Structural basis for mismatch surveillance by CRISPR–Cas9

Abstract CRISPR–Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage 1–4 , and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood 5–7 . Although Cas9 variants with greater discrimination against mismatches have been designed 8–10 , these suffer from substantially reduced rates of on-target DNA cleavage 5,11 . Here we used kinetics-guided cryo-electron microscopy to determine the structure of Cas9 at different stages of mismatch cleavage. We observed a distinct, linear conformation of the guide RNA–DNA duplex formed in the presence of mismatches, which prevents Cas9 activation. Although the canonical kinked guide RNA–DNA duplex conformation…

• CP
  Cancer Prevention and Research Institute of Texas

  Awards: F-1938, RR160088
• RJ
  Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation