Programmable Single RNA-Guided CRISPR/Cas9 Endonuclease Mediates Specific DNA Cleavage in Yeast

• 作 者: Xu Kun Ren Chonghua Liu Zhongtian Zhang Zhiying
• 机 构: College of Animal Science and Technology, Northwest A&F University, No.22 Xinong Road, Yangling 7121
• 出 处: 第七次全国动物生物技术学术研讨会暨新疆畜牧科学院第六次学术年会
• 发表日期: 2013年5期

　　Bacteria and archaea have evolved RNA-mediated adaptive defense systems called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) that protect organisms from invading viruses and plasmids.Here, we report the use of the programmed type II bacterial Streptococcus Thermophilus CRISPR/Cas9 system in yeast (Saccharomyces cerevisiae) for reporter DNA cleavage and genome engineering.The programmable CRISPR/Cas9 system is composed of Cas9 gene which was cloned from Streptococcus Thermophilus (Sth.Cas9) and a guide RNA (gRNA) with was designed according the bacterial defenses system.The system shows specific RNA-guided endonuclease activity at targets both designed in reporter plasmids and integrated in yeast genome.The designed wild type gRNA (WT.gRNA) consists of several domains including Target, Bulge, Match, Loop and TracrRNA (Fig 1).In our research, the domains were optimized and an Opti.gRNA was designed, which is much simpler to construct with higher activity.