Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair.
Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair.
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DOI：
10.1101/gr.161638.113
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发表时间：
2014-01
期刊：
影响因子：
7
通讯作者：
Del Bene F
中科院分区：
文献类型：
--
作者：
Auer TO;Duroure K;De Cian A;Concordet JP;Del Bene F
序列特异性核酸酶如TALEN和CRISPR/Cas9系统极大地扩展了模式生物如斑马鱼的基因组编辑可能性。这两种系统最近已被用于以极大的效率产生敲除等位基因，并且TALEN也已成功地用于通过同源重组（HR）在限定的基因座处敲入DNA盒。在这里，我们报告了CRISPR/Cas9介导的DNA盒通过同源非依赖性双链断裂（DSB）修复途径以非常高的速率敲入斑马鱼基因组。在供体质粒与短向导RNA（sgRNA）和Cas9核酸酶mRNA共注射后，供体质粒DNA和所选染色体整合位点的同时切割导致供体DNA的有效靶向整合。我们成功地采用这种方法将eGFP转化为Gal 4转基因系，并且相同的质粒和sgRNA可以应用于任何物种，其中产生eGFP系作为增强子和基因陷阱筛选的一部分。此外，我们显示了在内源基因座容易地靶向DNA整合的可能性，从而极大地促进了报告基因和功能丧失等位基因的创建。由于其简单性，灵活性和非常高的效率，我们的方法大大扩展了斑马鱼基因组编辑的库，并且可以很容易地适应许多其他生物。
Sequence-specific nucleases like TALENs and the CRISPR/Cas9 system have greatly expanded the genome editing possibilities in model organisms such as zebrafish. Both systems have recently been used to create knock-out alleles with great efficiency, and TALENs have also been successfully employed in knock-in of DNA cassettes at defined loci via homologous recombination (HR). Here we report CRISPR/Cas9-mediated knock-in of DNA cassettes into the zebrafish genome at a very high rate by homology-independent double-strand break (DSB) repair pathways. After co-injection of a donor plasmid with a short guide RNA (sgRNA) and Cas9 nuclease mRNA, concurrent cleavage of donor plasmid DNA and the selected chromosomal integration site resulted in efficient targeted integration of donor DNA. We successfully employed this approach to convert eGFP into Gal4 transgenic lines, and the same plasmids and sgRNAs can be applied in any species where eGFP lines were generated as part of enhancer and gene trap screens. In addition, we show the possibility of easily targeting DNA integration at endogenous loci, thus greatly facilitating the creation of reporter and loss-of-function alleles. Due to its simplicity, flexibility, and very high efficiency, our method greatly expands the repertoire for genome editing in zebrafish and can be readily adapted to many other organisms.
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影响因子：
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