The Effect of DNA Topology on Observed Rates of R-Loop Formation and DNA Strand Cleavage by CRISPR Cas12a.

Genes
Kara van AelstMark D Szczelkun

Abstract

Here we explored the mechanism of R-loop formation and DNA cleavage by type V CRISPR Cas12a (formerly known as Cpf1). We first used a single-molecule magnetic tweezers (MT) assay to show that R-loop formation by Lachnospiraceae bacterium ND2006 Cas12a is significantly enhanced by negative DNA supercoiling, as observed previously with Streptococcus thermophilus DGCC7710 CRISPR3 Cas9. Consistent with the MT data, the apparent rate of cleavage of supercoiled plasmid DNA was observed to be >50-fold faster than the apparent rates for linear DNA or nicked circular DNA because of topology-dependent differences in R-loop formation kinetics. Taking the differences into account, the cleavage data for all substrates can be fitted with the same apparent rate constants for the two strand-cleavage steps, with the first event >15-fold faster than the second. By independently following the ensemble cleavage of the non-target strand (NTS) and target strand (TS), we could show that the faster rate is due to NTS cleavage, the slower rate due to TS cleavage, as expected from previous studies.

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Citations

Mar 13, 2020·The Journal of Biological Chemistry·Karthik MuruganDipali G Sashital
Nov 2, 2019·Biochemical Society Transactions·Daan C Swarts
Mar 4, 2020·Proceedings of the National Academy of Sciences of the United States of America·Ivan E IvanovZev Bryant
Feb 1, 2021·The Journal of Biological Chemistry·Karthik MuruganDipali G Sashital
Mar 12, 2021·Science Advances·Isabel StrohkendlIlya J Finkelstein
Aug 22, 2021·Nucleic Acids Research·Oscar E Torres MontaguthMark D Szczelkun
Sep 9, 2021·Physical Chemistry Chemical Physics : PCCP·Marialucrezia LositoDavid S Rueda

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Methods Mentioned

BETA
electron microscopy
Fluorescence
FRET
in vitro transcription
density gradient centrifugation
PCR
electrophoresis
Assay

Software Mentioned

ImageQuant
NEBuilder HiFi DNA
NEBuilder

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