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Efficient genome engineering in eukaryotes using Cas9 from Streptococcus thermophilus.

Cellular and molecular life sciences : CMLS (2014-07-21)
Kun Xu, Chonghua Ren, Zhongtian Liu, Tao Zhang, Tingting Zhang, Duo Li, Ling Wang, Qiang Yan, Lijun Guo, Juncen Shen, Zhiying Zhang
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
2-Nitrophenyl β-D-galactopyranoside, ≥98% (enzymatic)
Histidine, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
Sodium trichloroacetate, 97%
Sigma-Aldrich
2-Nitrophenyl β-D-galactopyranoside, ≥99.0% (HPLC)
Sigma-Aldrich
DL-Leucine, ≥99% (HPLC)
Sigma-Aldrich
DL-Histidine, ≥99% (TLC)
Leucine, European Pharmacopoeia (EP) Reference Standard