title: Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Editas Medicine, Cambridge, MA, 02142, USA. Ari.Friedland@editasmed.com.
authors.forename: Ari E
authors.initials: AE
authors.lastname: Friedland
authors.affiliation: Editas Medicine, Cambridge, MA, 02142, USA.
authors.forename: Reshica
authors.initials: R
authors.lastname: Baral
authors.affiliation: Editas Medicine, Cambridge, MA, 02142, USA.
authors.forename: Pankhuri
authors.initials: P
authors.lastname: Singhal
authors.affiliation: Editas Medicine, Cambridge, MA, 02142, USA.
authors.forename: Katherine
authors.initials: K
authors.lastname: Loveluck
authors.affiliation: Editas Medicine, Cambridge, MA, 02142, USA.
authors.forename: Shen
authors.initials: S
authors.lastname: Shen
authors.affiliation: Editas Medicine, Cambridge, MA, 02142, USA.
authors.forename: Minerva
authors.initials: M
authors.lastname: Sanchez
authors.affiliation: Editas Medicine, Cambridge, MA, 02142, USA.
authors.forename: Eugenio
authors.initials: E
authors.lastname: Marco
authors.affiliation: Editas Medicine, Cambridge, MA, 02142, USA.
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Gotta
authors.affiliation: Editas Medicine, Cambridge, MA, 02142, USA.
authors.forename: Morgan L
authors.initials: ML
authors.lastname: Maeder
authors.affiliation: Department of Molecular Genetics and Microbiology and Center for Virology, Duke University Medical Center, Durham, North Carolina, 27710, USA.
authors.forename: Edward M
authors.initials: EM
authors.lastname: Kennedy
authors.affiliation: Department of Molecular Genetics and Microbiology and Center for Virology, Duke University Medical Center, Durham, North Carolina, 27710, USA.
authors.forename: Anand V R
authors.initials: AV
authors.lastname: Kornepati
authors.affiliation: Editas Medicine, Cambridge, MA, 02142, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Sousa
authors.affiliation: Editas Medicine, Cambridge, MA, 02142, USA.
authors.forename: McKensie A
authors.initials: MA
authors.lastname: Collins
authors.affiliation: Editas Medicine, Cambridge, MA, 02142, USA.
authors.forename: Hari
authors.initials: H
authors.lastname: Jayaram
authors.affiliation: Department of Molecular Genetics and Microbiology and Center for Virology, Duke University Medical Center, Durham, North Carolina, 27710, USA.
authors.forename: Bryan R
authors.initials: BR
authors.lastname: Cullen
authors.affiliation: Editas Medicine, Cambridge, MA, 02142, USA. David.Bumcrot@editasmed.com.
authors.forename: David
authors.initials: D
authors.lastname: Bumcrot
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems / Q000235:genetics*; D003850:Deoxyribonuclease I / Q000235:genetics; D000229:Dependovirus / Q000235:genetics; D004706:Endodeoxyribonucleases / Q000235:genetics*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D018014:Gene Transfer Techniques; D005818:Genetic Engineering; D017393:RNA Editing; D013211:Staphylococcus aureus / Q000235:genetics*; D013297:Streptococcus pyogenes / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004706:Endodeoxyribonucleases; D004720:Endonucleases; D003850:Deoxyribonuclease I