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The Development of SpCas9 Variants with High Specificity and Efficiency Based on the HH Theory

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Abstract

Streptococcus pyogenes Cas9 (SpCas9) is the most popular tool in gene editing; however, off-target mutagenesis is one of the biggest impediments in its application. In our previous study, we proposed the HH theory, which states that sgRNA/DNA hybrid (hybrid) extrusion-induced enhancement of hydrophobic interactions between the hybrid and REC3/HNH is a key factor in cleavage initiation. Based on the HH theory, we analyzed the interactions between the REC3 domain and hybrid and obtained 8 mutant sites. We designed 8 SpCas9 variants (V1–V8), used digital droplet PCR to assess SpCas9-induced DNA indels in human cells, and developed high-fidelity variants. Thus, the HH theory may be employed to further optimize SpCas9-mediated genome editing systems, and the resultant V3, V6, V7, and V8 SpCas9 variants may be valuable for applications requiring high-precision genome editing.

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Funding

This work was supported by the Characteristic Innovation Project in General Colleges and Universities of Guangdong Province (2022KTSCX230), National Natural Science Foundation of China (Grant no. 81601654), Natural Science Foundation of Guangdong Province, China (Grant nos. 2014A030310090 and 2016A030313578), Medical Scientific Research Foundation of Guangdong Province, China (Grant no. A2015207), and Pearl River S&T Nova Program of Guangzhou (201806010037), Medical Science and Technology Research Foundation of Guangdong Province (no. A2023283), Nanshan District Science and Technology Plan Project (no. NS2022077).

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Authors and Affiliations

  1. College of Life and Health Sciences, Guangdong Industry Polytechnic, 510300, Guangzhou, China

    G. H. Wang

  2. Department of Pharmacy, Nanfang Hospital, Southern Medical University, 510515, Guangzhou, China

    C. M. Wang

  3. Department of Pharmacy, Southern University of Science and Technology Hospital, 518000, Shenzhen, China

    C. M. Wang

  4. Lineberger Comprehensive Cancer Center, the University of North Carolina at Chapel Hill, Chapel Hill, 27517, North Carolina, USA

    X. J. Wu

  5. Sangon Biotech, 201611, Shanghai, China

    T. Chu

  6. Pharmaceutical and Material Engineering School, Jinhua Polytechnic, 321007, Jinhua, China

    D. W. Huang

  7. Department of Histology and Embryology, School of Basic Medical Sciences, Southern Medical University, 510515, Guangzhou, China

    J. Li

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Contributions

Guohua Wang: conceptualization, methodology, formal analysis, investigation, data curation, visualization, writing—original draft, review, and editing. Canmao Wang: methodology, conceptualization, investigation, writing— review and editing. Xinjun Wu: methodology, investigation, writing—review and editing. Teng Chu: methodology, writing—review and editing. Dongwei Huang: methodology and review. Juan Li: conceptualization, methodology, investigation, supervision, writing—original draft, review, and editing, funding acquisition.

Corresponding author

Correspondence to J. Li.

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CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

The article does not contain any studies involving humans or animals in experiments performed by any of the authors.

ADDITIONAL INFORMATION

SpCas9 structures (PDB ID: 4OO8, 6O0Z, 6O0Y) were analyzed using PyMOL 2.4 (Schrödinger). All 3D structure figures were generated using PyMOL2.4 (Schrödinger).

Strains and plasmids are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, and tables.

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These authors contributed equally to this work.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1134/S0026893324010187.

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Wang, G.H., Wang, C.M., Wu, X.J. et al. The Development of SpCas9 Variants with High Specificity and Efficiency Based on the HH Theory. Mol Biol 58, 133–146 (2024). https://doi.org/10.1134/S0026893324010187

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