Biological diversity of the CRISPR-Cas system. (A) The CRISPR-Cas defense mechanism has three stages, namely, adaptation, maturation, and interference. In the type 1-E system, adaption begins with the recognition of foreign DNA and an associated PAM by the Cas1-Cas2 complex. The host protein IHF, which is not part of the Cas operon, bends the host DNA, allowing the Cas1-Cas2 complex to integrate the newly acquired spacer sequence. During maturation, pre-crRNA transcribed from the CRISPR array is generated and processed into individual mature crRNAs. In the final step of interference, mature crRNAs guide type-specific Cas nucleases to their respective target nucleic acid sequences, resulting in cleavage of foreign DNA. (B) Maturation in the class 1 systems of type I-E and type III-A is carried out by Cas6, which cleaves the pre-crRNA into mature crRNA. In the type 1-E system, a Cas6 monomer remains bound to the crRNA; in the type III-A system, a Cas6 dimer dissociates from the crRNA. Maturation in class 2 systems is performed by the same protein as utilized in interference. In type II-A systems, maturation requires binding of a tracrRNA to the pre-crRNA, forming a tracrRNA-crRNA complex. Cas9 binds the tracrRNA-crRNA complex, and cleavage of the pre-crRNA to mature crRNA is performed by RNase III. Maturation is carried out by Cas12 in the type V-A system and by Cas13 in the type VI-A system. Interference in class 1 systems is mediated by a multiprotein complex, while a single nuclease mediates interference in class 2 systems. In the type I-E system, Cascade binds to a mature crRNA and the complex recognizes the protospacer and an associated PAM sequence on foreign DNA. Cascade binding generates conformational changes in target DNA, resulting in the R-loop structure and recruitment of Cas3 to cleave the nonbound strand of target DNA. In the type III-A system, the Csm complex binds to mature crRNA. This complex then recognizes target nucleic acid sequences, resulting in cleavage of ssRNA and dsDNA. DNA cleavage results in cyclic adenylate production, which activates Csm6 cleavage of nonspecific RNA. In the type II-A system, interference is mediated by Cas9 bound to mature crRNA-tracrRNA. The resulting complex recognizes the PAM on target DNA, resulting in double-stranded cleavage with blunt ends. The type V-A system utilizes Cas12 bound to mature crRNA to recognize foreign DNA and the associated PAM, inducing double-stranded cleavage with staggered ends. Type VI systems employ Cas13, which, along with its mature crRNA, recognizes target ssRNA with its associated PFS. Binding to ssRNA initiates cleavage of the target ssRNA, along with indiscriminate cleavage of nonspecific ssRNA.