Function and regulation of clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR associated (Cas) systems

Abstract

Phages are the most abundant biological entities on earth and pose a constant challenge to their bacterial hosts. Thus, bacteria have evolved numerous 'innate' mechanisms of defense against phage, such as abortive infection or restriction/modification systems. In contrast, the clustered regularly interspaced short palindromic repeats (CRISPR) systems provide acquired, yet heritable, sequence-specific 'adaptive' immunity against phage and other horizontally-acquired elements, such as plasmids. Resistance is acquired following viral infection or plasmid uptake when a short sequence of the foreign genome is added to the CRISPR array. CRISPRs are then transcribed and processed, generally by CRISPR associated (Cas) proteins, into short interfering RNAs (crRNAs), which form part of a ribonucleoprotein complex. This complex guides the crRNA to the complementary invading nucleic acid and targets this for degradation. Recently, there have been rapid advances in our understanding of CRISPR/Cas systems. In this review, we will present the current model(s) of the molecular events involved in both the acquisition of immunity and interference stages and will also address recent progress in our knowledge of the regulation of CRISPR/Cas systems.

Figure 1

Overview of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) adaptive immunity. (a) Adaptation. The CRISPR arrays are composed of short repeats and intervening sequences derived from foreign invaders. Upon infection with a foreign element (e.g., phages or plasmids), part of the genome is typically incorporated into the leader end of the CRISPR array and the repeat is duplicated. The CRISPR arrays are located adjacent to a cluster of cas genes; (b) crRNA generation. The CRISPRs are transcribed into pre-crRNAs that are then processed into mature crRNAs; (c) Interference. The crRNA, in a complex with Cas proteins, binds and degrades the target nucleic acid of the invading element.

Figure 2

Model of CRISPR/Cas adaptation (based on type I-E systems). (a) Naïve acquisition. Cas1 and Cas2 are required for acquisition of new spacers. The first repeat at the leader end of the CRISPR array is duplicated and incorporates a new spacer sequence from a protospacer. The final nucleotide of the repeat is not duplicated but is provided by the PAM nucleotide immediately adjacent to the protospacer; (b) Priming acquisition. Expression of the CRISPR array and generation of the crRNAs against the foreign DNA results in binding / targeting, which is hypothesized to aid in the generation of precursors for integration (possibly single- or double-stranded). Productive interference is not required for priming. Priming acquisition requires Cascade-crRNA, Cas3 and Cas1 and Cas2 and results in new spacers derived from the same strand as the initial spacer.

Figure 3

crRNA generation and target interference in type I, II and III CRISPR/Cas systems.(a) Transcription of the CRISPR array into a pre-crRNA; (b) Processing of the pre-crRNA into mature short crRNAs. In type I, RNA cleavage is performed by Cas6-homologues, which bind the repeat stem-loop and stay associated for Cascade formation. In type II, tracrRNA is required for binding and processing of the pre-crRNA by Cas9 and RNaseIII. In type III, Cas6 binds to non-structured repeats and processes the pre-crRNA into crRNA and then dissociates; (c) Target binding and cleavage. Type I Cascade binds the DNA target before recruiting Cas3 for degradation. In type II, Cas9 stays associated with the tracrRNA:crRNA complex after processing and subsequently binds and cleaves target DNA. The type III-B CMR-complex binds spacer sequence and targets RNA. It is hypothesized that a type III-A Csm complex forms and this system targets DNA.

Figure 4

Regulation of CRISPR/Cas activity. Network model summarizing the regulation of CRISPR/Cas activity. Triangular and flat arrowheads indicate positive and negative effects on CRISPR/Cas activity, respectively for E. coli (red), Salmonella (cyan), M. xanthus (green), Sulfolobus (black), T. thermophilus (purple) and S. thermophilus (blue). For details see text.