Conserved DNA motifs in the type II-A CRISPR leader region.

Mason J Van Orden,Rakhi Rajan,Fares Z Najar,Kesavan Babu,Peter Klein

doi:10.7717/peerj.3161

Abstract

The Clustered Regularly Interspaced Short Palindromic Repeats associated (CRISPR-Cas) systems consist of RNA-protein complexes that provide bacteria and archaea with sequence-specific immunity against bacteriophages, plasmids, and other mobile genetic elements. Bacteria and archaea become immune to phage or plasmid infections by inserting short pieces of the intruder DNA (spacer) site-specifically into the leader-repeat junction in a process called adaptation. Previous studies have shown that parts of the leader region, especially the 3′ end of the leader, are indispensable for adaptation. However, a comprehensive analysis of leader ends remains absent. Here, we have analyzed the leader, repeat, and Cas proteins from 167 type II-A CRISPR loci. Our results indicate two distinct conserved DNA motifs at the 3′ leader end: ATTTGAG (noted previously in the CRISPR1 locus of Streptococcus thermophilus DGCC7710) and a newly defined CTRCGAG, associated with the CRISPR3 locus of S. thermophilus DGCC7710. A third group with a very short CG DNA conservation at the 3′ leader end is observed mostly in lactobacilli. Analysis of the repeats and Cas proteins revealed clustering of these CRISPR components that mirrors the leader motif clustering, in agreement with the coevolution of CRISPR-Cas components. Based on our analysis of the type II-A CRISPR loci, we implicate leader end sequences that could confer site-specificity for the adaptation-machinery in the different subsets of type II-A CRISPR loci.

Highlights

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPRassociated (Cas) proteins constitute an RNA-based adaptive immune system that protects bacteria and archaea against phages and mobile genetic elements (Marraffini & Sontheimer, 2010; Sorek, Lawrence & Wiedenheft, 2013; Mojica et al, 2005; Barrangou et al, 2007; Makarova et al, 2006)
Group 1 has the 3 leader end conserved as ATTTGAG (Fig. 1) and Group 2 has the 3 -leader end conserved as CTRCGAG (Fig. 2A)
The CRISPR1 locus of Streptococcus thermophilus (Sth) DGCC7710 has the 3 leader end conserved as ATTTGAG while the CRISPR3 locus has the 3 leader end conserved as CTACGAG

Summary

Introduction

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPRassociated (Cas) proteins constitute an RNA-based adaptive immune system that protects bacteria and archaea against phages and mobile genetic elements (Marraffini & Sontheimer, 2010; Sorek, Lawrence & Wiedenheft, 2013; Mojica et al, 2005; Barrangou et al, 2007; Makarova et al, 2006). Though types I and III share certain similarities in the overall mechanism of action including crRNA association with multiple Cas proteins (Jackson & Wiedenheft, 2015; Reeks, Naismith & White, 2013; Makarova et al, 2011; Koonin & Makarova, 2013; Shmakov et al, 2015), types II, V, and VI use a single multi-domain protein (Cas, Cpf, or C2c2 respectively) along with cognate RNA components for activity (Abudayyeh et al, 2016; Jinek et al, 2012; Jinek et al, 2014; Nishimasu et al, 2014; Zetsche et al, 2015). Cas along with a guide RNA is widely being used for genome editing applications, and is being pursued for gene therapy and gene regulation applications (Sternberg & Doudna, 2015; Sontheimer & Barrangou, 2015)

Methods

Results

Discussion

Conclusion