Discovery and Engineering of Small SlugCas9 with Broad Targeting Range and High Specificity and Activity

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2021 Mar 15

PMID 33721016

Citations 27

Authors

Ziying Hu

Chengdong Zhang

Shuai Wang

Siqi Gao

Jingjing Wei

Miaomiao Li

Linghui Hou

Huilin Mao

YanYan Wei

Tao Qi

Hongmao Liu

Dong Liu

Feng Lan

Daru Lu

Hongyan Wang

Jixi Li

Yongming Wang

Affiliations

Soon will be listed here.

Abstract

The compact CRISPR/Cas9 system, which can be delivered with their gRNA and a full-length promoter for expression by a single adeno-associated virus (AAV), is a promising platform for therapeutic applications. We previously identified a compact SauriCas9 that displays high activity and requires a simple NNGG PAM, but the specificity is moderate. Here, we identified three compact Cas9 orthologs, Staphylococcus lugdunensis Cas9 (SlugCas9), Staphylococcus lutrae Cas9 (SlutrCas9) and Staphylococcus haemolyticus Cas9 (ShaCas9), for mammalian genome editing. Of these three Cas9 orthologs, SlugCas9 recognizes a simple NNGG PAM and displays comparable activity to SaCas9. Importantly, we generated a SlugCas9-SaCas9 chimeric nuclease, which has both high specificity and high activity. We finally engineered SlugCas9 with mutations to generate a high-fidelity variant that maintains high specificity without compromising on-target editing efficiency. Our study offers important minimal Cas9 tools that are ideal for both basic research and clinical applications.

Citing Articles

Engineering of SauriCas9 with enhanced specificity.

Zhang X, Tao C, Li M, Zhang S, Liang P, Huang Y Mol Ther Nucleic Acids. 2025; 36(1):102455.

PMID: 40027883 PMC: 11869866. DOI: 10.1016/j.omtn.2025.102455.

Enhanced genome editing with a Streptococcus equinus Cas9.

Liu J, Wang Y, Wei J, Wang S, Li M, Huang Z Commun Biol. 2025; 8(1):196.

PMID: 39920233 PMC: 11806022. DOI: 10.1038/s42003-025-07593-z.

Next-generation CRISPR technology for genome, epigenome and mitochondrial editing.

Lau C, Liang Q, Zhu H Transgenic Res. 2024; 33(5):323-357.

PMID: 39158822 DOI: 10.1007/s11248-024-00404-x.

Characterization of NiCas12b for In Vivo Genome Editing.

Zhang Y, Wei J, Wang H, Wang Y Adv Sci (Weinh). 2024; 11(36):e2400469.

PMID: 39076074 PMC: 11423069. DOI: 10.1002/advs.202400469.

Developing small Cas9 hybrids using molecular modeling.

Mangin A, Dion V, Menzies G Sci Rep. 2024; 14(1):17233.

PMID: 39060399 PMC: 11282279. DOI: 10.1038/s41598-024-68107-1.

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