CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR), and associated proteins (Cas) comprise the CRISPR-Cas system, which confers adaptive immunity against exogenic elements in many bacteria and most archaea. CRISPR-mediated immunization occurs through the uptake of DNA from invasive genetic elements such as plasmids and viruses, followed by its integration into CRISPR loci. These loci are subsequently transcribed and processed into small interfering RNAs that guide nucleases for specific cleavage of complementary sequences. Conceptually, CRISPR-Cas shares functional features with the mammalian adaptive immune system, while also exhibiting characteristics of Lamarckian evolution. Because immune markers spliced from exogenous agents are integrated iteratively in CRISPR loci, they constitute a genetic record of vaccination events and reflect environmental conditions and changes over time. Cas endonucleases, which can be reprogrammed by small guide RNAs have shown unprecedented potential and flexibility for genome editing and can be repurposed for numerous DNA targeting applications including transcriptional control.

Figure 1. The three stages of CRISPR immunity

CRISPR loci contain clusters of repeats (black diamonds) and spacers (colored boxes) that are flanked by a “leader” sequence (L) and CRISPR-associated (cas) genes. During adaptation new spacers derived from the genome of the invading virus are incorporated into the CRISPR array (Barrangou et al., 2007) by an unknown mechanism. The synthesis of a new Repeat is also required. During crRNA biogenesis a CRISPR precursor transcript is processed by Cas endoribonucleases within repeat sequences to generate small crRNAs (Brouns et al., 2008). During targeting the match between the crRNA spacer and target sequences (complementary protospacer) specifies the nucleolytic cleavage (red cross) of the invading nucleic acid (Garneau et al., 2010; Gasiunas et al., 2012; Jinek et al., 2012).

Figure 2. Mechanism of crRNA biogenesis and targeting in the three Types of CRISPR-Cas systems

Black arrowheads, primary processing sites of the crRNA precursor (pre-crRNA) to liberate intermediate crRNAs (int-crRNA). White arrowhead, further processing of the int-crRNA to yield mature crRNAs (mat-crRNA). Green line, target sequence (same sequence as crRNA spacer). Purple line, PAM (Mojica et al., 2009). (A) In Type I systems, primary processing of the pre-crRNA is achieved by the Cas6 endoribonuclease within the Cascade complex (Brouns et al., 2008). Cleavage occurs at the base of the stem-loop formed by the repeat RNA to release mat-crRNAs. The Cascade recruits the Cas3 nuclease to nick the DNA strand complementary to the proto-spacer, immediately downstream of the region of interaction with the crRNA spacer (Sinkunas et al., 2013). (B) In Type II systems primary processing requires the annealing of the tracrRNA to the repeat sequences of the pre-crRNA and the subsequent cleavage of the dsRNA by the host RNase III (Deltcheva et al., 2011). Primary processing occurs in the contex of Cas9 and it is followed by the trimming of the 5′-end repeat and spacer sequences of the int-crRNA to yield mat-crRNAs. Target cleavage requires the crRNA, the tracrRNA and the RuvC and HNH domains of Cas9, each of which cleaves one DNA strand of the proto-spacer region, 3-nt upstream of the PAM (Gasiunas et al., 2012; Jinek et al., 2012). (C) In Type III systems Cas6 cleaves the pre-crRNA to generate int-crRNAs that are incorporated into a Cmr/Cas10 or Csm/Cas10 complex, where further maturation occurs through the trimming of 3′-end sequences (Hale et al., 2012; Hale et al., 2009). While genetic evidence indicates that III-A subTypes cleave target DNA sequences (Hatoum-Aslan et al., 2014), biochemical data suggests that subType III-B cleave RNA molecules (Hale et al., 2009).

Figure 3. Cas9-based genetic applications

Wild-Type Cas9 loaded with a single guide RNA (sgRNA) generates dsDNA breaks that can be used to introduce target mutations. Chromosomal breaks can be repaired by non-homologous end joining (NHEJ), creating indels (Δ) that introduce knock-out frameshift mutations. If a sequence homologous to the Cas9 target is provided (the editing template; either linear dsDNA or a short oligonucleotide), the break can be repaired by homologous recombination (Cong et al., 2013; Mali et al., 2013b). In this case, site-specific mutations in the editing template are can also be incorporated in the genome. A catalytically dead Cas9 (dCas9, containing mutations in both the RuvC and HNH actives sites) can be used as an RNA-guided DNA binding protein that can repress both transcription initiation when bound to promoter sequences or transcription elongation when bound to the template strand within an open reading frame (Bikard et al., 2013; Qi et al., 2013). dCas9 can also be fused to different functional domains to bring enzymatic activities and/or reporters to specific sites of the genome (Gilbert et al., 2013b).