Mechanistic Insights into the cis- and trans-Acting DNase Activities of Cas12a

Mol Cell. 2019 Feb 7;73(3):589-600.e4. doi: 10.1016/j.molcel.2018.11.021. Epub 2019 Jan 10.


CRISPR-Cas12a (Cpf1) is an RNA-guided DNA-cutting nuclease that has been repurposed for genome editing. Upon target DNA binding, Cas12a cleaves both the target DNA in cis and non-target single-stranded DNAs (ssDNAs) in trans. To elucidate the molecular basis for both DNase cleavage modes, we performed structural and biochemical studies on Francisella novicida Cas12a. We show that guide RNA-target strand DNA hybridization conformationally activates Cas12a, triggering its trans-acting, non-specific, single-stranded DNase activity. In turn, cis cleavage of double-stranded DNA targets is a result of protospacer adjacent motif (PAM)-dependent DNA duplex unwinding, electrostatic stabilization of the displaced non-target DNA strand, and ordered sequential cleavage of the non-target and target DNA strands. Cas12a releases the PAM-distal DNA cleavage product and remains bound to the PAM-proximal DNA cleavage product in a catalytically competent, trans-active state. Together, these results provide a revised model for the molecular mechanisms of both the cis- and the trans-acting DNase activities of Cas12a enzymes, enabling their further exploitation as genome editing tools.

Keywords: CRISPR-Cas; Cas12a; Cas9; Cpf1; DNA cleavage; DNase; PAM; genetic engineering; genome editing; ssDNase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • CRISPR-Associated Proteins / chemistry
  • CRISPR-Associated Proteins / genetics
  • CRISPR-Associated Proteins / metabolism*
  • CRISPR-Cas Systems*
  • DNA, Single-Stranded / chemistry
  • DNA, Single-Stranded / genetics
  • DNA, Single-Stranded / metabolism*
  • Enzyme Activation
  • Francisella / enzymology*
  • Francisella / genetics
  • Gene Editing / methods*
  • Models, Molecular
  • Nucleic Acid Conformation
  • Protein Conformation
  • RNA, Guide, Kinetoplastida / chemistry
  • RNA, Guide, Kinetoplastida / genetics
  • RNA, Guide, Kinetoplastida / metabolism*
  • Structure-Activity Relationship
  • Substrate Specificity


  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • DNA, Single-Stranded
  • RNA, Guide