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




Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

Genome Biology

, 16:257

Genome editing

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.

KeywordsCRISPR Cas9 Genome engineering Nickases Staphylococcus aureus Adeno-associated virus GUIDE-seq AbbreviationsAAVadeno-associated virus

bpbase pair

DMEMDulbecco’s modified Eagle medium

DSBdouble-strand break

FACSfluorescence-activated cell sorting

FBSfetal bovine serum

gDNAgenomic DNA

GFPgreen fluorescent protein

gRNAguide RNA

indelinsertion and deletion

ITRinverted terminal repeat

NHEJnon-homologous end joining

PAMprotospacer adjacent motif

PBSphosphate-buffered saline

PCRpolymerase chain reaction

SaCas9Staphylococcus aureus Cas9

SpCas9Streptococcus pyogenes Cas9

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

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Autor: Ari E. Friedland - Reshica Baral - Pankhuri Singhal - Katherine Loveluck - Shen Shen - Minerva Sanchez - Eugenio Marco - G

Fuente: https://link.springer.com/







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