title: An integrated CRISPR Bombyx mori genome editing system with improved efficiency and expanded target sites.
abstract: 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.
authors.affiliation: 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.
authors.forename: Sanyuan
authors.initials: S
authors.lastname: Ma
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400716 PR China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400716 PR China.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400716 PR China.
authors.forename: Jiasong
authors.initials: J
authors.lastname: Chang
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400716 PR China.
authors.forename: Tong
authors.initials: T
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400716 PR China.
authors.forename: Xiaogang
authors.initials: X
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400716 PR China.
authors.forename: Run
authors.initials: R
authors.lastname: Shi
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400716 PR China.
authors.forename: Wei
authors.initials: W
authors.lastname: Lu
authors.affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400716 PR China.
authors.forename: Xiaojuan
authors.initials: X
authors.lastname: Xia
authors.affiliation: 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.
authors.forename: Ping
authors.initials: P
authors.lastname: Zhao
authors.affiliation: 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. Electronic address: xiaqy@swu.edu.cn.
authors.forename: Qingyou
authors.initials: Q
authors.lastname: Xia
mesh_terms: D000818:Animals; D012831:Bombyx / Q000235:genetics*; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D000072669:Gene Editing / Q000379:methods*
keywords: Bombyx mori; Cas9; Cpf1; Editing efficiency; Genome editing; Target site