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bioRxiv · 10.1101/2021.06.09.447528

Cas12a is a dynamic and precise RNA-guided nuclease without off-target activity on {lambda}-DNA

Paul, B.·Chaubet, L.·Verver, E.·Montoya, G.
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Abstract

Cas12a is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here we combined optical tweezers with fluorescence to monitor Cas12a binding onto {lambda}-DNA, providing insight into its DNA binding and cleavage mechanisms. At low forces Cas12a binds DNA specifically with two off-target sites, while at higher forces numerous binding events appear driven by the mechanical distortion of the DNA and partial matches to the crRNA. Despite the multiple binding events, cleavage is only observed on the target site at low forces, when the DNA is flexible. Activity assays show that the preferential off-target sites are not cleaved, and the {lambda}-DNA is severed at the target site. This precision is also observed in Cas12a variants where the specific dsDNA and the unspecific ssDNA cleavage are dissociated or nick the target DNA. We propose that Cas12a and its variants are precise endonucleases that efficiently scan the DNA for its target but only cleave the selected site in the {lambda}-DNA.

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Paul, B., Chaubet, L., Verver, E., Montoya, G.. 2021-06-09. Cas12a is a dynamic and precise RNA-guided nuclease without off-target activity on {lambda}-DNA. https://doi.org/10.1101/2021.06.09.447528

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