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Two high-fidelity variants: efSaCas9 and SaCas9-HF, which one is better?

Gene Therapy volume 29, pages 458–463 (2022)Cite this article

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Abstract

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated endonuclease Cas9) nucleases have been widely applied for genome engineering. Staphylococcus aureus Cas9 (SaCas9) is compact, which can be packaged in AAV (adeno-associated virus) vector for in vivo gene editing. While, wild-type SaCas9 can induce unwanted off-target mutations and substantially limits the applications. So far, there are two reported SaCas9 variants with high-fidelity, including efSaCas9 from our previous study and SaCas9-HF. However, it remains unknown which one possessing the better fidelity and higher activity. Here, we performed a parallel comparison of efSaCas9 and SaCas9-HF in human cells through fluorescent reporter system and target deep sequencing, respectively. The results demonstrated that efSaCas9 possesses higher cleavage activity and fidelity than SaCas9-HF at the most endogenous sites in human cells. Collectively, our study provides insights for the rational selection of suitable SaCas9 for human genome editing.

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Fig. 1: Activity and fidelity evaluation of efSaCas9 and SaCas9-HF using FCM.
Fig. 2: Activities and fidelities of efSaCas9 and SaCas9-HF at endogenous sites.
Fig. 3: The activities and fidelities of additional mutants based on efSaCas9.

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Data availability

The deep sequencing data are available at the NCBI Sequence Read Archive (SRA) under PRJNA749863 (SRA accession number, SRR15253970 to SRR15253977, sample accession number, SAMN20423045).

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Funding

This work was supported by grants from Basic research project of Henan Eye Hospital (20JCZD001), 23456 talent project of Henan Provincial People’s Hospital, National Natural Science Foundation of China (81201181), Natural Science Foundation of Zhejiang Province (2017C37176), Project of State Key Laboratory of Ophthalmology, Optometry and Visual Science, Wenzhou Medical University (J02-20190201) and Wenzhou City Grant, Zhejiang, China (2021Y1893).

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Authors and Affiliations

  1. School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, State Key Laboratory and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China

    Jineng Lv, Xiujuan Lv, Yue Zhou, Jiahua Wang, Haoran Chen, Tong Yan & Feng Gu

  2. Reproduction Center, Department of Obstetrics and Gynecology, The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China

    Haitao Xi & Junzhao Zhao

  3. Wenzhou People’s Hospital, The Third Clinical Institute Affiliated to Wenzhou Medical University, Wenzhou Medical University, Wenzhou, Zhejiang, China

    Jiang Jin

  4. Henan Eye Hospital, Henan Provincial People’s Hospital and People’s Hospital of Zhengzhou University, People’s Hospital of Henan University, Zhengzhou, Henan, China

    Zongming Song

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Contributions

FG and ZS designed research, JL, HX, XL, YZ, JW, HC, and TY performed research and JL, J.J, JZ, ZS, and FG performed data analyses, and JL, FG, and ZS wrote the manuscript. All authors have read and approved the final manuscript.

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Correspondence to Feng Gu or Zongming Song.

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Lv, J., Xi, H., Lv, X. et al. Two high-fidelity variants: efSaCas9 and SaCas9-HF, which one is better?. Gene Ther 29, 458–463 (2022). https://doi.org/10.1038/s41434-022-00319-4

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