Ma Sanyuan, Liu Yue, Liu Yuanyuan, Chang Jiasong, Zhang Tong, Wang Xiaogang, Shi Run, Lu Wei, Xia Xiaojuan, Zhao Ping, Xia Qingyou
State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400716 PR China; Chongqing Engineering and Technology Research Center for Novel Silk Materials, Southwest University, 2, Tiansheng Road, Beibei, Chongqing 400716, China.
State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400716 PR China.
Insect Biochem Mol Biol. 2017 Apr;83:13-20. doi: 10.1016/j.ibmb.2017.02.003. Epub 2017 Feb 9.
Genome editing enabled unprecedented new opportunities for targeted genomic engineering of a wide variety of organisms ranging from microbes, plants, animals and even human embryos. The serial establishing and rapid applications of genome editing tools significantly accelerated Bombyx mori (B. mori) research during the past years. However, the only CRISPR system in B. mori was the commonly used SpCas9, which only recognize target sites containing NGG PAM sequence. In the present study, we first improve the efficiency of our previous established SpCas9 system by 3.5 folds. The improved high efficiency was also observed at several loci in both BmNs cells and B. mori embryos. Then to expand the target sites, we showed that two newly discovered CRISPR system, SaCas9 and AsCpf1, could also induce highly efficient site-specific genome editing in BmNs cells, and constructed an integrated CRISPR system. Genome-wide analysis of targetable sites was further conducted and showed that the integrated system cover 69,144,399 sites in B. mori genome, and one site could be found in every 6.5 bp. The efficiency and resolution of this CRISPR platform will probably accelerate both fundamental researches and applicable studies in B. mori, and perhaps other insects.
基因组编辑为从微生物、植物、动物乃至人类胚胎等多种生物的靶向基因组工程带来了前所未有的新机遇。在过去几年中，基因组编辑工具的系列建立和快速应用显著加速了家蚕的研究。然而，家蚕中唯一的CRISPR系统是常用的SpCas9，它只识别含有NGG PAM序列的靶位点。在本研究中，我们首先将之前建立的SpCas9系统的效率提高了3.5倍。在BmNs细胞和家蚕胚胎的几个位点也观察到了提高后的高效率。然后，为了扩大靶位点范围，我们发现两种新发现的CRISPR系统SaCas9和AsCpf1也能在BmNs细胞中诱导高效的位点特异性基因组编辑，并构建了一个整合的CRISPR系统。我们进一步对可靶向位点进行了全基因组分析，结果表明该整合系统覆盖了家蚕基因组中的69,144,399个位点，平均每6.5个碱基对中就有一个位点。这个CRISPR平台的效率和分辨率可能会加速家蚕以及其他昆虫的基础研究和应用研究。
