Abasic CRISPR RNAs inherently harness fidelity of SpCas9 for genome editing

초록

CRISPR-Cas9, an RNA-guided immune system, functions specifically in bacteria while controlling autoimmunity. However, its application to genome editing often causes deleterious off-target cleavages. Here, by sequencing CRISPR RNAs (crRNAs), we discovered abasic modifications that naturally suppress off-target self-cleavages from activated Cas9 in Streptococcuspyogenes (SpCas9). Bacteriophage infection induces oxidative stress, preferentially oxidizing the 5 ' end of crRNAs into abasic modifications. Mechanistically, abasic substitutions at the 5 ' end reduce off-target effects by limiting base pairing while preserving SpCas9-interacting backbones to maintain on-target efficiency. Abasic extensions at the 5 ' end reduce off-target effects by sterically constraining SpCas9 but retain on-target activity by avoiding extra base pairs. Moreover, these approaches can be combined (abasic substitution and extension), enhancing SpCas9 fidelity by increasing mismatch intolerance at the protospacer-adjacent motif-distal region and outperforming SpCas9 variants. Biologically inspired, we developed abasic chemical modifications for guide RNAs that improve CRISPR-Cas9 genome-editing specificity, demonstrating potential for in vivo application.

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