Mechanisms of improved specificity of engineered Cas9s revealed by single molecule analysis

Digvijay Singh, Yanbo Wang, John Mallon, Olivia Yang, Jingyi Fei, Anustup Poddar, Damon Ceylan, Scott Bailey, Taekjip Ha

Research output: Contribution to journalArticlepeer-review


In microbes, CRISPR-Cas systems provide adaptive immunity against invading genetic elements. Cas9 in complex with a guide-RNA targets complementary DNA for cleavage and has been repurposed for wide-ranging biological applications. New Cas9s have been engineered (eCas9 and Cas9-HF1) to improve specificity, but how they help reduce off-target cleavage is not known. Here, we developed single molecule DNA unwinding assay to show that sequence mismatches affect cleavage reactions through rebalancing the internal unwinding/rewinding equilibrium. Increasing PAM-distal mismatches facilitate rewinding, and the associated cleavage impairment shows that cleavage proceeds from the unwound state. Engineered Cas9s depopulate the unwound state more readily upon mismatch detection. Intrinsic cleavage rate is much lower for engineered Cas9s, preventing cleavage from transiently unwound off-targets. DNA interrogation experiments showed that engineered Cas9s require about one additional base pair match for stable binding, freeing them from sites that would otherwise sequester them. Therefore, engineered Cas9s achieve their improved specificity (1) by inhibiting stable DNA binding to partially matching sequences, (2) by making DNA unwinding more sensitive to mismatches, and (3) by slowing down intrinsic cleavage reaction.

Original languageEnglish (US)
JournalUnknown Journal
StatePublished - Sep 22 2017

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)
  • Pharmacology, Toxicology and Pharmaceutics(all)

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