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

Kara Van Aelst,Mark Szczelkun,Stephen Cross,Carlos Martínez-Santiago

doi:10.3390/genes10020169

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.

Highlights

IntroductionCRISPR (clustered regularly interspersed short palindromic repeats)-Cas (CRISPR-associated) systems evolved to defend microbes against bacteriophages and have been classified into different types based on their cas genes [1,2]
CRISPR-Cas (CRISPR-associated) systems evolved to defend microbes against bacteriophages and have been classified into different types based on their cas genes [1,2]
By comparing cleavage kinetics on plasmid, nicked and linear DNA, we show that the microscopic DNA cleavage rate constants for the first cleavage event is >15-fold faster than the second, and that the observed cleavage of nicked and linear DNA were slower by at least 50-fold compared to negatively-supercoiled DNA

Summary

Introduction

CRISPR (clustered regularly interspersed short palindromic repeats)-Cas (CRISPR-associated) systems evolved to defend microbes against bacteriophages and have been classified into different types based on their cas genes [1,2]. Due to their RNA-based programmable DNA-targeting capability, the type-II Cas effector nucleases have been widely adapted as tools for gene editing, and beyond [3]. The type V Cas12a effectors (formerly known as Cpf1) have been shown to be active for gene editing [4,5]. We sought to understand the nuclease mechanism of Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a) by examining the kinetics of DNA cleavage and the effect of DNA topology on the observed rates

Methods

Results

Conclusion