Dong Zhanqi, Qin Qi, Zhang Xinling, Li Kejie, Chen Peng, Pan Minhui
State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China.
Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural and Rural Affairs Ministry, Southwest University, Chongqing 400716, China.
Sheng Wu Gong Cheng Xue Bao. 2021 Dec 25;37(12):4342-4350. doi: 10.13345/j.cjb.210126.
The CRISPR/Cas9 gene editing system has been widely used in basic research, gene therapy and genetic engineering due to its high efficiency, fast speed and convenience. Meanwhile, the discovery of novel CRISPR/Cas systems in the microbial community also accelerated the emergence of novel gene editing tools. CRISPR/Cpf1 is the second type (V type) CRISPR system that can edit mammalian genome. Compared with the CRISPR/Cas9, CRISPR/Cpf1 can use 5'T-PAM rich region to increase the genome coverage, and has many advantages, such as sticky end of cleavage site and less homologous recombination repair. Here we constructed three CRISPR/Cpf1 (AsCpf1, FnCpf1 and LbCpf1) expression vectors in silkworm cells. We selected a highly conserved BmHSP60 gene and an ATPase family BmATAD3A gene to design the target gRNA, and constructed gHSP60-266 and gATAD3A-346 knockout vectors. The efficiency for editing the target genes BmATAD3A and BmHSP60 by AsCpf1, FnCpf1 and LbCpf1 were analyzed by T7E1 analysis and T-clone sequencing. Moreover, the effects of target gene knockout by different gene editing systems on the protein translation of BmHSP60 and BmATAD3A were analyzed by Western blotting. We demonstrate the CRISPR/Cpf1 gene editing system developed in this study could effectively edit the silkworm genome, thus providing a novel method for silkworm gene function research, genetic engineering and genetic breeding.
CRISPR/Cas9基因编辑系统因其高效、快速和便捷，已在基础研究、基因治疗和基因工程中得到广泛应用。与此同时，微生物群落中新型CRISPR/Cas系统的发现也加速了新型基因编辑工具的出现。CRISPR/Cpf1是第二种能够编辑哺乳动物基因组的CRISPR系统（V型）。与CRISPR/Cas9相比，CRISPR/Cpf1可利用富含5'T-PAM的区域来增加基因组覆盖范围，并且具有许多优势，如切割位点的粘性末端和较少的同源重组修复。在此，我们在蚕细胞中构建了三种CRISPR/Cpf1（AsCpf1、FnCpf1和LbCpf1）表达载体。我们选择了一个高度保守的BmHSP60基因和一个ATPase家族BmATAD3A基因来设计靶向gRNA，并构建了gHSP60-266和gATAD3A-346敲除载体。通过T7E1分析和T克隆测序分析了AsCpf1、FnCpf1和LbCpf1对靶基因BmATAD3A和BmHSP60的编辑效率。此外，通过蛋白质免疫印迹分析了不同基因编辑系统对靶基因敲除对BmHSP60和BmATAD3A蛋白质翻译的影响。我们证明了本研究中开发的CRISPR/Cpf1基因编辑系统能够有效地编辑家蚕基因组，从而为家蚕基因功能研究、基因工程和遗传育种提供了一种新方法。
