via the non-homologous end joining pathway have been more widely reported and successfully accomplished using electroporation than have gene knock-ins. Delivering large DNA plasmids into the zygote is hindered by the zona pellucida (ZP), and the majority of gene knock-ins accomplished by electroporation have been using short single stranded DNA (ssDNA) repair templates, typically less than 1 kb. The most promising approach to deliver larger donor repair templates of up to 4.9 kb along with genome editing reagents into zygotes, without using cytoplasmic injection, is to use recombinant adeno-associated viruses (rAAVs) in combination with electroporation. However, similar to other methods used to deliver clustered regularly interspaced palindromic repeat (CRISPR) genome-editing reagents, this approach is also associated with high levels of mosaicism. Recent developments complementing germline ablated individuals with edited germline-competent cells offer an approach to avoid mosaicism in the germline of genome edited founder lines. Even with electroporation-mediated delivery of genome editing reagents to mammalian zygotes, there remain additional chokepoints in the genome editing pipeline that currently hinder the scalable production of non-mosaic genome edited livestock.
... We developed a new method, called genome editing via oviductal nucleic acid delivery (GONAD), which was subsequently renamed "improved GONAD (i-GONAD)", for the production of genome-edited mice [16][17][18], rats [19,20], and hamsters [21]. This technology is based on the injection of a solution (1-1.5 µL) containing genome editing reagents into the lumen of an oviduct via the oviductal wall of pregnant female animals at the late zygote to two-cell stage following in vivo EP of the entire oviduct using tweezer-type electrodes under a dissecting microscope [22,23]. ...
... According to Gurumurthy et al. [22], the optimal electric conditions for B6 involve in vivo EP being carried out under a constant current of 100 mA. To our knowledge, two electroporators, NEPA21 (NEPA GENE Co. Ltd., Chiba, Japan) and CUY21EditII, are those that have been most frequently used for successful GONAD/i-GONAD [16][17][18][19][20][21]. CUY21EditII can provide a constant current, whereas NEPA21 cannot. ...
Improved genome editing via oviductal nucleic acid delivery (i-GONAD) is a novel method for producing genome-edited mice in the absence of ex vivo handling of zygotes. i-GONAD involves the intraoviductal injection of clustered regularly interspaced short palindromic repeats (CRISPR) ribonucleoproteins via the oviductal wall of pregnant females at 0.7 days post-coitum, followed by in vivo electroporation (EP). Unlike outbred Institute of Cancer Research (ICR) and hybrid mouse strains, genome editing of the most widely used C57BL/6J (B6) strain with i-GONAD has been considered difficult but, recently, setting a constant current of 100 mA upon EP enabled successful i-GONAD in this strain. Unfortunately, the most widely used electroporators employ a constant voltage, and thus we explored conditions allowing the generation of a 100 mA current using two electroporators: NEPA21 (Nepa Gene Co., Ltd.) and GEB15 (BEX Co., Ltd.). When the current and resistance were set to 40 V and 350-400 Ω, respectively, the current was fixed to 100 mA. Another problem in using B6 mice for i-GONAD is the difficulty in obtaining pregnant B6 females consistently because estrous females often fail to be found. A single intraperitoneal injection of low-dose pregnant mare's serum gonadotrophin (PMSG) led to synchronization of the estrous cycle of these mice. Consequently, approximately 51% of B6 females had plugs upon mating with males 2 days after PMSG administration, which contrasts with the case (≈26%) when B6 females were subjected to natural mating. i-GONAD performed on PMSG-treated pregnant B6 females under conditions of average resistance of 367 Ω and average voltage of 116 mA resulted in the production of pregnant females at a rate of 56% (5/9 mice), from which 23 fetuses were successfully delivered. Nine (39%) of these fetuses exhibited successful genome editing at the target locus.
Complex chromosomal rearrangements (CCRs) are often observed in clinical samples from patients with cancer and congenital diseases but are difficult to induce experimentally. Here, we report the first success in establishing animal models for CCRs. Mutation in Recql5, a crucial member of the DNA helicase RecQ family involved in DNA replication, transcription, and repair, enabled CRISPR/Cas9-mediated CCRs, establishing a mouse model containing triple fusion genes and megabase-sized inversions. Some of these structural features of individual chromosomal rearrangements use template switching and microhomology-mediated break-induced replication mechanisms and are reminiscent of the newly described phenomenon “chromoanasynthesis.” These data show that Recql5-mutant mice could be a powerful tool to analyze the pathogenesis of CCRs (particularly chromoanasynthesis) whose underlying mechanisms are poorly understood. The Recql5 mutants generated in this study are to be deposited at key animal research facilities, thereby making them accessible for future research on CCRs.
The clustered regularly interspaced short palindromic repeats (CRISPR) technology has made it possible to produce genome-edited (GE) animals more easily and rapidly than before. In most cases, GE mice are produced by microinjection (MI) or by in vitro electroporation (EP) of CRISPR reagents into fertilized eggs (zygotes). Both of these approaches require ex vivo handling of isolated embryos and their subsequent transfer into another set of mice (called recipient or pseudopregnant mice). Such experiments are performed by highly skilled technicians (especially for MI). We recently developed a novel genome editing method, called "GONAD (Genome-editing via Oviductal Nucleic Acids Delivery)," which can completely eliminate the ex vivo handling of embryos. We also made improvements to the GONAD method, termed "improved-GONAD (i-GONAD)." The i-GONAD method involves injection of CRISPR reagents into the oviduct of an anesthetized pregnant female using a mouthpiece-controlled glass micropipette under a dissecting microscope, followed by EP of the entire oviduct allowing the CRISPR reagents to enter into the zygotes present inside the oviduct, in situ. After the i-GONAD procedure, the mouse recovered from anesthesia is allowed to continue the pregnancy to full term to deliver its pups. The i-GONAD method does not require pseudopregnant female animals for embryo transfer, unlike the methods relying on ex vivo handling of zygotes. Therefore, the i-GONAD method can reduce the number of animals used, compared to the traditional methods. In this chapter, we describe some newer technical tips about the i-GONAD method. Additionally, even though the detailed protocols of GONAD and i-GONAD have been published elsewhere (Gurumurthy et al., Curr Protoc Hum Genet 88:15.8.1-15.8.12, 2016 Nat Protoc 14:2452-2482, 2019), we provide all the protocol steps of i-GONAD in this chapter so that the reader can find most of the information, needed for performing i-GONAD experiments, in one place.
As the efficiency of the clustered regularly interspaced short palindromic repeats/Cas system is extremely high, creation and maintenance of homozygous lethal mutants are often difficult. Here, we present an efficient in vivo electroporation method called improved genome editing via oviductal nucleic acid delivery (i-GONAD), wherein one of two alleles in the lethal gene was selectively edited in the presence of a non-targeted B6.C3H-In(6)1J inversion identified from the C3H/HeJJcl strain. This method did not require isolation, culture, transfer, or other in vitro handling of mouse embryos. The edited lethal genes were stably maintained in heterozygotes, as recombination is strongly suppressed within this inversion interval. Using this strategy, we successfully generated the first Tprkb null knockout strain with an embryonic lethal mutation and showed that B6.C3H-In(6)1J can efficiently suppress recombination. As B6.C3H-In(6)1J was tagged with a gene encoding the visible coat color marker, Mitf, the Tprkb mutation could be visually recognized. We listed the stock balancer strains currently available as public bioresources to create these lethal gene knockouts. This method will allow for more efficient experiments for further analysis of lethal mutants.
