Cut Site Selection by the Two Nuclease Domains of the Cas9 RNA-guided Endonuclease

Scott Bailey,Jihoon Choi,Hongfan Chen

doi:10.1074/jbc.m113.539726

Abstract

Cas9, the RNA-guided DNA endonuclease from the CRISPR-Cas (clustered regularly interspaced short palindromic repeat-CRISPR-associated) system, has been adapted for genome editing and gene regulation in multiple model organisms. Here we characterize a Cas9 ortholog from Streptococcus thermophilus LMG18311 (LMG18311 Cas9). In vitro reconstitution of this system confirms that LMG18311 Cas9 together with a trans-activating RNA (tracrRNA) and a CRISPR RNA (crRNA) cleaves double-stranded DNA with a specificity dictated by the sequence of the crRNA. Cleavage requires not only complementarity between crRNA and target but also the presence of a short motif called the PAM. Here we determine the sequence requirements of the PAM for LMG18311 Cas9. We also show that both the efficiency of DNA target cleavage and the location of the cleavage sites vary based on the position of the PAM sequence.

Highlights

The Cas9 RNA-guided endonuclease has been adapted for genome manipulation and regulation
Identifying the protospacer adjacent motif (PAM) for LMG18311 Cas9—The genome of S. thermophilus LMG18311 contains two CRISPR-Cas systems, of type II-A and III-A, each associated with a CRISPR loci: CRISPR-1 and CRISPR-2, respectively
All of the transformation efficiencies were comparable with those previously reported [35]. These results indicate that the identified PAM is functional in vivo and that the type II CRISPR-Cas system of S. thermophilus LMG18311 protects E. coli cells from transformation by plasmid DNA

Summary

Background

The Cas RNA-guided endonuclease has been adapted for genome manipulation and regulation. The RNA-guided DNA endonuclease from the CRISPRCas (clustered regularly interspaced short palindromic repeat– CRISPR-associated) system, has been adapted for genome editing and gene regulation in multiple model organisms. A short signature sequence, named the protospacer adjacent motif (PAM), is characteristic of the invading DNA targeted by the type I and type II CRISPR-Cas systems. Characterization of S. thermophilus LMG18311 Cas or activation, with its nickase (generated by inactivating one of its two nuclease domains) or nuclease null variants [15, 17, 18, 29] Another appealing possibility for the Cas system is to target different Cas9-mediated activities to multiple target sites, for example transcriptional repression of one gene but activation of another [30]. The HNH domain catalyzes cleavage of the complementary strand at a fixed position, whereas the RuvC-like domain catalyzes cleavage of the noncomplementary strand using a ruler mechanism

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

C T C A AC GTTTTACTCAA AC GTTTTAC

C AA AA CGTTTTAC