Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements

Genome Biol. 2015 Nov 19:16:253. doi: 10.1186/s13059-015-0818-7.

Abstract

To expand the repertoire of Cas9s available for genome targeting, we present a new in vitro method for the simultaneous examination of guide RNA and protospacer adjacent motif (PAM) requirements. The method relies on the in vitro cleavage of plasmid libraries containing a randomized PAM as a function of Cas9-guide RNA complex concentration. Using this method, we accurately reproduce the canonical PAM preferences for Streptococcus pyogenes, Streptococcus thermophilus CRISPR3 (Sth3), and CRISPR1 (Sth1). Additionally, PAM and sgRNA solutions for a novel Cas9 protein from Brevibacillus laterosporus are provided by the assay and are demonstrated to support functional activity in vitro and in plants.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Base Sequence
  • Brevibacillus / enzymology
  • Brevibacillus / genetics
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Endonucleases / genetics
  • Endonucleases / metabolism
  • Escherichia coli / genetics
  • Gene Targeting / methods*
  • Molecular Sequence Data
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • Streptococcus / enzymology
  • Streptococcus / genetics

Substances

  • Bacterial Proteins
  • RNA, Guide, CRISPR-Cas Systems
  • Endonucleases