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The CRISPR/Cas systems have been classified into three distinct types: 

  • type I
  • type II
  • type III

The top level of the current CRISPR-Cas classification hierarchy includes the three major types (I, II, and III) and the less common but clearly distinct type IV. The distinction between the CRISPR-Cas types is based on the respective signature genes and the typical organization of the respective loci.

Type I CRISPR-Cas Systems

All type I loci has the signature gene cas3 which encodes a large protein with a helicase having a single-stranded DNA (ssDNA)-stimulated ATPase activity coupled to unwinding of DNA-DNA and RNA-DNA duplexes. Often, but not always, the helicase domain is fused to an HD family domain which has an endonuclease activity and is involved in the cleavage of the targeted DNA. 

Usually type I systems are encoded by a single operon containing the cas1 and cas2 genes, genes for the subunits of the Cascade or effector complex, including large subunit, small subunit (often fused to the large subunit), cas5 and cas7 genes, and cas6 gene that is directly responsible for pre-crRNA transcript processing. Each gene in the type I system operons is usually present in a single copy. 

Type II CRISPR-Cas Systems

The signature gene for type II CRISPR-Cas systems is cas9, which encodes a multi-domain protein that combines all the functions of effector complexes and the target DNA cleavage and is essential for the maturation of the crRNA. The type II systems are also known as the “HNH” systems, Streptococcus-like or Nmeni subtype. Every CRISPR-Cas locus of this subtype, in addition to the cas9 gene, also contains the ubiquitous cas1 and cas2 genes. In addition to these three protein-coding genes, the vast majority of type II loci also encompass one or two genes for tracrRNA, an RNA that is partially homologous to the cognate CRISPR. 

Type III CRISPR- Cas Systems

All type III systems have the signature gene Cas10 which encodes a multi-domain protein having a palm domain similar to that in cyclases and polymerases of the PolB family. Thus, this protein originally was predicted to be a polymerase. Recently, the structure of Cas10 has been solved and four distinct domains have been identified: the N-terminal cyclase-like domain that adopts the same RRM fold as the palm domain but is not predicted to possess enzymatic activity, a helical domain containing the Zn-binding treble clef motif, the palm domain that retains the catalytic residues and is predicted to be active, and the C-terminal alpha helical domain resembling the thumb domain of A-family DNA polymerase and Cmr5, a small alpha helical protein present in some of the type III CRISPR-Cas systems. Each type III locus also encodes other subunits of effector complexes such as one gene for the small subunit, one gene for a Cas5 group RAMP protein, and usually several genes for Type III CRISPR-Cas systems often do not encode their own cas1 and cas2 genes but use crRNAs produced from CRISPR arrays associated with type I or type II systems. 

The composition and organization of type III CRISPR-Cas systems are much more diverse compared with type I systems. The diversity is gained by gene duplications and deletions, domain insertions and fusions, and the presence of additional, poorly characterized domains that presumably are involved in either effector complexes or associated immunity. At least two of the type III variants (one of type III-A and the other of type III-B) are relatively common. 

Type IV CRISPR- Cas Systems

Type IV CRISPR-Cas systems, present in many bacterial genomes, often on plasmids, can be typified by the CRISPR-Cas locus in Acidithiobacillus ferrooxidans. Similar to subtype III-A, this system does not have cas1 and cas2 genes and often is not associated with CRISPR arrays. Moreover, in many bacteria, this is the only CRISPR-Cas system, with no CRISPR cassette detectable in the genome. The type IV systems has an effector complex that is consisted of a highly reduced large subunit (csf1), two genes for RAMP proteins of the Cas5 (csf3) and Cas7 (csf2) groups, and, in some cases, a gene for a predicted small subunit. The csf1 gene could be considered a signature gene for the system. There are two distinct subtypes of type IV systems;

  • one of which has a DinG family helicase csf4
  • the second subtype do not have DinG but typically has it 

Type IV CRISPR-Cas systems could be mobile modules that, similar to type III systems, could use crRNA from different CRISPR arrays once these become available. 

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