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Nat Commun ; 15(1): 5471, 2024 Jun 28.
Article in English | MEDLINE | ID: mdl-38942756

ABSTRACT

The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise, with a negligible affinity for mismatched substrates, but its low cellular targeting efficiency limits therapeutic use. Here, we rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants and broaden their accessibility across human genomic sites by ~3.5-fold. The enFnCas9 proteins with single mismatch specificity expanded the target range of FnCas9-based CRISPR diagnostics to detect the pathogenic DNA signatures. They outperform Streptococcus pyogenes Cas9 (SpCas9) and its engineered derivatives in on-target editing efficiency, knock-in rates, and off-target specificity. enFnCas9 can be combined with extended gRNAs for robust base editing at sites which are inaccessible to PAM-constrained canonical base editors. Finally, we demonstrate an RPE65 mutation correction in a Leber congenital amaurosis 2 (LCA2) patient-specific iPSC line using enFnCas9 adenine base editor, highlighting its therapeutic utility.


Subject(s)
CRISPR-Associated Protein 9 , CRISPR-Cas Systems , Francisella , Gene Editing , Humans , Gene Editing/methods , CRISPR-Associated Protein 9/metabolism , CRISPR-Associated Protein 9/genetics , CRISPR-Cas Systems/genetics , Francisella/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Leber Congenital Amaurosis/genetics , Streptococcus pyogenes/genetics , HEK293 Cells , Mutation , RNA, Guide, CRISPR-Cas Systems/genetics , RNA, Guide, CRISPR-Cas Systems/metabolism , Protein Engineering/methods , Genome, Human
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