Abstract
Cas12a (also called Cpf1) is a representative type V-A CRISPR effector RNA-guided DNA endonuclease, which provides an alternative to type II CRISPR–Cas9 for genome editing. Previous studies have revealed that Cas12a has unique features distinct from Cas9, but the detailed mechanisms of target searching and DNA cleavage by Cas12a are still unclear. Here, we directly observe this entire process by using single-molecule fluorescence assays to study Cas12a from Acidaminococcus sp. (AsCas12a). We determine that AsCas12a ribonucleoproteins search for their on-target site by a one-dimensional diffusion along elongated DNA molecules and induce cleavage in the two DNA strands in a well-defined order, beginning with the non-target strand. Furthermore, the protospacer-adjacent motif (PAM) for AsCas12a makes only a limited contribution of DNA unwinding during R-loop formation and shows a negligible role in the process of DNA cleavage, in contrast to the Cas9 PAM.
Highlights
Cas12a is a representative type V-A CRISPR effector RNA-guided DNA endonuclease, which provides an alternative to type II CRISPR–Cas[9] for genome editing
CRISPR-Cas systems provide adaptive immune responses in bacteria and archaea[1,2,3]. These systems are presently divided into two major classes on the basis of their sub-components and function: Class 1 CRISPR-Cas members utilize multi-subunit effector complexes, whereas Class 2 members work with a single multidomain effector protein for DNA or RNA cleavage[4,5]
We perform a single-molecule fluorescence imaging assay with a long DNA molecule (21 kb) to reveal the mechanism of target DNA searching and a single-molecule fluorescence resonance energy transfer (FRET) assay with a short DNA fragment (50 bp), which allows us to observe the DNA cleavage mechanism of AsCas12a in detail
Summary
Cas12a ( called Cpf1) is a representative type V-A CRISPR effector RNA-guided DNA endonuclease, which provides an alternative to type II CRISPR–Cas[9] for genome editing. In contrast to Cas[9], Cas12a has the ability to process its own pre-crRNA into crRNA17, facilitating multiplex genome editing[18] Taken together, these distinguishing features may imply major differences between Cas[9] and Cas12a during both target DNA searching and cleavage. We perform a single-molecule fluorescence imaging assay with a long DNA molecule (21 kb) to reveal the mechanism of target DNA searching and a single-molecule fluorescence resonance energy transfer (FRET) assay with a short DNA fragment (50 bp), which allows us to observe the DNA cleavage mechanism of AsCas12a in detail With these two assays, we can directly observe the entire dynamic process comprising target searching, recognition, and cleavage by Cas12a ribonucleoproteins (RNPs)
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