The CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein system) is a bona fide adaptive (acquired) immunity system that is present in most archaea and many bacteria. CRISPR-Cas system is stored in the form of spacer sequences derived from foreign genomes and inserted into CRISPR arrays, providing sequence-specific protection against foreign DNA or, in some cases, RNA.
CRISPR-Cas is a programmable form of immunity that can adapt to target any sequence and therefore is not under pressure to evolve an immense diversity of specificities as is the case for restriction-modification enzymes, the most abundant form of innate immunity in prokaryotes. Nevertheless, like any defense system, CRISPR-Cas is engaged in an incessant arms race with viruses, which results in rapid evolution of cas genes and notable diversity of the gene repertoires and architectures of the CRISPR-cas loci translating into diversification of the actual defense mechanisms. More specifically, the diversification of the CRISPR-Cas systems is likely to be partly driven by their competitive coevolution with virus-encoded dedicated anti-CRISPR proteins.
The CRISPR-Cas adaptive immune systems of prokaryotes split into two distinct classes based on effector module organization. Class 1 CRISPR-Cas systems utilize multi-protein effector complexes, whereas class 2 CRISPR-Cas systems utilize single-protein effectors. Owing to the complexity of the gene composition and genomic architecture of the CRISPR-Cas systems, any single, all-encompassing classification criterion is rendered impractical, and thus a 'polythetic' approach based on combined evidence from phylogenetic, comparative genomic and structural analysis was developed. Based on the two distinct classes, the CRISPR-Cas system is further divided into six main types (type I–type VI). Within each type of CRISPR-Cas system, several subtypes have been delineated based on additional signature genes and characteristic gene arrangements.
Class 1 CRISPR-Cas systems are defined by the presence of a multisubunit crRNA-effector complex and are divided into 3 types and 15 subtypes. Class 1 systems represent about 90% of the CRISPR-Cas loci and are found in diverse bacterial and archaeal phyla. It includes the most common and diversified type I, type III that is represented in numerous archaea but is less frequent in bacteria, as well as the rare type IV that includes rudimentary CRISPR-cas loci lacking the adaptation module.
Fig 1. Classification of Class 1 CRISPR-Cas systems
In addition to the effector genes, most of the class 1 loci encode adaptation module proteins, Cas1 and Cas2, and multiple accessory proteins, such as Cas4, reverse transcriptase, CARF (CRISPR-associated Rossmann fold) domain-containing protein, and others. Type III and type IV systems often lack adaptation module genes and/or CRISPR arrays in their respective loci. All type I systems also encode DNA helicase Cas3, which is often fused to an HD family nuclease domain. The repeats of class 1 systems are usually palindromic. In type I systems, a protospacer adjacent motif (PAM), which varies between subtypes and is located either 5′ or 3′ of the (proto)spacer, is required for both adaptation and interference.
Class 2 CRISPR–Cas systems are defined by the presence of a single subunit crRNA-effector module, and are divided into 3 types and 18 subtypes. The effector modules of Class 2 consist of a single, large, multidomain protein each, such that the respective CRISPR-cas loci have a much simpler and more uniform organization than those of Class 1. Class 2 systems represent about 10% of the CRISPR-Cas loci and are found in diverse bacterial phyla but virtually absent in archaea. In addition to the effector proteins, most of the Class 2 genomic loci encode adaptation module protein, Cas1 and Cas2, and accessory proteins, such as Cas4. Type II and type V-B loci also include tracrRNA (trans-activating CRISPR RNA), which is partially complementary to the repeats and is involved in CRISPR (cr) RNA processing and interference. Some Class 2 systems, however, especially those of type VI, consist only of a CRISPR array and an effector protein.
Fig 2. Classification of Class 2 CRISPR-Cas systems
CRISPR-Cas Systems is a hot research area for scientists, and has made outstanding contributions to agriculture and medical care, etc,. With the rapid development of CRISPR–Cas Systems, its potential application value will be further explored. The recent progress in the study of the diversity of CRISPR-Cas systems has led to the discovery of not only novel effector protein and locus architectures but also fundamentally new molecular mechanisms. These include the exclusive RNA-targeting by type VI systems that appear to directly couple CRISPR immunity with dormancy induction or PCD; pre-crRNA processing by the effector proteins of types V-A and VI-A; differential regulation type VI-B effectors by small Cas proteins; and also the use of RNA for adaptation via CRISPR-associated reverse transcriptases by some type III systems. With the continuous development of science and technology, CRISPR–Cas systems will continue to be improved.
1. Makarova et al. An updated evolutionary classification of CRISPR–Cas systems. Nat Rev Microbiol. 2015 November ; 13(11): 722–736.
2. Koonin et al. Diversity, classification and evolution of CRISPR-Cas systems. Curr Opin Microbiol. 2017 June ; 37: 67–78.
3. Makarova and Koonin. Annotation and Classification of CRISPR-Cas Systems. Methods Mol Biol. 2015 ; 1311: 47–75.