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

TL;DR

This paper introduces a new method to improve CRISPR-Cas9 precision by designing guide RNAs with intentional mismatches, enhancing the ability to edit specific mutant alleles in cancer-related genes.

Contribution

The study introduces a systematic strategy for designing gRNAs with mismatches to enhance allele-specific editing and reduce off-target effects.

Findings

Introducing mismatches into gRNAs reduces Cas9 sequence tolerance and increases specificity for mutant alleles.

The method successfully edited cancer-related mutations in EGFR L858R and KRAS G12V with minimal impact on wild-type alleles.

Strategic mismatch incorporation improves the precision and safety of CRISPR-based genome editing for cancer therapy.

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.

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