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

Open access
Friedland, Ari E., Baral, Reshica, Singhal, Pankhuri, Loveluck, Katherine, Shen, Shen, Sanchez, Minerva, Marco, Eugenio, Gotta, Gregory M., Maeder, Morgan L., Kennedy, Edward M., Kornepati, Anand V. R., Sousa, Alexander, Collins, McKensie A., Jayaram, Hari, Cullen, Bryan R., Bumcrot, David·2015·Genome Biology

Abstract

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.

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Metadata

Authors
Friedland, A. E., Baral, R., Singhal, P., Loveluck, K., Shen, S., Sanchez, M., Marco, E., Gotta, G. M., Maeder, M. L., Kennedy, E. M., Kornepati, A. V. R., Sousa, A., Collins, M. A., Jayaram, H., Cullen, B. R., & Bumcrot, D.
Venue
Genome Biology
Published
2015-11-24
Volume
16
Issue
1
Pages
257-257
ISSN
  • 1465-6906 (ISSN-L)
  • 1474-760X
OpenAlex
W2172425918
Cited by
278
References
10
FWCI
12.09
Type
journal article
Language
en
Open access
gold

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CoChat. (2026). Citation check: Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications. https://app.cochat.ai/papers/10.1186/s13059-015-0817-8
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Friedland, A. E., Baral, R., Singhal, P., Loveluck, K., Shen, S., Sanchez, M., Marco, E., Gotta, G. M., Maeder, M. L., Kennedy, E. M., Kornepati, A. V. R., Sousa, A., Collins, M. A., Jayaram, H., Cullen, B. R., & Bumcrot, D. (2015). Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications. Genome Biology. https://doi.org/10.1186/s13059-015-0817-8

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