SMOOT libraries and phage-induced directed evolution of Cas9 to engineer reduced off-target activity.

RNA-guided endonucleases such as Cas9 provide efficient on-target genome editing in cells but may also cleave at off-target loci throughout the genome. Engineered variants of Streptococcus pyogenes Cas9 (SpCas9) 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 evolve against activity at specific off-targets while maintaining strong on-target editing, we developed a novel M13 bacteriophage-mediated selection method. Using this method, sequential rounds of positive and negative selection are used to identify mutations to Cas9 that enhance or diminish editing activity at particular genomic sequences. We also introduce scanning mutagenesis of oligo-directed targets (SMOOT), a comprehensive mutagenesis method to create highly diverse libraries of Cas9 variants that can be challenged with phage-based selection. Our platform identifies novel SpCas9 mutants which mitigate cleavage against off-targets both in biochemical assays and in T-cells while maintaining higher on-target activity than previously described variants. We describe an evolved variant, S. pyogenes Adapted to Reduce Target Ambiguity Cas9 (SpartaCas), composed of the most enriched mutations, each of unknown function. This evolved Cas9 mutant reduces off-target cleavage while preserving efficient editing at multiple therapeutically relevant targets. Directed evolution of Cas9 using our system demonstrates an improved structure-independent methodology to effectively engineer nuclease activity.

Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications.

BACKGROUND: CRISPR-Cas systems have been broadly embraced as effective tools for genome engineering applications, with most studies to date utilizing the Streptococcus pyogenes Cas9. Here we characterize and manipulate the smaller, 1053 amino acid nuclease Staphylococcus aureus Cas9. RESULTS: We find that the S. aureus Cas9 recognizes an NNGRRT protospacer adjacent motif (PAM) and cleaves target DNA at high efficiency with a variety of guide RNA (gRNA) spacer lengths. When directed against genomic targets with mutually permissive NGGRRT PAMs, the S. pyogenes Cas9 and S. aureus Cas9 yield indels at comparable rates. We additionally show D10A and N580A paired nickase activity with S. aureus Cas9, and we further package it with two gRNAs in a single functional adeno-associated virus (AAV) vector. Finally, we assess comparative S. pyogenes and S. aureus Cas9 specificity using GUIDE-seq. CONCLUSION: Our results reveal an S. aureus Cas9 that is effective for a variety of genome engineering purposes, including paired nickase approaches and all-in-one delivery of Cas9 and multiple gRNA expression cassettes with AAV vectors.