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

Jan 15, 2019Molecular cell

Understanding the two ways Cas12a cuts DNA within and outside its target sites

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Abstract

CRISPR-Cas12a is shown to cleave both target and non-target DNA strands through distinct mechanisms.

Cas12a is activated by the binding of guide RNA to the target DNA, which prompts its non-specific single-stranded DNA cutting activity.
The cleavage of double-stranded DNA targets occurs through the unwinding of the DNA duplex, which relies on specific DNA sequences known as protospacer adjacent motifs (PAM).
Electrostatic interactions help stabilize the non-target DNA strand after it is displaced during the cleavage process.
Sequential cleavage of both the target and non-target DNA strands is ordered and contributes to Cas12a's activity.
Cas12a stays attached to the portion of DNA it cleaves closest to the PAM site, indicating a state ready for further action.

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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.

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

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