Type II CRISPR-Cas systems are considered to be the minimal CRISPR-Cas systems that include the CRISPR repeat spacer array and only four (but often three) cas genes, although additional bacterial factors, in particular trans-activating crRNA (tracrRNA) and RNase III, contribute to the function of this system. Similar to type I, type II CRISPR-Cas systems require a well-defined short PAM that is located immediately downstream of the protospacer on the non-target DNA strand. The PAM sequence is important both for spacer acquisition and for target recognition and cleavage. Type II CRISPR systems are the rarest, missing in archaea, and represented in ∼5% of bacterial genomes, with an over-representation among pathogens and commensals. Recently, type II systems have been developed into a powerful gene editing and engineering tool with a major biotechnological potential.
Type II CRISPR-Cas systems incorporate sequences from invading nucleic acid between CRISPR repeat sequences thanks to the Cas1-Cas2 complex. The two proteins, that are present in the great majority of the known CRISPR-Cas systems are sufficient for the insertion of spacers into the CRISPR arrays. The endonuclease activity of Cas1 is required for spacer integration whereas Cas2 appears to perform a nonenzymatic function. The Cas1-Cas2 complex represents the highly conserved information processing module of CRISPR that appears to be quasi-autonomous from the rest of the system.
The minimal type II CRISPR-Cas systems employ an elaborate, unique processing mechanism of pre-crRNA. Unlike most other systems of types I and III that use a dedicated Cas endoribonuclease to cleave pre-crRNA by recognizing the repeat units, type II systems use endogenous RNase III and a second RNA called trans-activating CRISPR RNA (tracrRNA). The tracrRNAs have been identified in most genomes encoding type II systems and are now considered to be an integral component of this CRISPR type. The processing event involves base-pairing of tracrRNA with the pre-crRNA repeats in the presence of a nuclease and helicase protein, called Cas9, to form RNA duplexes that are cleaved by the endogenous RNase III. The intermediate crRNAs undergo further maturation resulting in mature individual crRNAs that remain duplexed with tracrRNA in a complex with the Cas9 protein.
Fig 1. The mechanism of Type Ⅱ CRISPR-Cas Systems mediated gene engineering.
Cas9 nuclease, the signature of type II CRISPR-Cas systems, is a large multidomain protein that combines all the functions of effector complexes and the target DNA cleavage and is essential for the crRNA maturation. Protospacer-encoded portion of the crRNA directs Cas9 to cleave complementary target-DNA sequences, if they are adjacent to short sequences known as protospacer adjacent motifs (PAMs). PAMs are very important to the recognition of self and non-self DNA because they are presents only in the foreign DNA sparing the CRISPR mechanism to delete itself. Indeed, protospacer sequences incorporated into the CRISPR locus are not cleaved presumably because they are not next to a PAM sequence.
20 nucleotides at the 5' end of the gRNA direct Cas9 to a specific target DNA site using standard RNA-DNA complementarity Watson-Crick base-pairing rules. Cas9-induced DSBs are commonly repaired exploiting the NHEJ mediating indel mutations as well as inducing HDR by providing single-stranded oligonucleotide acting as a donor template. In the error-prone NHEJ pathway, the ends of DSB are processed by endogenous DNA repair machinery and rejoined, which results in random indel mutations at the site of junction which can result in the frameshifts within the coding region of a gene and can cause the creation of a premature stop codon, knocking out the target gene. Alternatively, when a repair template in the form of a single-stranded oligodeoxynucleotide is supplied, the HDR pathway allows high fidelity and precise editing.
Type II CRISPR-Cas systems are currently classified into three subtypes(II-A, II-B, and II-C). In the Cas9 phylogeny, subtypes II-A and II-B are monophyletic whereas subtype II-C is paraphyletic with respect to II-A (that is, subtype II-A originates from within II-C). Nevertheless, II-C was retained as a single subtype given the minimalist architecture of the effector modules shared by all II-C loci. The three distinct Type II subtypes each contain three Cas proteins (Cas1, Cas2, and Cas9), a trans-activating crRNA (tracrRNA), and the CRISPR array. Interestingly, a fourth Cas protein is found in Type II-A (Csn2) and Type II-B (Cas4) systems but not Type II-C systems.
Fig 2. Classification of Type Ⅱ CRISPR-Cas Systems.
The Csn2 protein is not required for interference but apparently has an unclear role in spacer integration. The long and short variants of Csn2 form compact clusters when superimposed over the Cas9 phylogenyand seem to correspond to two distinct variants of subtype II-A. Type II-B system lacks csn2 but possess a distinct fourth gene that belongs to the Cas4 family, which is otherwise typical of type I systems. The Cas4 proteins possess 5′-single-stranded DNA exonuclease activity and belong to the PD-EDxK family of nucleases. The actual role of the Cas4 proteins in the CRISPR Cas systems remains unknown. Type II-C CRISPR-Cas systems are the most common type II system in sequenced bacterial genomes. A notable example of a subtype II-C system is the crRNA-processing-independent system found in Neisseria meningitidis.
Type II CRISPR-Cas system has been adapted to be used in vitro, merging the crRNA with a part of the tracrRNA in a hybrid called guide RNA (gRNA). Thus, a variety of guide RNAs can be designed to direct the Cas9 endonuclease for site-specific DNA cleavage and further genetic manipulations such as gene editing, insertion or deletions. The easy conversion of Cas9 into a nickase was utilized to facilitate homology directed repair in mammalian genomes with reduced mutagenic activity and reported increased specificity. Furthermore, the DNA-binding capacity of a catalytically inactive Cas9 mutant can be exploited to engineer diverse RNA-programmable devices that can be used to mediate transcriptional silencing or activation, or as DNA modification tools. The unprecedented versatility in alternatives of genome engineering and modulation of gene expression makes RNA-programmable Cas9 a unique technology in molecular biology.
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