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

• Published in: Molecular cell Vol. 73; no. 3; pp. 589 - 600.e4
• Main Authors: Swarts, Daan C., Jinek, Martin
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 07.02.2019
• Subjects: Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Cas12a; Cas9; Cpf1; CRISPR-Associated Proteins - chemistry; CRISPR-Associated Proteins - genetics; CRISPR-Associated Proteins - metabolism; CRISPR-Cas; CRISPR-Cas Systems; DNA cleavage; DNA, Single-Stranded - chemistry; DNA, Single-Stranded - genetics; DNA, Single-Stranded - metabolism; DNase; Enzyme Activation; Francisella - enzymology; Francisella - genetics; Gene Editing - methods; genetic engineering; genome editing; Models, Molecular; Nucleic Acid Conformation; PAM; Protein Conformation; RNA, Guide, CRISPR-Cas Systems - chemistry; RNA, Guide, CRISPR-Cas Systems - genetics; RNA, Guide, CRISPR-Cas Systems - metabolism; ssDNase; Structure-Activity Relationship; Substrate Specificity; genome editing; ssDNase; CRISPR-Cas; Cpf1; DNA cleavage; DNase; Cas12a; Cas9; PAM; genetic engineering
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2018.11.021

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. [Display omitted] •Target ssDNA binding allosterically induces unblocking of the RuvC active site•PAM binding facilitates strand separation in dsDNA targets•Non-target DNA strand cleavage precedes target DNA strand cleavage•Cas12a releases the PAM-distal end of cleaved dsDNA targets Swarts et al. elucidated two structures of Cas12a in complex with a guide RNA and with either single-stranded or double-stranded DNA targets. Together with biochemical assays, these structures reveal the mechanisms of Cas12a-mediated cis and trans cleavage of DNA substrates.