In-depth assessment of the PAM compatibility and editing activities of Cas9 variants.

Zhang, Weiwei; Yin, Jianhang; Zhang-Ding, Zhengrong; Xin, Changchang; Liu, Mengzhu; Wang, Yuhong; Ai, Chen; Hu, Jiazhi

Zhang, Weiwei; Yin, Jianhang; Zhang-Ding, Zhengrong; Xin, Changchang; Liu, Mengzhu; Wang, Yuhong; Ai, Chen; Hu, Jiazhi.

Afiliación

Zhang W; The MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Center for Life Sciences, Genome Editing Research Center, Peking University, Beijing 100871, China.
Yin J; The MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Center for Life Sciences, Genome Editing Research Center, Peking University, Beijing 100871, China.
Zhang-Ding Z; The MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Center for Life Sciences, Genome Editing Research Center, Peking University, Beijing 100871, China.
Xin C; The MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Center for Life Sciences, Genome Editing Research Center, Peking University, Beijing 100871, China.
Liu M; The MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Center for Life Sciences, Genome Editing Research Center, Peking University, Beijing 100871, China.
Wang Y; The MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Center for Life Sciences, Genome Editing Research Center, Peking University, Beijing 100871, China.
Ai C; The MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Center for Life Sciences, Genome Editing Research Center, Peking University, Beijing 100871, China.
Hu J; The MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Center for Life Sciences, Genome Editing Research Center, Peking University, Beijing 100871, China.

Nucleic Acids Res ; 49(15): 8785-8795, 2021 09 07.

Article en En | MEDLINE | ID: mdl-34133740

RESUMEN

A series of Cas9 variants have been developed to improve the editing fidelity or targeting range of CRISPR-Cas9. Here, we employ a high-throughput sequencing approach primer-extension-mediated sequencing to analyze the editing efficiency, specificity and protospacer adjacent motif (PAM) compatibility of a dozen of SpCas9 variants at multiple target sites in depth, and our findings validate the high fidelity or broad editing range of these SpCas9 variants. With regard to the PAM-flexible SpCas9 variants, we detect significantly increased levels of off-target activity and propose a trade-off between targeting range and editing specificity for them, especially for the near-PAM-less SpRY. Moreover, we use a deep learning model to verify the consistency and predictability of SpRY off-target sites. Furthermore, we combine high-fidelity SpCas9 variants with SpRY to generate three new SpCas9 variants with both high fidelity and broad editing range. Finally, we also find that the existing SpCas9 variants are not effective in suppressing genome instability elicited by CRISPR-Cas9 editing, raising an urgent issue to be addressed.

Asunto(s)

Sistemas CRISPR-Cas/genética; Edición Génica; Oryza/genética; Streptococcus pyogenes/enzimología; Proteína 9 Asociada a CRISPR/genética; Genoma de Planta/genética; Mutación/genética

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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Oryza / Streptococcus pyogenes / Sistemas CRISPR-Cas / Edición Génica Tipo de estudio: Prognostic_studies Idioma: En Revista: Nucleic Acids Res Año: 2021 Tipo del documento: Article País de afiliación: China Pais de publicación: Reino Unido

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