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Minimal PAM specificity of a highly similar SpCas9 ortholog.
Chatterjee, Pranam; Jakimo, Noah; Jacobson, Joseph M.
Afiliación
  • Chatterjee P; Center for Bits and Atoms, Massachusetts Institute of Technology, Cambridge, MA, USA.
  • Jakimo N; Media Lab, Massachusetts Institute of Technology, Cambridge, MA, USA.
  • Jacobson JM; Center for Bits and Atoms, Massachusetts Institute of Technology, Cambridge, MA, USA.
Sci Adv ; 4(10): eaau0766, 2018 10.
Article en En | MEDLINE | ID: mdl-30397647
RNA-guided DNA endonucleases of the CRISPR-Cas system are widely used for genome engineering and thus have numerous applications in a wide variety of fields. CRISPR endonucleases, however, require a specific protospacer adjacent motif (PAM) flanking the target site, thus constraining their targetable sequence space. In this study, we demonstrate the natural PAM plasticity of a highly similar, yet previously uncharacterized, Cas9 from Streptococcus canis (ScCas9) through rational manipulation of distinguishing motif insertions. To this end, we report affinity to minimal 5'-NNG-3' PAM sequences and demonstrate the accurate editing capabilities of the ortholog in both bacterial and human cells. Last, we build an automated bioinformatics pipeline, the Search for PAMs by ALignment Of Targets (SPAMALOT), which further explores the microbial PAM diversity of otherwise overlooked Streptococcus Cas9 orthologs. Our results establish that ScCas9 can be used both as an alternative genome editing tool and as a functional platform to discover novel Streptococcus PAM specificities.
Asunto(s)

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Streptococcus / Biología Computacional / Sistemas CRISPR-Cas / Edición Génica / Proteína 9 Asociada a CRISPR Límite: Humans Idioma: En Revista: Sci Adv Año: 2018 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Streptococcus / Biología Computacional / Sistemas CRISPR-Cas / Edición Génica / Proteína 9 Asociada a CRISPR Límite: Humans Idioma: En Revista: Sci Adv Año: 2018 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos