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

Abstract

BackgroundCRISPR-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.ResultsWe 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.ConclusionOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0817-8) contains supplementary material, which is available to authorized users.

CRISPR/Cas9 with single guide RNA expression driven by small tRNA promoters showed reduced editing efficiency compared to a U6 promoter

“…However, the use of two different AAVs for separate delivery of Cas9 and two sgRNAs rendered a relatively low efficiency of successful excision. One approach to increase the efficiency would be to have SaCas9 and two or even more different sgRNAs delivered by one AAV vector (Friedland et al 2015). As AAV vectors have a DNA packaging limit of ∼5 kb, and the standard AAV-SaCRISPR system almost fills the cargo limitation, it would be helpful to further reduce the total length of the SaCRISPR system in order to make extra space for more sgRNA cassettes and to improve the packaging efficiency.…”

Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications

“…94 In another study, scientists packaged SaCas9 and multiple sgRNAs in to AAV and showed approximately 60% gene-editing efficiency. 95 …”

Technical considerations for the use of CRISPR/Cas9 in hematology research

“…These include SaCas9, AsCpf1, LbCpf1, or other CRISPR modalities such as CRISPR-mediated repression or activation (Friedland et al, 2015, Kleinstiver et al, 2016, Gilbert et al, 2014). Additionally, use of such complementary tools can validate positive hits further, and help to resolve mechanistic details through increasing genomic targeting space and direct regulatory control.…”