What is a CRISPR-Cas system ?

CRISPR are repeat arrays found in the DNA of many bacteria and archaea. The name is an acronym for Clustered Regularly Interspaced Short Palindromic Repeats.
The repeats or DR, ranging in size from 23 to 47 base pairs, are separated by spacers of similar length. Repeats often show some dyad symmetry but are not truly palindromic. Spacers are usually unique in a genome. They match sequences in genomes of phage, plasmid or mobile genetic elements. Inside a species, the CRISPR repeat array may show polymorphism.

Cas genes stand for CRISPR-associated genes. Together with the CRISPR array they constitute the CRISPR-Cas defense mechanism. Cas function as clusters of 3 to more than 10 genes and can be distributed into 6 types (I to VI) and more than 30 subtypes.

CRISPR –Cas systems

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) are specific structures found in many prokaryotic genomes that show characteristics of both tandem and interspaced repeats. They have been described in a wide range of prokaryotes, including the majority of Archae and many Eubacteria. A CRISPR locus is characterized by:

  • Repeats and Spacers : A CRISPR is a succession of 23-47bp sequences called repeats separated by unique sequences of a similar length (spacers). Sometimes, at one end of the CRISPR, the repeat is not totally conserved, it is called degenerate repeat.
  • A leader sequence : the CRISPR locus is generally flanked on one side by an AT-rich leader sequence of 100-350 bp, acting as a promoter for the pre-crRNA synthesis.
Together with a set of genes called cas for “CRISPR-associated”, they constitute an immune system.
  • Cluster of cas genes : CRISPR-associated genes are genes found closely linked to the repetitive sequences.

Repeats and Spacers

In a given strain several CRISPRs can be found with a single or different repeat sequences but only one of each kind is associated with the cas genes. The spacers in the different CRISPRs are different.
The unique sequences or spacers correspond mostly to fragments of foreign DNA, ie. viruses, plasmids or mobile genetic elements.

Cas Genes

Several genes called cas for CRISPR-associated are found in the vicinity of CRISPRs and perform the three different functions of the immune system: adaptation, crRNA maturation and interference. Their number varies from one type to another. Phylogenetic studies performed on the CAS protein suggest that CRISPRs are acquired by horizontal transfer. This is further shown by their presence on megaplasmids.

Leader sequence

CRISPR loci are transcribed into a pre-crRNA from the leader acting as a promoter. This precursor is then matured into small crRNA that play a role in the targeting and destruction of homologous foreign sequences.

Classification of CRISPR–Cas systems

All cas genes (13 plus many variants) are not shared by all CRISPR–Cas systems, leading to an enormous diversity. We present here the classification of Kira Makarova & al. (*) based on the identification of signature genes for CRISPR–Cas types and subtypes. CRISPR-Cas systems are thus classified into 2 classes, 6 types and 33 subtypes.
  • Class 1 includes types I, III and IV. They have different effector modules, composed of multiple Cas proteins.
  • Class 2 includes types II, V and VI. Their effector module is a single multidomain protein (Cas9 in type II).
(*) Makarova KS, Wolf YI, Iranzo J, Shmakov SA, Alkhnbashi OS, Brouns SJJ, Charpentier E, Cheng D, Haft DH, Horvath P, Moineau S, Mojica FJM, Scott D, Shah SA, Siksnys V, Terns MP, Venclovas Č, White MF, Yakunin AF, Yan W, Zhang F, Garrett RA, Backofen R, van der Oost J, Barrangou R, Koonin EV. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nat Rev Microbiol. 2020 Feb;18(2):67-83. doi: 10.1038/s41579-019-0299-x. Epub 2019 Dec 19. PMID: 31857715.