High-fidelity CRISPR genome editing of single-nucleotide mutation with near-complementary guide RNA via enhanced target binding kinetics

Abstract

The CRISPR-Cas9 system is a powerful genome editing tool capable of precisely recognizing and cleaving specific DNA sequences, and has been extensively investigated as a strategy for correcting mutations associated with genetic diseases and cancer. However, conventional CRISPR genome engineering often fail to discriminate single-nucleotide mutations from wild-type alleles when the mutation is located outside the protospacer adjacent motif (PAM) sequence. To address this limitation, we developed a RNA engineering approach for designing near-complementary single guide RNA (sgRNA) that contain intentional mismatches within the seed region of the sgRNA. Single molecule kinetic analyses showed that the near-complementary sgRNA selectively reduces the binding affinity of CRISPR ribonucleoprotein complex by via differentiated increment in the dissociation rates to the wild-type target DNA compared to the mutant allele. The engineered kinetic characteristics of near-complementary sgRNAs enable highly specific genome editing of single-base mutations without reliance on PAM proximity. We demonstrate the application of the strategy to the a cancer-specific single-nucleotide G228A (-124C > T) mutation in the TERT promoter, frequently found in glioblastomas and other tumors, that does not generate a canonical PAM sequence. Our near-complementary sgRNA successfully induced selective editing of the mutant allele while sparing the wild-type sequence. Furthermore, single-molecule fluorescence resonance energy transfer (smFRET) analyss revealed distinct differences in binding kinetics between mutant and wild-type DNA, providing kinetic insight into the discrimination process. We conclude that the near-complementary sgRNA CRISPR editing strategy facilitates precise PAM-independent targeting of single-nucleotide mutations without protein engineering and offers a molecular framework for expanding the specificity and applicability of CRISPR-based genome and epigenome editing technologies.