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

Kara van Aelst, Carlos Martinez-Santiago, Stephen Cross, Mark Szczelkun

Research output: Contribution to journalArticle (Academic Journal)peer-review

20 Citations (Scopus)
321 Downloads (Pure)

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.
Original languageEnglish
Article number169
Number of pages14
JournalGenes
Volume10
Issue number2
Early online date22 Feb 2019
DOIs
Publication statusPublished - Feb 2019

Keywords

  • rapid reaction kinetics
  • endonuclease mechanism
  • DNA topology

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