Minimal PAM specificity of a highly similar SpCas9 ortholog.

Journal Article (Journal Article)

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.

Full Text

Duke Authors

Cited Authors

  • Chatterjee, P; Jakimo, N; Jacobson, JM

Published Date

  • October 2018

Published In

Volume / Issue

  • 4 / 10

Start / End Page

  • eaau0766 -

PubMed ID

  • 30397647

Pubmed Central ID

  • PMC6200363

Electronic International Standard Serial Number (EISSN)

  • 2375-2548

International Standard Serial Number (ISSN)

  • 2375-2548

Digital Object Identifier (DOI)

  • 10.1126/sciadv.aau0766

Language

  • eng