Mechanism of spacer integration links the CRISPR/Cas system to transposition as a form of mobile DNA.

Fred Dyda,Alison B Hickman

doi:10.1186/s13100-015-0039-3

Abstract

It has recently become clear that many bacterial and archaeal species possess adaptive immune systems. These are typified by multiple copies of DNA sequences known as clustered regularly interspaced short palindromic repeats (CRISPRs). These CRISPR repeats are the sites at which short spacers containing sequences of previously encountered foreign DNA are integrated, and the spacers serve as the molecular memory of previous invaders. In vivo work has demonstrated that two CRISPR-associated proteins - Cas1 and Cas2 - are required for spacer integration, but the mechanism by which this is accomplished remained unclear. Here we review a recent paper describing the in vitro reconstitution of CRISPR spacer integration using purified Cas1 and Cas2 and place the results in context of similar DNA transposition reactions and the crystal structure of the Cas1/Cas2 complex.

Highlights

The recent in vitro reconstitution of the crucial spacer integration step using purified Cas1 and Cas2 proteins, synthetic oligonucleotides, and plasmid substrates represents a major step forward in defining the fundamental biochemistry of the process [3]
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system, present in approximately 90% of archaea and approximately 50% of bacteria, is an adaptive immune system that generates small RNAs transcribed from chromosomally integrated foreign DNA fragments and uses them to direct the degradation of invading DNA that contains the same sequence
The cas genes associated with CRISPR systems are quite variable, with over 45 different gene families identified in various organisms

Summary

Background

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system, present in approximately 90% of archaea and approximately 50% of bacteria, is an adaptive immune system (reviewed in [1]) that generates small RNAs (known as crRNAs) transcribed from chromosomally integrated foreign DNA fragments and uses them to direct the degradation of invading DNA that contains the same sequence. The foreign DNA fragments, called ‘spacers,’ are integrated into a chromosomal CRISPR locus composed of an array of short palindromic repeats forming a repeat-spacer-repeat pattern (Figure 1A). The phenomenon of CRISPR/Cas immunity can be divided into three processes: adaptation, crRNA biogenesis, and crRNA-based interference. Pre-crRNA is produced as a long transcript from the promoter in the leader and subsequently matured into short crRNAs. The exact process of crRNA biogenesis differs significantly in the three different major types of CRISPR/Cas systems. A recent report by Nuñez et al [3] describes the in vitro reconstitution of protospacer insertion, shedding light onto many aspects of adaptation

Main text

Findings

Conclusions