CRISPR-Cas immunity in prokaryotes

Journal name:
Nature
Volume:
526,
Pages:
55–61
Date published:
DOI:
doi:10.1038/nature15386
Received
Accepted
Published online

Abstract

Prokaryotic organisms are threatened by a large array of viruses and have developed numerous defence strategies. Among these, only clustered, regularly interspaced short palindromic repeat (CRISPR)-Cas systems provide adaptive immunity against foreign elements. Upon viral injection, a small sequence of the viral genome, known as a spacer, is integrated into the CRISPR locus to immunize the host cell. Spacers are transcribed into small RNA guides that direct the cleavage of the viral DNA by Cas nucleases. Immunization through spacer acquisition enables a unique form of evolution whereby a population not only rapidly acquires resistance to its predators but also passes this resistance mechanism vertically to its progeny.

At a glance

Figures

  1. Stages of CRISPR-Cas immunity.
    Figure 1: Stages of CRISPR-Cas immunity.

    CRISPR loci are a cluster of short DNA repeats (white boxes) separated by equally short spacer sequences of phage and plasmid origin (coloured, numbered boxes). This repeat/spacer array is flanked by an operon of CRISPR-associated (cas) genes (blue-tone arrows) that encode the machinery for the immunization and immunity stages of the system. The CRISPR array is preceded by a leader sequence (grey box) containing the promoter for its expression. a, In the immunization stage, spacer sequences are captured upon entry of the foreign DNA into the cell and integrated into the first position of the CRISPR array. b, In the immunity stage the spacer is used to target invading DNA that carries a cognate sequence for destruction. Spacers are transcribed and processed into small CRISPR RNAs (crRNAs) in the ‘crRNA biogenesis’ phase. These small RNAs act as antisense guides for Cas RNA-guided nucleases (which usually form a complex) that locate and cleave the target sequence (black arrowhead) in the invader’s genome during the ‘targeting’ phase.

  2. Immunity mechanisms of the different CRISPR-Cas types.
    Figure 2: Immunity mechanisms of the different CRISPR-Cas types.

    a, Type I systems. A Cas protein complex known as Cascade cleaves at the base of the stem–loop structure of each repeat in the long precursor crRNA (pre-crRNA, black arrowheads), which generates short crRNA guides. The Cascade–crRNA complex scans the target DNA for a matching sequence (known as protospacer), which is flanked by a protospacer-adjacent motif (PAM, in green). Annealing of the crRNA to the target strand forms an R-loop; the Cas3 nuclease is recruited and cleaves the target downstream of the PAM (red arrowhead) and also degrades the opposite strand. b, Type II systems. These systems encode another small RNA known as trans-encoded crRNA (tracrRNA) which is bound by Cas9 and has regions of complementarity to the repeat sequences in the pre-crRNA. The repeat/tracrRNA dsRNA is cleaved by RNase III to generate crRNA guides for the Cas9 nuclease (black arrowheads). This nuclease cleaves both strands of the protospacer/crRNA R-loop (red arrowhead). A PAM (in green) is located downstream of the target sequence. c, Type III systems. Cas6 is a repeat-specific endoribonuclease that cleaves the pre-crRNA at the base of the stem–loop structure of each repeat (black arrowhead). The crRNA is loaded into the Cas10 complex where it is further trimmed at the 3′ end to generate a mature crRNA (white arrowhead). The Cas10 complex requires target transcription to cleave the non-template strand of the protospacer DNA and it is also capable of crRNA-guided transcript cleavage (red arrowheads).

  3. Mechanism of CRISPR immunization.
    Figure 3: Mechanism of CRISPR immunization.

    a, The first step of CRISPR immunization is the sampling of the spacer sequences. These are believed to be generated from non-specific DNA breaks that occur during replication of the virus or plasmid. The fragments generated are captured by the Cas1–Cas2 complex, with the participation of the targeting machinery for the recognition of DNA sequences carrying a functional PAM. b, The Cas1–Cas2 complex catalyses the integration of the spacer into the first position of the CRISPR array. Cas1 performs two concerted cleavage-ligation reactions whereby the 5′ end of each repeat strand is cleaved (blue arrowhead) and ligated to the 3′ ends of the spacer. This mechanism generates two ssDNA gaps on the repeat sequences that flank the inserted spacer, which presumably are filled by DNA polymerase (dotted arrow). L, leader; R1, first repeat; R2, second repeat; Rnew, new repeat; S1, first spacer; Snew, new spacer.

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  1. Laboratory of Bacteriology, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA

    • Luciano A. Marraffini

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