In a 2011 paper, Makarova KS1, Haft DH, Barrangou R, Brouns SJ, Charpentier E, Horvath P, Moineau S, Mojica FJ, Wolf YI, Yakunin AF, van der Oost J, Koonin EV proposed a new system for classifying various CRISPR/cas systems in bacteria and archaea. The genetically engineered CRISPR/cas9 is from Type II.
The following figure is from a different paper of Makarova and Koonin, but it presents the differences between type I, II and III the best. Also note that each of I, II and III have many subdivisions.
Details below -
A new CRISPRCas classification
Here, we propose a new, polythetic classification of CRISPRCas systems in which the cas1 and cas2 genes constitute the core of three distinct types of system (FIG. 2; TABLE 2). Cas1 and Cas2 are present in all CRISPRCas systems that are predicted to be active, and are thought to be the information processing subsystem that is involved in spacer integration during the adaptation stage.
Type I CRISPRCas systems
Typical type I loci contain the cas3 gene, which encodes a large protein with separate helicase and DNase activities35, in addition to genes encoding proteins that probably form Cascade-like complexes with different compositions24,26. These complexes contain numerous proteins that have been included in the RAMP superfamily, which encompasses the large Cas5 and Cas6 families, on the basis of extensive sequence and structure comparisons14 (see TABLE 2 for the available structures). Furthermore, the Cas7 (COG1857) proteins represent another distinct, large family within the RAMP superfamily, as detected by the HHPred method, which can detect distant sequence and structure similarities between proteins36 (Supplementary Information S2 (figure)). In addition, the complexes involved in the CRISPRCas function may contain large proteins such as Cse1 and BH0338-like families, as well as small ?-helical proteins such as Cse2, or other, less conserved subunits. In the Cascade complex, a RAMP protein with RNA endonuclease activity has been
identified as the main enzyme that catalyses the processing of the long spacerrepeatcontaining transcript into a mature crRNA24,26. In most cases, the catalytic RAMP proteins (Cas6, Cas6e and Cas6f; see TABLE 2) do not belong to the most prevalent Cas5 or Cas7 families of RAMPs and are often encoded in the periphery of the respective operon. However, the subtype I-C system (also known as Dvulg or CASS1) (FIG. 2; TABLE 2) might be an exception in which either Cas5 or Cas7 possesses RNase activity. The type I CRISPRCas systems seem to target DNA; target cleavage is catalysed by the HD nuclease domains of Cas3 (REF. 35). As the RecB nuclease domain of Cas4 is fused to Cas1 in several type I CRISPRCas systems, Cas4 could potentially play a part in spacer acquisition instead.
Type II CRISPRCas systems
The type II systems include the `HNH’-type system (Streptococcus-like; also known as the Nmeni subtype, for Neisseria meningitidis serogroup A str. Z2491, or CASS4), in which Cas9, a single, very large protein, seems to be sufficient for generating crRNA and cleaving the target DNA, in addition to the ubiquitous Cas1 and Cas2. Cas9 contains at least two nuclease domains, a RuvC- like nuclease domain near the amino terminus and the HNH (or McrA-like) nuclease domain in the middle of the protein, but the function of these domains remains to be elucidated. However, as the HNH nuclease domain is abundant in restriction enzymes and possesses endonuclease activity37,38, it is likely to be responsible for target cleavage. Furthermore, for the S. thermophilus type II CRISPRCas system, targeting of plasmid and phage DNA has been demonstrated in vivo20 and inactivation of Cas9 has been shown to abolish interference16.
Type II systems cleave the pre-crRNA through an unusual mechanism that involves duplex formation between a tracrRNA and part of the repeat in the pre-crRNA; the first cleavage in the pre-crRNA processing pathway subsequently occurs in this repeat region. This cleavage is catalysed by the housekeeping, double-stranded RNA-specific RNase III in the presence of Cas925.
Type III CRISPRCas systems
The type III CRISPRCas systems contain polymerase and RAMP modules in which at least some of the RAMPs seem to be involved in the processing of the spacerrepeat transcripts, analogous to the Cascade complex. Type III systems can be further divided into sub-types III-A (also known as Mtube or CASS6) and III-B (also known as the polymeraseRAMP module). Subtype III-A systems can target plasmids, as has been demonstrated in vivo for S. epidermidis31, and it seems plausible that the HD domain of the polymerase-like protein encoded in this subtype (COG1353) might be involved in the cleavage of target DNA. There is strong evidence that, at least in vitro, the type III-B CRISPRCas systems can target RNA, as shown with a subtype III-B system from P. furiosus28. It is intriguing that these two type III systems seem to target different nucleic acids, and this finding will require further study.
The only identified ribonucleases in the type III CRISPRCas systems, apart from the universal Cas2 protein, are RAMP proteins. Type III systems include at least two RAMPs in addition to Cas6, which is involved in CRISPR transcript processing. In many organisms, type III CRISPRcas operons lack the cas1cas2 gene pair; in all these cases, an additional CRISPR locus (of either type I or type II) is also present in the respective genome, indicating that Cas1 and Cas2 are probably provided in trans. In other organisms, the polymeraseRAMP modules are present in a single operon with cas1 and cas2, forming a module with the typical architecture in S. epidermidis and Mycobacterium tuberculosis (a type III-A module) and forming a distinct version in Halorhodospira halophila (a type III-B module). In these organisms, the type III operon is the only CRISPRcas locus, suggesting that the polymeraseRAMP module forms a fully functional, autonomous type III system when combined with Cas1 and Cas2, which are likely to be involved in the incorporation of new spacers.
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