crRNA, specifically, is found in the CRISPR repeat/spacer library (loci) of prokaryotes as one long string of crRNAs. The crRNA is made of the spacer (complementary to the target) and a structural piece (the repeat) that complements with the tracrRNA. In type II CRISPR-Cas system, the crRNAs confer target specificity to Cas9 protein, but they can't bind to Cas9 alone. The crRNAs have to complex with tracrRNAs, also known as the handle or scaffold region. Only then can the RNA:RNA duplex properly fit into Cas9. In the interference step, crRNAs combine with Cas proteins to form an effector complex which recognizes the target sequence in the invasive nucleic acid by base pairing to the complementary strand and induces sequence-specific cleavage, thereby preventing proliferation and propagation of foreign genetic elements.
Short mature crRNAs are key elements in the interference step of the immune pathway. The precursor crRNA (pre-crRNA) usually undergoes one or two maturation steps to generate the mature crRNAs that function as guides in destruction of invading DNA or RNA. Generally, primary cleavage of the pre-crRNA occurs at a specific site within the repeats to yield crRNAs that consist of the entire spacer sequence flanked by partial repeat sequences. In some cases, an additional secondary cleavage step is required to generate the active mature crRNAs. In the interference step. CRISPR-Cas systems are categorized into three major types (I, II and III) and several subtypes. Types I (except for I-C) and III employ the Cas6-mediated crRNAs processing while the type II CRISPR-Cas systems use the tracrRNA-guided processing mechanism with endogenous RNase III. Type I-C is associated with the Cas5d-mediated processing.
Single guide RNA (sgRNA), also known as gRNA. Consisting of crRNA- and tracrRNA-derived sequences connected by an artificial tetraloop, sgRNAs were artificially made by humans and don't exist in nature. The sgRNA can form a functional complex with CRISPR-associated nuclease (Cas9) and guide the nuclease to genomic loci matching a 20bp complementary invading DNA, cleaving it immediately upstream of a required 5'-NGG PAM. Crystal structure reveals that the sgRNA binds the target DNA to form a T-shaped architecture comprising a guide:target heteroduplex, a repeat:anti-repeat duplex, and stem loops 1-3. The guide and target DNA form the guide:target heteroduplex via 20 Watson-Crick base pairs. The repeat and the anti-repeat form the repeat:anti-repeat duplex via nine base pairs. The repeat:anti-repeat duplex and stem loop 1 are connected by a single nucleotide, while stem loops 1 and 2 are connected by a 5nt single-stranded linker.
Comparing with the original functional structure of guide RNA, which is composed of crRNA and tracrRNA, the widely used chimeric sgRNA has shorter crRNA and tracrRNA sequence. The sgRNA is functionally equivalent to the crRNA-tracrRNA complex. Furthermore, the deleted RNA sequence could form extra loop structure, which might enhance the stability of the guide RNA structure and subsequently the genome editing efficiency. Chimeric sgRNA is much simpler as a research tool for mammalian genome editing. By modifying the guide sequence, it is possible to create sgRNAs with different target specificities. For example, a key advantage of the CRISPR-Cas9 technology is that the Cas9 protein remains invariant yet can be readily reprogrammed to cleave novel DNA target sites by expressing an sgRNA with a different variable domain. Moreover, we can try to alter the composition of sgRNA to reduce the off-target effects.
Fig 1. The sequence structures of the single guide RNA (sgRNA)
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