caa a22 4500 606177140 CHVBK 20210128100750.0 cr unu---uuuuu 210128e20150101xx s 000 0 eng 10.1007/s00018-014-1679-z doi (NATIONALLICENCE)springer-10.1007/s00018-014-1679-z Efficient genome engineering in eukaryotes using Cas9 from Streptococcus thermophilus [Elektronische Daten] [Kun Xu, Chonghua Ren, Zhongtian Liu, Tao Zhang, Tingting Zhang, Duo Li, Ling Wang, Qiang Yan, Lijun Guo, Juncen Shen, Zhiying Zhang] 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. Springer Basel, 2014 S. thermophilus Cas9 nationallicence Single guide RNA nationallicence Surrogate reporter nationallicence DNA repair nationallicence Genome targeting nationallicence Xu Kun Key Laboratory of Molecular Biology of Shaanxi Agriculture, College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut Ren Chonghua Key Laboratory of Molecular Biology of Shaanxi Agriculture, College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut Liu Zhongtian Key Laboratory of Molecular Biology of Shaanxi Agriculture, College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut Zhang Tao Key Laboratory of Molecular Biology of Shaanxi Agriculture, College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut Zhang Tingting Key Laboratory of Molecular Biology of Shaanxi Agriculture, College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut Li Duo Key Laboratory of Molecular Biology of Shaanxi Agriculture, College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut Wang Ling Key Laboratory of Molecular Biology of Shaanxi Agriculture, College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut Yan Qiang Key Laboratory of Molecular Biology of Shaanxi Agriculture, College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut Guo Lijun Key Laboratory of Molecular Biology of Shaanxi Agriculture, College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut Shen Juncen Key Laboratory of Molecular Biology of Shaanxi Agriculture, College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut Zhang Zhiying Key Laboratory of Molecular Biology of Shaanxi Agriculture, College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut Cellular and Molecular Life Sciences Springer Basel 72/2(2015-01-01), 383-399 1420-682X 72:2<383 2015 72 18 https://doi.org/10.1007/s00018-014-1679-z text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00018-014-1679-z text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Xu Kun Key Laboratory of Molecular Biology of Shaanxi Agriculture, College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut NATIONALLICENCE 700 1- Ren Chonghua Key Laboratory of Molecular Biology of Shaanxi Agriculture, College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut NATIONALLICENCE 700 1- Liu Zhongtian Key Laboratory of Molecular Biology of Shaanxi Agriculture, College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut NATIONALLICENCE 700 1- Zhang Tao Key Laboratory of Molecular Biology of Shaanxi Agriculture, College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut NATIONALLICENCE 700 1- Zhang Tingting Key Laboratory of Molecular Biology of Shaanxi Agriculture, College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut NATIONALLICENCE 700 1- Li Duo Key Laboratory of Molecular Biology of Shaanxi Agriculture, College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut NATIONALLICENCE 700 1- Wang Ling Key Laboratory of Molecular Biology of Shaanxi Agriculture, College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut NATIONALLICENCE 700 1- Yan Qiang Key Laboratory of Molecular Biology of Shaanxi Agriculture, College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut NATIONALLICENCE 700 1- Guo Lijun Key Laboratory of Molecular Biology of Shaanxi Agriculture, College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut NATIONALLICENCE 700 1- Shen Juncen Key Laboratory of Molecular Biology of Shaanxi Agriculture, College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut NATIONALLICENCE 700 1- Zhang Zhiying Key Laboratory of Molecular Biology of Shaanxi Agriculture, College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China aut NATIONALLICENCE 773 0- Cellular and Molecular Life Sciences Springer Basel 72/2(2015-01-01), 383-399 1420-682X 72:2<383 2015 72 18