Jun 19, 2016

High-throughput biochemical profiling reveals Cas9 off-target binding and unbinding heterogeneity

BioRxiv : the Preprint Server for Biology
Evan A BoyleWilliam J Greenleaf

Abstract

The bacterial adaptive immune system CRISPR-Cas9 has been appropriated as a versatile tool for editing genomes, controlling gene expression, and visualizing genetic loci. To analyze Cas9′s ability to bind DNA rapidly and specifically, we measured the kinetics of catalytically dead Cas9 (dCas9) interactions with a library of potential binding partners. Using a massively parallel assay of protein-DNA interactions derived from a high-throughput sequencing flow cell (HiTS-FLIP) and building on the established importance of protospacer adjacent motif (PAM) and seed recognition, we identify two PAM-distal regions, proximal and distal to the seed region, with distinct behaviors: multiple mismatches in the seed-proximal region work in a highly synergistic manner to reduce Cas9 association whereas seemingly tolerated mismatches in the distal region precipitate comparatively rapid dissociation of Cas9. Together, these observations support a model for Cas9 specificity wherein gRNA-DNA mismatches at distinct domains of PAM-distal bases modulate different biophysical parameters of association and dissociation, opening the possibility of kinetic and thermodynamic tuning of the Cas9-DNA interaction and quantitative prediction of off-target bi...Continue Reading

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Mentioned in this Paper

Immune System
Genome
Gene Expression
Protein-Protein Interaction
Biophysics
Sequencing
High Throughput Analysis
MYCBP2 gene
Binding (Molecular Function)
Protein Domain

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