Diversity, classification and evolution of CRISPR-Cas systems

Abstract

The bacterial and archaeal CRISPR-Cas systems of adaptive immunity show remarkable diversity of protein composition, effector complex structure, genome locus architecture and mechanisms of adaptation, pre-CRISPR (cr)RNA processing and interference. The CRISPR-Cas systems belong to two classes, with multi-subunit effector complexes in Class 1 and single-protein effector modules in Class 2. Concerted genomic and experimental efforts on comprehensive characterization of Class 2 CRISPR-Cas systems led to the identification of two new types and several subtypes. The newly characterized type VI systems are the first among the CRISPR-Cas variants to exclusively target RNA. Unexpectedly, in some of the class 2 systems, the effector protein is additionally responsible for the pre-crRNA processing. Comparative analysis of the effector complexes indicates that Class 2 systems evolved from mobile genetic elements on multiple, independent occasions.

Figure 1. Updated classification of CRISPR-Cas systems

(A) Class 1 (B) Class 2 The organization of the CRISPR-cas loci and domain architectures of the effector proteins as well as the (predicted) target (DNA or RNA, or both) are shown for each subtype. For subtype III-D, a locus with reverse transcriptase fused to cas1 is included; other reverse transcriptase-containing variants, from subtypes III-A and III-D, are not shown. The genes for Class 1 effector subunits are shaded. SS, small subunit; TM, predicted transmembrane segment.

Figure 1. Updated classification of CRISPR-Cas systems

(A) Class 1 (B) Class 2 The organization of the CRISPR-cas loci and domain architectures of the effector proteins as well as the (predicted) target (DNA or RNA, or both) are shown for each subtype. For subtype III-D, a locus with reverse transcriptase fused to cas1 is included; other reverse transcriptase-containing variants, from subtypes III-A and III-D, are not shown. The genes for Class 1 effector subunits are shaded. SS, small subunit; TM, predicted transmembrane segment.

Figure 2. Cryo-electron microscopic structures of type I and type III effector complexes

The Electron Microscopy Data Bank (EMDB) codes are indicated for each structure. The structures are from Refs. [21] (I-C), [20] (I-E), [23] (III-A), [22] (III-B).

Figure 3. The Class 2 effector proteins

(A) Schematic representation of the complexes of effector proteins with the target DNA or RNA, guide RNA and (for type II) tracrRNA (B) Domain architectures of the effector proteins and their transposon-encoded evolutionary ancestors IscB and TnpB are the inferred ancestors of the type II (Cas9) and type V (Cas12) effectors, respectively. The catalytic residues of the effector nuclease domain and, for Cas12a and Cas13a, the residues shown to be required for pre-crRNA processing are indicated in red. The Protein Data Bank (PDB) codes are included for proteins with solved structures. HTH, helix-turn-helix DNA-binding domain. The tracrRNA, the pre-crRNA processing catalytic sites and the nicking, target strand-cleaving nuclease are denoted by asterisks to indicate that they are each present only in subsets of the type II and type V effectors 9see text for details). The catalytic amino acid residues of the target-cleaving nucleases are shown in red, and those of the pre-crRNA processing nuclease are shown in blue. RuvCI, II and III stand for the three conserved motifs of the RuvC-like nuclease domain that contain the catalytic residues. In the motif signature, “x” stands for amino acid, and “..” indicated that the catalytic residues are separated by a small, variable number of non-conserved residues.