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bioRxiv · 10.1101/2025.09.02.671676

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

Lee, H. K.·Kim, S.·Hong, J.·Bae, T.·An, Y.·Sohn, C. H.·Hwang, W. C.·Park, C.-K.·Lee, S. H.·Koh, H. R.·Hur, J. K.
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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.

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Lee, H. K., Kim, S., Hong, J., Bae, T., An, Y., Sohn, C. H., Hwang, W. C., Park, C.-K., Lee, S. H., Koh, H. R., Hur, J. K.. 2025-09-04. High-fidelity CRISPR genome editing of single-nucleotide mutation with near-complementary guide RNA via enhanced target binding kinetics. https://doi.org/10.1101/2025.09.02.671676

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