Clustered, regularly interspaced short palindromic repeat (CRISPR)-Cas systems provide rapid and robust adaptation to the rapidly evolving viruses of archaea and bacteria. Upon viral injection or plasmid invasion, a small DNA sequence from invasive genetic elements, known as a spacer, is integrated into the CRISPR loci to immunize the host cell. CRISPR loci consist of an array of short and partially palindromic, repetitive sequences interspaced by equally short 'spacer' sequences. Spacers are transcribed into small RNA guides that direct the cleavage of the viral DNA by Cas nucleases. Spacers are at the centre of CRISPR defence as they specify immunity against phages or plasmids that contain a complementary sequence. 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.
CRISPR-Cas immune systems function in three stages. The first stage is spacer acquisition, also known as adaptation, in which new sequences from exogenous nucleic acid are integrated into the CRISPR locus. The spacer acquisition stage is followed by crRNA biogenesis, in which CRISPR loci are transcribed and processed to generate small interfering CRISPR RNAs (crRNAs) containing one spacer sequence. Spacer crRNA transcripts provide the means for R-loop-based interference, when crRNA base pairs with the complimentary DNA sequence in an mobile genetic element (MGE). The final stage is targeting, in which the spacer sequences in these crRNAs are used as guides to direct the specific cleavage of the viral genome by the Cas nucleases. CRISPR-Cas systems have been classified into three major types, namely Type I, Type II and Type III. We will briefly introduce the immune mechanism of the three CRISPR-Cas systems below.
Genetically, Cas1 and Cas2 which are central to the immunization process of universally occur across types and subtypes, whereas Cas3, Cas9 and Cas10 have been defined as the signature genes for Type I, Type II and Type III, respectively. All types use the same basic molecular mechanism to achieve immunity, that is, through crRNA-guided nucleases, while they differ in the biogenesis of crRNAs and the targeting requirements.
Type I CRISPR-Cas immunity is mediated by Cas3 nuclease and Cascade complex. Cas6e, one of the subunits of Cascade complex, is a repeat-specific endoribonuclease that cleaves the precursor crRNA which is generated by transcription of the full CRISPR loci. The cleavage can generate short crRNAs that remain associated with Cascade and that are used by the complex to locate a complementary sequence in the target DNA, also known as the protospacer. Another subunit, Cas8, also called as Cas A or Cse1, recognizes a short sequence motif located immediately upstream of the target sequence recognized by the crRNA. Sequence motifs adjacent to the targets specified by CRISPR spacers were first identified in type II systems and subsequently named as protospacer adjacent motif (PAM). PAM recognition is required for type I CRISPR-Cas immunity, and the absence of a PAM in the repeat sequences prevents the targeting of the spacers within the CRISPR loci by their complementary crRNAs.
Fig 1. (A) Stages of CRISPR-Cas immunity. (B) Immunity mechanisms of the different CRISPR-Cas types.
Type II CRISPR-Cas systems require only one cas gene, cas9, to execute immunity in the presence of an existing targeting spacer sequence. However, as opposed to the other CRISPR types, two small RNAs are needed for immunity: the crRNA and the trans-encoded crRNA (tracrRNA). The tracrRNA forms a secondary structure that mediates its association with Cas9 protein but also has a region that is complementary to the repeat sequences of the CRISPR array. The dsRNA formed between the tracrRNA and the precursor crRNA is cleaved by RNase III, resulting in the cleavage of each repeat and the processing of the long CRISPR transcript into small crRNA guides. Type II immunity also requires a PAM, and mutations in this motif are the most common mechanism of escape from CRISPR immunity by the targeted viruses.
Type II CRISPR-Cas immunity results in the introduction of crRNA-specific dsDNA breaks in the invading DNA that require two nuclease domains: HNH and RuvC. Each of these domains cleaves one DNA strand of the protospacer sequence and the tracrRNA is absolutely required for cleavage. The first step in target recognition is the transient binding of Cas9 to PAM sequences within the target DNA, which promotes the melting of the two DNA strands immediately upstream of the PAM. A productive interaction in this region of the target, between 6-8 bases of the spacer sequence of the crRNA guide and the melted DNA, triggers the formation of an R-loop and target cleavage.
In type III CRISPR-Cas systems, the precursor crRNA is cleaved by Cas6, a repeat-specific endoribonuclease, which is not a subunit of the complex. As a result of the cleavage, 8 nucleotides of the repeat sequence remain at the 5' end of the spacer sequence in crRNA, a sequence known as the crRNA tag. Then the resulting small crRNAs are transferred to a larger complex, the Cas10-Csm or Cas10-Cmr complex for type III-A or III-B systems, respectively. Within these complexes the crRNAs undergo a mature process whereby the 3' end is trimmed at 6-nucleotide intervals. Type III CRISPR-Cas immunity requires target transcription and a crRNA complementary to the non-template strand of the DNA target and to the transcript. Both DNA and RNA are targeted by type III CRISPR-Cas systems, resulting in the co-transcriptional crRNA-guided cleavage of the target DNA and its transcripts.
The Cas10 complex contains both nucleolytic activities: the palm domain of Cas10 is required for cleavage of the non-template DNA strand, and backbone subunits Csm3 or Cmr4, for type III-A or III-B systems, respectively, are responsible for cleavage of the RNA transcripts. To date, no PAM requirements have been observed for type III CRISPR-Cas targeting. To avoid targeting of the CRISPR locus, type III systems rely on the differential base pairing between the crRNA tag and the sequences flanking the protospacer. Whereas the absence of complete complementarity between these sequences licenses DNA targeting, full complementarity between the crRNA tag and the repeat sequence in the CRISPR locus prevents DNA targeting and thus autoimmunity.
1. Luciano A. Marraffini. CRISPR-Cas immunity in prokaryotes. Nature. 2015 October 1; 526: 55–61.
2. Cubbon et al. CRISPR-Cas Immunity, DNA repair and Genome Stability. Bioscience Reports. 2018 October 31; 38(5): BSR20180457.
3. Barrangou and Marraffini. CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity. Mol Cell. 2014 April 24; 54(2): 234–244.
4. Tom Killelea and Edward L. Bolt. CRISPR-Cas adaptive immunity and the three Rs. Bioscience Reports. 2017 Aug 31; 37(4): BSR20160297.