Improving the on-target activity of high-fidelity Cas9 editors by combining rational design and random mutagenesis

被引:6
作者
Spasskaya, Daria S. [1 ]
Davletshin, Artem I. [2 ]
Bachurin, Stanislav S. [3 ]
Tutyaeva, Vera V. [1 ]
Garbuz, David G. [1 ]
Karpov, Dmitry S. [1 ]
机构
[1] Russian Acad Sci, Ctr Precis Genome Editing & Genet Technol Biomed, Engelhardt Inst Mol Biol, Vavilov St 32, Moscow 119991, Russia
[2] Russian Acad Sci, Engelhardt Inst Mol Biol, Vavilov St 32, Moscow 119991, Russia
[3] FSBEI HE Rostov State Med Univ, Minist Hlth, Nakhichevanskiy Lane 29, Rostov Na Donu 344022, Russia
基金
俄罗斯科学基金会;
关键词
CRISPR; Cas9; Genome editing; High-fidelity Cas9 variants; On-target activity; GENOME-WIDE ANALYSIS; DNA CLEAVAGE; CRISPR-CAS9; NUCLEASES; RNA; ENDONUCLEASE; COMPLEX; YEAST; KINETICS; DYNAMICS; VARIANT;
D O I
10.1007/s00253-023-12469-5
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Genomic and post-genomic editors based on CRISPR/Cas systems are widely used in basic research and applied sciences, including human gene therapy. Most genome editing tools are based on the CRISPR/Cas9 type IIA system from Streptococcus pyogenes. Unfortunately, a number of drawbacks have hindered its application in therapeutic approaches, the most serious of which is the relatively high level of off-targets. To overcome this obstacle, various high-fidelity Cas9 variants have been created. However, they show reduced on-target activity compared to wild-type Cas9 possibly due to increased sensitivity to eukaryotic chromatin. Here, we combined a rational approach with random mutagenesis to create a set of new Cas9 variants showing high specificity and increased activity in Saccharomyces cerevisiae yeast. Moreover, a novel mutation in the PAM (protospacer adjacent motif)-interacting Cas9 domain was found, which increases the on-target activity of high-fidelity Cas9 variants while retaining their high specificity. The obtained data suggest that this mutation acts by weakening the eukaryotic chromatin barrier for Cas9 and rearranging the RuvC active center. Improved Cas9 variants should further advance genome and post-genome editing technologies.
引用
收藏
页码:2385 / 2401
页数:17
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