Deleting specific residues from the HNH linkers creates a CRISPR-SpCas9 variant with high fidelity and efficiency

Wang，Guohua1; Wang，Canmao2,3; Chu，Teng4; Wu，Xinjun5; Anderson，Christopher M.6; Huang，Dongwei7; Li，Juan8

2023-05-20

10.1016/j.jbiotec.2023.04.008

Journal of Biotechnology   影响因子和分区

0168-1656

1873-4863

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems are immunological defenses used in archaea and bacteria to recognize and destroy DNA from external invaders. The CRISPR-SpCas9 system harnessed from Streptococcus pyogenes (SpCas9) has become the most widely utilized genome editing tool and shows promise for clinical application. However, the off-target effect is still the major challenge for the genome editing of CRISPR-SpCas9. Based on analysis of the structure and cleavage procedures, we proposed two strategies to modify the SpCas9 structure and reduce off-target effects. Shortening the HNH or REC3 linkers (Strategy #1) aimed to move the primary position of HNH or REC3 far away from the single-guide RNA (sgRNA)/DNA hybrid (hybrid), while elongating the helix around the sgRNA (Strategy #2) aimed to strengthen the contacts between SpCas9 and the sgRNA/DNA. We designed 11 SpCas9 variants (variant No.1– variant No.11) and verified their efficiencies on the classic genome site EMX1–1, EMX1–1-OT1, and EMX1–1-OT2. The top three effective SpCas9 variants, variant No.1, variant No.2, and variant No.5, were additionally validated on other genome sites. The further selected variant No.1 was compared with two previous SpCas9 variants, HypaCas9 (a hyper-accurate Cas9 variant released in 2017) and eSpCas9 (1.1) (an “enhanced specificity” SpCas9 variant released in 2016), on two genome sites, EMX1–1 and FANCF-1. The results revealed that the deletion of Thr769 and Gly906 could substantially decrease off-target effects, while maintaining robust on-target efficiency in most of the selected genome sites.

CRISPR Design Efficiency High fidelity HNH linkers SpCas9

SCI

英语

其他

Pearl River S and T Nova Program of Guangzhou[201806010037];National Natural Science Foundation of China[81601654];

WOS:000989817000001

BIOLOGY & BIOCHEMISTRY

2-s2.0-85153569884

Scopus

1.School of Food and Biotechnology,Guangdong Industry Polytechnic,Guangzhou,510300,China
2.Department of Pharmacy,Nanfang Hospital,Southern Medical University,Guangzhou,510515,China
3.Department of Pharmacy,Southern University of Science and Technology Hospital (SUS Tech Hospital),Shenzhen,518000,China
4.Sangon Biotech (Shanghai) Co.,Ltd,Shanghai,201611,China
5.Lineberger Comprehensive Cancer Center,the University of North Carolina at Chapel Hill,Chapel Hill,27517,United States
6.Department of Biology,East Carolina University,Greenville,27517,United States
7.Pharmaceutical and Material Engineering School,Jinhua Polytechnic,Jinhua,321007,China
8.Department of Histology and Embryology,School of Basic Medical Sciences,Southern Medical University,Guangzhou,510515,China

Wang，Guohua,Wang，Canmao,Chu，Teng,et al. Deleting specific residues from the HNH linkers creates a CRISPR-SpCas9 variant with high fidelity and efficiency[J]. Journal of Biotechnology,2023,368:42-52.

Wang，Guohua.,Wang，Canmao.,Chu，Teng.,Wu，Xinjun.,Anderson，Christopher M..,...&Li，Juan.(2023).Deleting specific residues from the HNH linkers creates a CRISPR-SpCas9 variant with high fidelity and efficiency.Journal of Biotechnology,368,42-52.

Wang，Guohua,et al."Deleting specific residues from the HNH linkers creates a CRISPR-SpCas9 variant with high fidelity and efficiency".Journal of Biotechnology 368(2023):42-52.