Types of CRISPR-Cas systems (Part 22- CRISPR in gene editing and beyond)
Welcome to the 22nd part of the multi-part series on applications of CRISPR in gene editing and beyond.
Though the CRISPR-Cas system’s general mechanism remains the same, the CRISPR-Cas system can be classified into six major types- Type 1, 2, 3, 4, 5, and 6. This classification is based on differences in the processing of pre-crRNA, interference step, and the requirement of different Cas proteins.
Type 1 CRISPR-Cas system: In the Type 1 CRISPR-Cas system, pre-crRNA cleavage is carried out by Cas endoribonuclease Cas6 to form mature crRNA. The mature crRNA then interacts with the Cas protein complex consisting of 5 Cas proteins Cse1, Cse2, Cas7, Cas5e, and Cas6e subunits. This Cas proteins-crRNA complex is called Cascade, which locates foreign genetic material. If Cascade binds to foreign DNA, its conformation changes and causes the recruitment of a Cas nuclease Cas3 during the interference step, which then degrades the target DNA.
Type II CRISPR-Cas9 system: In type I CRISPR-Cas system, Cas endoribonuclease Cas6 carries out the pre-crRNA cleavage. But in the case of the type II CRISPR-Cas system, this process involves the expression of a transactivation RNA or tracrRNA. TracrRNA includes an “anti-repeat” region that hybridizes with repeat sequences in the pre-crRNA transcript. The resulting duplex is then cleaved in the repeat sequences by RNase III in a Cas9-dependent reaction.
Then crRNA and tracrRNA form a complex with Cas9 protein to form a complete search complex. Cas9 is literally a programmable protein because it has a program defined by the crRNA guide sequence, which is a 20-letter nucleotide sequence derived from the CRISPR spacer sequence. It means, the 20-letter nucleotide sequence directs the Cas9 protein to recognize a piece of viral DNA that matches the 20-letter sequences of crRNA. If the sequence of crRNA matches the invading virus’s DNA sequence, then Cas9 cuts the viral DNA by introducing a double-stranded break. Like type 1, in the type II system, the target nucleic acid is dsDNA. The Cas9 protein has two lobes; one lobe is for target recognition. And the other lobe contains nuclease activity. The recognition lobe is essential for binding crRNA and target DNA.
On the other hand, the nuclease lobe cleaves the target DNA by generating double-stranded breaks (DSBs). Additionally, the nuclease lobe contains HNH and RuvC nuclease domains, and these domains cause double-stranded breaks in the target DNA. The HNH domain cleaves the DNA strand complementary to the crRNA guide, while the RuvC domain cleaves the non-complementary or coding strand of target DNA.
Type III CRISPR-Cas system: Like Type I systems, in the case of Type III CRISPR-Cas system, pre-crRNA cleavage is carried out by Cas endoribonuclease, Cas6 to form mature crRNA. In addition, the crRNA generated from type III CRISPR locus is composed of eight nucleotides at its 5′ end, termed as the 5′ handle. These eight nucleotides are derived from the repeat sequence of the CRISPR. Following the 5' handle is the guide sequence, which is 30–45 nucleotides and is derived from a CRISPR spacer sequence.
Like Type I systems, in Type III systems, the mature crRNA interacts with the Cas protein complex. The Type III system is further divided into two subtypes, Type III-A and Type III-B systems. The Cas proteins-crRNA complex of the Type III-A CRISPR-Cas system is called the Csm complex, containing a single crRNA and five proteins Csm2, Csm3, Csm4, Csm5, and Cas10 (also called Csm1). On the other hand, the Cas proteins-crRNA complex of Type III-B CRISPR-Cas system is called Cmr complex, containing a single crRNA and six proteins Cmr1, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6.
The mechanism of targeting invading nucleic acids by the type III system is different from type- I and II systems. In Type III-A, invading DNA targeting requires directional transcription across the target sequence or protospacer sequences to produce an RNA transcript that is complementary to the crRNA. While transcription, denaturation of the target double‐stranded DNA occurs in the transcription bubble. Then during the interference step, the crRNA binds to the targeted RNA transcript; this binding leads to the activation of Cas10 and Csm3 nucleases of the Csm complex required to cleave denatured target nucleic acid and the RNA transcript. Cas10 cleaves the non-template or coding DNA strand, and Csm3 protein chops RNA within the protospacer region. The Csm complex also recruits Csm6 protein that degrades nonspecific transcripts in the vicinity.
Cas proteins-crRNA complex of Type III-B CRISPR-Cas system, also called Cmr, can cleave an ssRNA target that is complementary to the CRISPR RNA. The Cmr effector complex consists of six proteins Cmr1–6 and a crRNA. Cmr complex can cleave the target RNA at multiple sites within the region of base-pairing.
Thus, target nucleic acid is usually dsDNA in type I, type II, and type III-A systems. In contrast, the type III-B system targets complementary single-stranded RNA or ss DNA of the invading virus.
Type 4 CRISPR-Cas system: Type IV CRISPR-Cas system is a newly discovered system and is not well characterized. Type 4 systems occur within plasmids and lack the genes like Cas1 and Cas2 that encode the CRISPR-Cas mediated defense system’s first step adaptation. The type IV CRISPR–Cas system has Cas proteins like Cas7 (also called Csf2), Cas5 (also called Csf3), and a smaller version of Cas8 referred to as Csf1. Moreover, Type IV CRISPR–Cas also encodes a DinG family helicase Csf4 and a type IV-specific Cas6-like protein Csf5. A recent structural and biochemical analysis of a type-4 CRISPR–Cas system demonstrated the role of the Cas6-like protein in both the maturation of crRNAs and in the subsequent formation of a Cascade-like crRNA-guided effector complex, composed of Csf1, Csf3, Csf5, and multiple copies of Csf2. As in other CRISPR–Cas systems, the effector complex survey the cellular environment searching for matching nucleic acid targets.
Type 5 CRISPR-Cas12 system: Type 5 CRISPR-Cas system uses the Cas12 enzyme. Cas12 has several critical differences from Cas9: Cas12 causes a ‘staggered’ cut in double-stranded DNA, producing ends with a single-stranded overhang instead of the ‘blunt’ cut made by the Cas9 enzyme. Also, Cas12 requires only a CRISPR RNA (crRNA) for successful targeting. By contrast, Cas9 requires both crRNA and a transactivating rRNA to target the invading virus’s DNA successfully.
Type 6 CRISPR-Cas13 system: The type 6 CRISPR-Cas system utilizes the Cas13 enzyme. Cas13 is an RNA-guided RNA endonuclease, meaning it does not cleave DNA but only single-stranded RNA. Cas13 is guided by its crRNA to the target ssRNA, and once bound, it cleaves the target RNA.
An additional interesting feature of Cas12 and Cas13 enzymes is that they show trans or collateral-cutting activity. It means that on finding the target, the cleavage activity of Cas12 and Cas13 enzymes is not just restricted to the target DNA or RNA, but they can also cut any single-stranded non-targeted nucleic acid molecules in the vicinity. For instance, after binding and cutting the target DNA, Cas12 becomes further active and chops any single-stranded DNA molecule present in its vicinity. On the other hand, after cleaving the target RNA, the activated Cas13 enzyme chops any single-stranded RNA molecule present in its vicinity.
Although this collateral DNAse and RNase activity might appear to be a disadvantage in terms of specific gene editing, but it has made these enzymes a powerful tool for the development of CRISPR-based diagnostics. We will study about this in detail later in the following parts of the series.
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