Abstract
Using optical tweezers, we investigate target search and cleavage by CRISPR–Cas12a on force-stretched λ-DNA. Cas12a uses fast, one-dimensional hopping to locate its target. Binding and cleavage occur rapidly and specifically at low forces (≤5 pN), with a 1.8 nm rate-limiting conformational change. Mechanical distortion slows diffusion, increases off-target binding but hinders cleavage.
Highlights
We investigate target search and cleavage by CRISPR–Cas12a on force-stretched k-DNA
At low forces (Fig. 4A, r20 pN), we obtained primarily on-target binding with only a few off-target bound molecules that diffuse randomly on the stretched DNA (Supplementary Movie, Electronic supplementary information (ESI)†)
Cas12a6 has emerged as a salient alternative for genome editing applications to Cas[9] because it exhibits fewer off-target effects in cells[10,11] and staggered cleavage.[6,12]
Summary
Rueda et al Cas12a target search and cleavage on force-stretched DNA We investigate target search and cleavage by CRISPR–Cas12a on force-stretched k-DNA. We use our previously developed optical tweezers assay[24] to further investigate the Cas12a target search and cleavage mechanism on mechanically-stretched l-DNA.
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