Abasic CRISPR RNAs inherently harness fidelity of SpCas9 for genome editing.

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 Streptococcus pyogenes (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.