Phage-assisted evolution of compact Cas9 variants targeting a simple NNG PAM.

Tao Qi, Yao Wang, Yuan Yang, Siqi Gao, Jingtong Liu, Qiang Huang, Yuwen Tian, Junnan Tang, Wei V Zheng, Yongming Wang

Nature chemical biology 2024 Mar

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Compact Cas9 nucleases hold great promise for therapeutic applications. Although several compact Cas9 nucleases have been developed, many genomic loci still could not be edited due to a lack of protospacer adjacent motifs (PAMs). We previously developed a compact SlugCas9 recognizing an NNGG PAM. Here we demonstrate that SlugCas9 displays comparable activity to SpCas9. We developed a simple phage-assisted evolution to engineer SlugCas9 for unique PAM requirements. Interestingly, we generated a SlugCas9 variant (SlugCas9-NNG) that could recognize an NNG PAM, expanding the targeting scope. We further developed a SlugCas9-NNG-based adenine base editor and demonstrated that it could be delivered by a single adeno-associated virus to disrupt PCSK9 splice donor and splice acceptor. These genome editors greatly enhance our ability for in vivo genome editing. © 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.

Citation

Tao Qi, Yao Wang, Yuan Yang, Siqi Gao, Jingtong Liu, Qiang Huang, Yuwen Tian, Junnan Tang, Wei V Zheng, Yongming Wang. Phage-assisted evolution of compact Cas9 variants targeting a simple NNG PAM. Nature chemical biology. 2024 Mar;20(3):344-352