ARROW: Allele-Specific Recombined gRNA Design for Reduced Off-Target with Enhanced Specificity

Abstract

Background/Objectives: Allele-specific genome editing using the CRISPR-Cas9 system is crucial for achieving precise therapeutic interventions in dominant inherited diseases that are otherwise difficult to treat with conventional approaches. However, Cas9-guide RNA (gRNA) complexes often tolerate single-base mismatches in target sequences, making it challenging to discriminate between wild-type and mutant alleles differing by only one nucleotide. Although previous studies have attempted to improve specificity by introducing mismatches into gRNAs, none has systematically investigated the impact of different mismatch types and positions on editing outcomes. In this study, we developed an effective strategy to enhance specificity and minimize off-target effects by deliberately introducing mismatches into gRNAs and comprehensively evaluating their editing performance. Results: We established an efficient strategy for the selective editing of mutant alleles that reduces Cas9 sequence tolerance and enhances specificity through the intentional introduction of mismatches into gRNAs. The efficacy of this approach was demonstrated by successful allele-specific editing of cancer-associated heterozygous point mutations in EGFR L858R and KRAS G12V, while minimizing editing of the corresponding wild-type alleles. Conclusion: Compared with perfectly matched gRNAs, the strategic incorporation of mismatches into gRNAs enhanced editing specificity for single-base mutant alleles. Our findings substantially improve the precision and safety of CRISPR-based genome editing for cancer therapy, particularly in cases involving mutant alleles.