SMART Libraries and Phage-Induced Directed Evolution of Cas9 to Engineer Reduced Off-Target Activity

摘要

RNA-guided endonucleases such as Cas9 often show efficient editing in cells but can cleave at off-target loci in the genome. Engineered variants of S. pyogenes Cas9 (Sp. Cas9) have been developed to globally reduce off-target activity, but individual off-targets may remain or on-target activity may be compromised. In order to engineer decreased editing at specific off-targets and maintain on-target editing, we created a phage-based selection system in which we can negatively select for cleavage of specific sequences in a competitive pool and positively select for cleavage at our desired on-target. We introduce Scanning Mutagenesis At Random Targets (SMART) to create comprehensive libraries of Sp. Cas9 to challenge with phage-based directed evolution. Enriched residues are synthesized, purified, and evaluated in cells. Our platform identifies novel Sp. Cas9 mutants which mitigate cleavage against off-targets biochemically and in T-cells while maintaining higher activ ity than previously engineered variants. We describe an evolved variant, S. pyogenes Adapted to Reduce Target Ambiguity cas9 (SPARTACas), composed of the most enriched mutations of unknown function which reduces off-target cleavage while maintaining efficient editing ability at multiple sites. Directed evolution of Cas9 using our system demonstrates a structure-independent methodology to effectively engineer nuclease activity.