Assessing Temperature-Dependent DNA Cleavage by CRISPR-Cas9.

Abstract

The RNA-guided CRISPR-Cas9 endonuclease has been a transformative tool for laboratory biochemistry with huge potential as a precision therapeutic. This tool site-specifically cleaves double-stranded DNA following the recognition of a unique protospacer-adjacent motif (PAM). Activation of the protein-nucleic acid Cas complex has also been widely recognized to feature an allosteric mechanism dependent on structural remodeling and interdomain crosstalk. Biophysical methods have probed the impact of allosteric perturbations on cleavage and specificity of Cas9, with the aim of engineering enhanced Cas effectors. These studies include Cas9 from thermophilic organisms that edit at higher temperatures and are active in human plasma. Validation of biophysical insights has necessitated the quantitation of DNA cleavage in vitro and, subsequently, the adaptation of established protocols to encompass temperature-dependent function that is evident in extremophilic Cas systems, such as Cas9 from Geobacillus stearothermophilus and the mesophilic SpCas9. This protocol is advantageous for probing functional temperature ranges of DNA cleavage that can theoretically be applied to any Cas-RNP system. Key features • Builds upon the original Cas9 cleavage assays reported by Jinek et al. [1] to include the active temperature range of thermophiles. • Validated for assessing the cleavage activity of both mesophilic and thermophilic Cas9 systems. • Allows for qualitative and quantitative assessment of DNA cleavage across multiple physiological regimes. • Can be adapted to assess cleavage at multiple genomic loci or with different PAM requirements. • Assay can be completed in a single day.