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Article

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

1
School of Biochemistry, Faculty of Life Sciences, University of Bristol, Bristol BS8 1TD, UK
2
DNA-Protein Interactions Unit, School of Biochemistry, Faculty of Life Sciences, University of Bristol, Bristol BS8 1TD, UK
3
Wolfson Bioimaging Facility, Faculty of Life Sciences, University of Bristol, Bristol BS8 1TD, UK
*
Author to whom correspondence should be addressed.
Genes 2019, 10(2), 169; https://doi.org/10.3390/genes10020169
Received: 30 January 2019 / Revised: 14 February 2019 / Accepted: 15 February 2019 / Published: 22 February 2019
(This article belongs to the Special Issue CRISPR-Cas: Interactions with Genome and Physiological Maintenance)
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. View Full-Text
Keywords: rapid reaction kinetics; endonuclease mechanism; DNA topology rapid reaction kinetics; endonuclease mechanism; DNA topology
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MDPI and ACS Style

van Aelst, K.; Martínez-Santiago, C.J.; Cross, S.J.; Szczelkun, M.D. The Effect of DNA Topology on Observed Rates of R-Loop Formation and DNA Strand Cleavage by CRISPR Cas12a. Genes 2019, 10, 169. https://doi.org/10.3390/genes10020169

AMA Style

van Aelst K, Martínez-Santiago CJ, Cross SJ, Szczelkun MD. The Effect of DNA Topology on Observed Rates of R-Loop Formation and DNA Strand Cleavage by CRISPR Cas12a. Genes. 2019; 10(2):169. https://doi.org/10.3390/genes10020169

Chicago/Turabian Style

van Aelst, Kara, Carlos J. Martínez-Santiago, Stephen J. Cross, and Mark D. Szczelkun 2019. "The Effect of DNA Topology on Observed Rates of R-Loop Formation and DNA Strand Cleavage by CRISPR Cas12a" Genes 10, no. 2: 169. https://doi.org/10.3390/genes10020169

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