Structure- and Content-Dependent Efficiency of Cas9-Assisted DNA Cleavage in Genome-Editing Systems

Svetlana V. Baranova, Polina V. Zhdanova, Alexander A. Lomzov, Vladimir V. Koval, Alexander A. Chernonosov

Research output: Contribution to journalArticlepeer-review


Genome-editing systems, being some of the key tools of molecular biologists, represent a reasonable hope for progress in the field of personalized medicine. A major problem with such systems is their nonideal accuracy and insufficient selectivity. The selectivity of CRISPR-Cas9 systems can be improved in several ways. One efficient way is the proper selection of the consensus sequence of the DNA to be cleaved. In the present work, we attempted to evaluate the effect of formed non-Watson–Crick pairs in a DNA duplex on the efficiency of DNA cleavage in terms of the influence of the structure of the formed partially complementary pairs. We also studied the effect of the location of such pairs in DNA relative to the PAM (protospacer-adjacent motif) on the cleavage efficiency. We believe that the stabilization of the Cas9-sgRNA complex with a DNA substrate containing noncomplementary pairs is due to loop reorganization in the RuvC domain of the enzyme. In addition, PAM-proximal mismatches in the DNA substrate lower enzyme efficiency because the “seed” region is involved in binding and cleavage, whereas PAM-distal mismatches have no significant impact on target DNA cleavage. Our data suggest that in the case of short duplexes with mismatches, the stages of recognition and binding of dsDNA substrates by the enzyme determine the reaction rate and time rather than the thermodynamic parameters affected by the “unwinding” of DNA. The results will provide a theoretical basis for predicting the efficiency and accuracy of CRISPR-Cas9 systems at cleaving target DNA.

Original languageEnglish
Article number13889
JournalInternational Journal of Molecular Sciences
Issue number22
Publication statusPublished - Nov 2022


  • Cas9 activity
  • cleavage
  • CRISPR-Cas system
  • oligonucleotide mismatch
  • thermodynamics
  • DNA/chemistry
  • CRISPR-Cas Systems
  • Gene Editing/methods
  • DNA Cleavage
  • Endonucleases/metabolism




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