title: Efficient genome engineering in eukaryotes using Cas9 from Streptococcus thermophilus.
abstract: The Streptococcus thermophilus CRISPR3-Cas (StCas9) system has been shown to mediate DNA cleavage in its original host and in E. coli as well as in vitro. Here, we have reconstituted the StCas9 system in yeast and conducted a systematic optimization of the sgRNA structure, including the minimal length of tracrRNA, loop structure, Match II region, Bulge motif, the minimal length of guide sequence within the crRNA, tolerance of mismatches and target sequence preference. The optimal sgRNA design for the StCas9 system achieved up to 12 and 40 % targeting efficiencies in yeast and human cells, respectively. This study provides important insight into the sequence and structural requirements necessary to develop a targeted and highly efficient eukaryotic gene editing platform using CRISPR-Cas systems.
authors.affiliation: Key Laboratory of Molecular Biology of Shaanxi Agriculture, College of Animal Science and Technology, Northwest A&F University, Yangling, 712100, Shaanxi, China.
authors.forename: Kun
authors.initials: K
authors.lastname: Xu
authors.forename: Chonghua
authors.initials: C
authors.lastname: Ren
authors.forename: Zhongtian
authors.initials: Z
authors.lastname: Liu
authors.forename: Tao
authors.initials: T
authors.lastname: Zhang
authors.forename: Tingting
authors.initials: T
authors.lastname: Zhang
authors.forename: Duo
authors.initials: D
authors.lastname: Li
authors.forename: Ling
authors.initials: L
authors.lastname: Wang
authors.forename: Qiang
authors.initials: Q
authors.lastname: Yan
authors.forename: Lijun
authors.initials: L
authors.lastname: Guo
authors.forename: Juncen
authors.initials: J
authors.lastname: Shen
authors.forename: Zhiying
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D020029:Base Pairing; D064113:CRISPR-Cas Systems / Q000235:genetics / Q000502:physiology*; D003001:Cloning, Molecular; D005818:Genetic Engineering / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017393:RNA Editing / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D012441:Saccharomyces cerevisiae; D048249:Streptococcus thermophilus / Q000235:genetics*
chemical_list: D012333:RNA, Messenger