Guo Xiaochen, Geng Lishuang, Jiang Chaoqian, Yao Wang, Jin Junxue, Liu Zhonghua, Mu Yanshuang
Key Laboratory of Animal Cellular and Genetic Engineering of Heilongjiang Province, College of Life Science, Northeast Agricultural University, Harbin, China.
Anim Biotechnol. 2023 Dec;34(9):4703-4712. doi: 10.1080/10495398.2023.2187402. Epub 2023 Mar 22.
Multiplex gene modifications are highly required for various fields of porcine research. In many species, the CRISPR/Cas9 system has been widely applied for genomic editing and provides a potential tool for introducing multiplex genome mutations simultaneously. Here, we present a CRISPR-Cas9 gRNA-tRNA array (GTR-CRISPR) for multiplexed engineering of porcine fetal fibroblasts (PFFs). We successfully produced multiple sgRNAs using only one Pol III promoter by taking advantage of the endogenous tRNA processing mechanism in porcine cells. Using an all-in-one construct carrying GTR and Cas9, we disrupted the , , , and genes in multiple codon regions in one PFF cell simultaneously. This technique allows the simultaneous disruption of four genes with 5.5% efficiency. As a result, this approach may effectively target multiple genes at the same time, making it a powerful tool for establishing multiple genes mutant cells in pigs.
猪研究的各个领域都高度需要多重基因修饰。在许多物种中，CRISPR/Cas9系统已被广泛应用于基因组编辑，并为同时引入多重基因组突变提供了一个潜在工具。在此，我们展示了一种用于猪胎儿成纤维细胞（PFFs）多重工程改造的CRISPR-Cas9 gRNA-tRNA阵列（GTR-CRISPR）。我们利用猪细胞中的内源性tRNA加工机制，仅使用一个Pol III启动子成功产生了多个sgRNA。使用携带GTR和Cas9的一体化构建体，我们在一个PFF细胞的多个密码子区域同时破坏了、、和基因。该技术以5.5%的效率同时破坏四个基因。因此，这种方法可能有效地同时靶向多个基因，使其成为在猪中建立多个基因突变细胞的强大工具。
