Evaluation of multiple gene targeting in porcine embryos by the CRISPR/Cas9 system using electroporation
Evaluation of multiple gene targeting in porcine embryos by the CRISPR/Cas9 system using electroporation
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DOI：
10.1007/s11033-020-05576-3
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发表时间：
2020-06-09
影响因子：
2.8
通讯作者：
Otoi, Takeshige
中科院分区：
文献类型：
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作者：
Hirata, Maki;Wittayarat, Manita;Otoi, Takeshige
CRISPR/Cas9系统现在允许前所未有的基因组编辑可能性。然而，存在一些限制，包括实现高效的一步多基因组靶向以节省成本、时间并确保高质量。在本研究中，我们研究了通过电穿孔在猪受精卵中使用靶向CMAH、GHR、GGTA 1和PDX 1的混合引导RNA（gRNA）进行一步多基因修饰的效率。我们首先根据对胚胎发育和突变效率的影响，从每个基因的三种不同设计中选择表现最好的gRNA。三种gRNA对每个靶基因的囊胚形成率显示出等同的作用;然而，gRNA CMAH#2、GHR#3、GGTA 1#3和PDX1#3显示出最高的双等位基因突变率，尽管PDX1#3的总突变率显著低于PDX1#1。因此，在电穿孔中使用CMAH#2、GHR#3、GGTA1#3和PDX1#1作为混合物，以进一步阐明是否可以同时靶向多个基因。在每个基因的靶位点对43个囊胚进行单独测序，分别在24个（55.8%）和9个（20.9%）囊胚中显示一个和两个靶基因突变。在10个（23.3%）囊胚中未检测到任何靶基因突变，并且没有囊胚具有三个或更多靶基因突变。这些结果表明，电穿孔可以有效地将多种gRNA和Cas9蛋白递送到猪受精卵中，以在一步过程中靶向多个基因。然而，该技术还需要进一步发展，以提高多重基因修饰的成功率。
The CRISPR/Cas9 system now allows for unprecedented possibilities of genome editing. However, there are some limitations, including achieving efficient one-step multiple genome targeting to save costs, time, and ensure high quality. In the present study, we investigated the efficiency of one-step multiple gene modification by electroporation in porcine zygotes using pooled guide RNAs (gRNAs) targetingCMAH,GHR,GGTA1, andPDX1. We first selected the best-performing gRNA from three different designs for each gene based on the effect on embryo development and mutation efficiency. The three gRNAs showed equivalent effects on the rates of blastocyst formation in each targeted gene; however, gRNAsCMAH#2,GHR#3,GGTA1#3, andPDX1#3 showed the highest biallelic mutation rate, although the total mutation rate ofPDX1#3 was significantly lower than that ofPDX1#1. Therefore,CMAH#2,GHR#3,GGTA1#3, andPDX1#1 were used as a mixture in electroporation to further clarify whether multiple genes can be targeted simultaneously. Individual sequencing of 43 blastocysts at the target sites of each gene showed mutations in one and two target genes in twenty-four (55.8%) and nine (20.9%) blastocysts, respectively. No mutation was detected in any target gene in ten (23.3%) blastocysts and no blastocysts had a mutation in three or more target genes. These results indicate that electroporation could effectively deliver multiple gRNAs and Cas9 protein into porcine zygotes to target multiple genes in a one-step process. However, the technique requires further development to increase the success rate of multiple gene modification.