GONAD: A Novel CRISPR/Cas9 Genome Editing Method that Does Not Require Ex Vivo Handling of Embryos
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Abstract Transgenic technologies used for creating a desired genomic change in animals involve three critical steps: isolation of fertilized eggs, microinjection of transgenic DNA into them and their subsequent transfer to recipient females. These ex vivo steps have been widely used for over 3 decades and they were also readily adapted for the latest genome editing technologies such as ZFNs, TALENs, and CRISPR/Cas9 systems. We recently developed a method called GONAD ( G enome editing via O viductal N ucleic A cids D elivery) that does not require all the three critical steps of transgenesis and therefore relieves the bottlenecks of widely used animal transgenic technologies. Here we provide protocols for the GONAD system. © 2016 by John Wiley & Sons, Inc.Keywords:
Transgenesis
Zinc finger nuclease
Ex vivo
Morpholino
In a seminal study in 1996 , Kim et, al [1] demonstrated that a fusion of the C-terminal endonuclease domain of the FokI protein to a single zinc finger protein induces DNA cuts (ZFNs). This also applied to transcription activator-like effector proteins (TALEN). Later studies suggested that FokI needs to dimerize in order to cleave, without ever repeating the simple experiment mentioned above. Dimerization might increase efficiency, but the single monomer of FokI contains the catalytic site to cleave. If the monomeric FokI cleaves, the off-target problems with ZFN/TALENS would be significant in a large genome (especially with mismatches), since each component (left and right ZFN/TALEN) are typically 18 nucleotides or less. Previously, I had shown an unreported off-target in hornless TALEN-edited cattle [2]. Here, I show multiple off-targets in an ZFN study that edited two loci (AAVS1 and IL2RG) in stem cells [3]. The plasmid integrates at a loci in the HBB gene, which was not edited, across multiple(11) samples, with significant number of reads corroborating this fact. This also highlights the problem of plasmid integration in such gene-therapies, including the bacterial nuclease, which ought to be unacceptable. I also provide information on pre-clinical studies using ZFN that are now in clinical trials. Thus, more studies are needed to demonstrate safety in ZFN/TALEN studies with respect to off-targets.
Zinc finger nuclease
Nuclease
FokI
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Cleave
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Zinc finger nuclease
Genome Engineering
Guide RNA
Homing endonuclease
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Gene targeting in mice, first reported 25 years ago, has led to monumental advances in the understanding of basic biology and human disease. The ability to employ a similarly straightforward method for gene manipulation in other experimental organisms would make their already significant contributions all the more powerful. Here, we briefly outline the strengths and weaknesses of reverse genetics techniques in non-murine model organisms, ending with a more detailed description of two that promise to bring targeted gene modification to the masses: zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Dana Caroll, a forefather of zinc finger technology, and Bo Zhang, among the first to introduce TALEN-targeted mutagenesis to zebrafish, discuss their experience with these techniques, and speculate about the future of the field.
Zinc finger nuclease
Genome Engineering
Reverse Genetics
Gene targeting
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The pig is an important livestock for food supply and an ideal model for various human diseases. Efficient and precise genetic engineering in pigs holds great promise in agriculture and biomedicine1. Using currently available approach, generating specific gene modifications in pigs requires two steps. First, site-specific nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are used to generate targeted mutations in pig somatic cells. Then the engineered somatic nucleus is used to generate cloned animals using somatic cell nuclear transfer (SCNT) technology2,3. The complex design and generation of ZFNs and TALENs, as well as the technical challenges of SCNT, greatly limit the application of this method.
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Genome Engineering
Gene targeting
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Zinc-finger nucleases have proven to be successful as reagents for targeted genome manipulation in Drosophila melanogaster and many other organisms. Their utility has been limited, however, by the significant failure rate of new designs, reflecting the complexity of DNA recognition by zinc fingers. Transcription activator-like effector (TALE) DNA-binding domains depend on a simple, one-module-to-one-base-pair recognition code, and they have been very productively incorporated into nucleases (TALENs) for genome engineering. In this report we describe the design of TALENs for a number of different genes in Drosophila, and we explore several parameters of TALEN design. The rate of success with TALENs was substantially greater than for zinc-finger nucleases , and the frequency of mutagenesis was comparable. Knockout mutations were isolated in several genes in which such alleles were not previously available. TALENs are an effective tool for targeted genome manipulation in Drosophila.
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Genome Engineering
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Zinc finger nuclease
Genome Engineering
Gene targeting
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Zinc finger nuclease
Genome Engineering
Gene targeting
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Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been successfully used to knock out endogenous genes in stem cell research. However, the deficiencies of current gene-based delivery systems may hamper the clinical application of these nucleases. A new delivery method that can improve the utility of these nucleases is needed. In this study, we utilized a cell-penetrating peptide-based system for ZFN and TALEN delivery. Functional TAT-ZFN and TAT-TALEN proteins were generated by fusing the cell-penetrating TAT peptide to ZFN and TALEN, respectively. However, TAT-ZFN was difficult to purify in quantities sufficient for analysis in cell culture. Purified TAT-TALEN was able to penetrate cells and disrupt the gene encoding endogenous human chemokine (C-C motif) receptor 5 (CCR5, a co-receptor for HIV-1 entry into cells). Hypothermic treatment greatly enhanced the TAT-TALEN-mediated gene disruption efficiency. A 5% modification rate was observed in human induced pluripotent stem cells (hiPSCs) treated with TAT-TALEN as measured by the Surveyor assay. TAT-TALEN protein-mediated gene disruption was applicable in hiPSCs and represents a promising technique for gene knockout in stem cells. This new technique may advance the clinical application of TALEN technology.
Zinc finger nuclease
Gene knockout
Gene targeting
Genome Engineering
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For a number of decades, attempts have been made to successfully produce transgenic animals which have numerous applications in the biotechnology industry with the foremost emphasis on production of monoclonal antibodies and recombinant proteins of human welfare. Different techniques are adopted in order to produce transgenic farm animals which could be further used as bioreactors. The most common traditional transgenesis technique employed is Somatic Cell Nuclear Transfer (SCNT) using genetically modified somatic cells or stem cells as nuclear donors. This review article summarizes the merits and demerits of the techniques currently used to produce transgenic livestock with major emphasis on somatic cell nuclear transfer. In the end, a brief discussion is done about the novel methods adopted to produce transgenic animals like Zinc Finger Nucleases (ZFN), Transcription Activator-like Effector Nuclease (TALEN) and Clustered regularly interspaced short palindromic repeats (CRISPR). It is expected that the new techniques developed would overcome the problems faced with existing traditional transgenesis methods.
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Genome Engineering
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Genome editing technologies are important for functional genomics study and application. Zinc finger nucleases(ZFNs), transcription activitor-like effector nucleases(TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR associated protein(CRISPR/Cas) system are three major genome editing technologies established in recent years. Mutagenesis induced by these three techniques is mainly through making double strand break(DSB) at a specific site and followed by DSB repair process. ZFNs is the first established genome editing technology which could be used to operate site-specific knock out and knock in. However, the ZFNs technology suffers from construction complexity, high cost and other problems. The TALENs technology, which was developed based on the ZFNs technology, is much better than ZFNs technology for higher flexibility and lower cost. The CRISPR/Cas system is different from ZFNs and TALENs technologies for its unique targeting mechanism which makes this technology more suitable for multiplexed targeting. Until now, all these technologies have been successfully tested in a number of organisms, e.g., mouse, zebrafish, fruit fly, nematode, silkworm. These genome editing tools will play important roles in future functional genomics study in the post genome era.
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Genome Engineering
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