title: Efficient Production of Gene-Modified Mice using Staphylococcus aureus Cas9.
abstract: The CRISPR/Cas system is an efficient genome-editing tool to modify genes in mouse zygotes. However, only the Streptococcus pyogenes Cas9 (SpCas9) has been systematically tested for generating gene-modified mice. The protospacer adjacent motif (PAM, 5'-NGG-3') recognized by SpCas9 limits the number of potential target sites for this system. Staphylococcus aureus Cas9 (SaCas9), with its smaller size and unique PAM (5'-NNGRRT-3') preferences, presents an alternative for genome editing in zygotes. Here, we showed that SaCas9 could efficiently and specifically edit the X-linked gene Slx2 and the autosomal gene Zp1 in mouse zygotes. SaCas9-mediated disruption of the tyrosinase (Tyr) gene led to C57BL/6J mice with mosaic coat color. Furthermore, multiplex targeting proved efficient multiple genes disruption when we co-injected gRNAs targeting Slx2, Zp1, and Tyr together with SaCas9 mRNA. We were also able to insert a Flag tag at the C-terminus of histone H1c, when a Flag-encoding single-stranded DNA oligo was co-introduced into mouse zygotes with SaCas9 mRNA and the gRNA. These results indicate that SaCas9 can specifically cleave the target gene locus, leading to successful gene knock-out and precise knock-in in mouse zygotes, and highlight the potential of using SaCas9 for genome editing in preimplantation embryos and producing gene-modified animal models.
authors: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510275, China.|Key Laboratory of Gene Engineering of the Ministry of Education, Guangzhou Key Laboratory of Healthy Aging Research and State Key Laboratory of Biocontrol, SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.
authors: Xiya
authors: X
authors: Zhang
authors: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510275, China.|Key Laboratory of Gene Engineering of the Ministry of Education, Guangzhou Key Laboratory of Healthy Aging Research and State Key Laboratory of Biocontrol, SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.|Key Laboratory of Reproductive Medicine of Guangdong Province, School of Life Sciences and the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510275, China.
authors: Puping
authors: P
authors: Liang
authors: Key Laboratory of Reproductive Medicine of Guangdong Province, School of Life Sciences and the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510275, China.
authors: Chenhui
authors: C
authors: Ding
authors: Key Laboratory of Gene Engineering of the Ministry of Education, Guangzhou Key Laboratory of Healthy Aging Research and State Key Laboratory of Biocontrol, SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.
authors: Zhen
authors: Z
authors: Zhang
authors: Department of Pathology, First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, China.
authors: Jianwen
authors: J
authors: Zhou
authors: Key Laboratory of Gene Engineering of the Ministry of Education, Guangzhou Key Laboratory of Healthy Aging Research and State Key Laboratory of Biocontrol, SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.
authors: Xiaowei
authors: X
authors: Xie
authors: Key Laboratory of Gene Engineering of the Ministry of Education, Guangzhou Key Laboratory of Healthy Aging Research and State Key Laboratory of Biocontrol, SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.
authors: Rui
authors: R
authors: Huang
authors: Key Laboratory of Gene Engineering of the Ministry of Education, Guangzhou Key Laboratory of Healthy Aging Research and State Key Laboratory of Biocontrol, SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.
authors: Ying
authors: Y
authors: Sun
authors: Key Laboratory of Gene Engineering of the Ministry of Education, Guangzhou Key Laboratory of Healthy Aging Research and State Key Laboratory of Biocontrol, SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.
authors: Hongwei
authors: H
authors: Sun
authors: Key Laboratory of Gene Engineering of the Ministry of Education, Guangzhou Key Laboratory of Healthy Aging Research and State Key Laboratory of Biocontrol, SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.
authors: Jinran
authors: J
authors: Zhang
authors: Key Laboratory of Reproductive Medicine of Guangdong Province, School of Life Sciences and the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510275, China.
authors: Yanwen
authors: Y
authors: Xu
authors: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510275, China.|Key Laboratory of Gene Engineering of the Ministry of Education, Guangzhou Key Laboratory of Healthy Aging Research and State Key Laboratory of Biocontrol, SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.|Key Laboratory of Reproductive Medicine of Guangdong Province, School of Life Sciences and the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510275, China.
authors: Zhou
authors: Z
authors: Songyang
authors: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510275, China.|Key Laboratory of Gene Engineering of the Ministry of Education, Guangzhou Key Laboratory of Healthy Aging Research and State Key Laboratory of Biocontrol, SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.|Key Laboratory of Reproductive Medicine of Guangdong Province, School of Life Sciences and the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510275, China.
authors: Junjiu
authors: J
authors: Huang
mesh_terms: D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D004260:DNA Repair; D004622:Embryo, Mammalian / Q000378:metabolism; D018703:Founder Effect; D055879:Gene Knock-In Techniques; D018390:Gene Targeting; D005818:Genetic Engineering / Q000379:methods*; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D008822:Mice, Transgenic; D009030:Mosaicism; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D013211:Staphylococcus aureus / Q000235:genetics*
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems