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TitleMolecular Basis and Genome Editing Applications of a Compact CRISPR-Cas9 System.
Journal, issue, pagesACS Synth Biol, Vol. 13, Issue 1, Page 269-281, Year 2024
Publish dateJan 19, 2024
AuthorsNa Tang / Zhaowei Wu / Yan Gao / Weizhong Chen / Zixiao Wang / Mengjiao Su / Wenxin Ji / Quanjiang Ji /
PubMed AbstractCRISPR-Cas9 systems have been widely harnessed for diverse genome editing applications because of their ease of use and high efficiency. However, the large molecular sizes and strict PAM requirements ...CRISPR-Cas9 systems have been widely harnessed for diverse genome editing applications because of their ease of use and high efficiency. However, the large molecular sizes and strict PAM requirements of commonly used CRISPR-Cas9 systems restrict their broad applications in therapeutics. Here, we report the molecular basis and genome editing applications of a novel compact type II-A CRISPR-Cas9 system (EvCas9) with 1107 residues and distinct 5'-NNGDGN-3' (where D represents A, T, or G) PAM specificity. We determine the cryo-EM structure of EvCas9 in a complex with an sgRNA and a target DNA, revealing the detailed PAM recognition and sgRNA and target DNA association mechanisms. Additionally, we demonstrate the robust genome editing capacity of EvCas9 in bacteria and human cells with superior fidelity compared to SaCas9 and SpCas9, and we engineer it to be efficient base editors by fusing a cytidine or adenosine deaminase. Collectively, our results facilitate further understanding of CRISPR-Cas9 working mechanisms and expand the compact CRISPR-Cas9 toolbox.
External linksACS Synth Biol / PubMed:38061052
MethodsEM (single particle)
Resolution3.43 Å
Structure data

EMDB-35035, PDB-8hud:
Cryo-EM structure of the EvCas9-sgRNA-target DNA ternary complex
Method: EM (single particle) / Resolution: 3.43 Å

Source
  • eubacterium ventriosum atcc 27560 (bacteria)
  • synthetic construct (others)
KeywordsRNA BINDING PROTEIN/RNA/DNA / RNA / DNA / RNA BINDING PROTEIN-RNA-DNA complex

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