A Biophysical Model of CRISPR/Cas9 Activity for Rational Design of Genome Editing and Gene Regulation

• DOI: 10.1371/journal.pcbi.1004724
• Publication Date: 2016-01-29T18:39:03Z
• Authors: Iman Farasat; Howard M. Salis
• Keywords: Trans-activating crRNA; Guide RNA

The ability to precisely modify genomes and regulate specific genes will greatly accelerate several medical engineering applications. CRISPR/Cas9 (Type II) system binds cuts DNA using guide RNAs, though the variables that control its on-target off-target activity remain poorly characterized. Here, we develop parameterize a system-wide biophysical model of Cas9-based genome editing gene regulation predict how changing RNA sequences, superhelical densities, Cas9 crRNA expression levels, organisms growth conditions, experimental conditions collectively dynamics dCas9-based binding cleavage at all sites with both canonical non-canonical PAMs. We combine statistical thermodynamics kinetics Cas9:crRNA complex formation, diffusion, site selection, reversible R-loop cleavage, large amounts structural, biochemical, expression, next-generation sequencing data determine kinetic parameters free energy models. Our results identify supercoiling as novel mechanism controlling binding. Using model, frequencies across lambdaphage human genomes, explain why Cas9's can be so high. With this improved understanding, propose rules for designing experiments minimizing activity. also discuss implications genetic circuits.