Type VI CRISPR-Cas system, termed REPAIR (RNA editing for programmable A to I (G) replacement) is the first CRISPR tool for RNA editing, and it displays high specificity and targeting flexibility. This system contains the programmable single-effector RNA-guided RNases Cas13, which is the core of the precise and flexible RNA base editing technology. Cas13 enzymes are quickly becoming major players in the RNA editing field. The CRISPR RNA editor, composed of a modified Cas13 enzyme which can bind RNA but doesn't cut it, and an RNA adenosine deaminase (ADAR), can effectively edit an adenosine (A) to the nonstandard nucleotide inosine (I) in double-stranded RNAs. The cell's protein synthesis machinery then reads the I as a G.
This CRISPR RNA editor would permit point mutations in RNA which could recapitulate or rescue known pathogenic alleles, or introduce a premature stop codon to render an RNA nonfunctional. REPAIR has no strict sequence constraints, can be used to edit full-length transcripts containing pathogenic mutations. REPAIR presents a promising RNA editing platform with broad applicability for biotechnology research and therapeutics. CRISPR RNA editing has multiple advantages over more traditional DNA editing systems. The temporary nature of REPAIR-mediated edits will likely be useful for treating diseases caused by temporary alterations. For instance, Cas13b could be fused to a variety of editing enzymes that would allow a range of di-erent sequence modifications.
Type VI CRISPR system has the possibility of editing RNA transcripts to alter their coding potential in a programmable manner, named "REPAIR". This modification is mediated by two functional ADAR orthologs, ADAR1 (targeting mainly repetitive regions) and ADAR2 (mainly targeting non-repetitive coding regions), which consist of N-terminal double stranded RNA-binding domains and a C-terminal catalytic deamination domain. Endogenous target sites of ADAR1 and ADAR2 contain substantial double stranded identity, and the catalytic domains require duplexed regions for efficient editing in vitro and in vivo. Importantly, the ADAR catalytic domain is capable of deaminating target adenosines without any protein co-factors in vitro. As I is read as guanosine by the splicing and translation apparatuses, ADARs can also amend splicing patterns and modify amino-acid sequences.
Fig 2. CRISPR-Cas13 system for RNA editing.
For the bacterial CRISPR-Cas13 system, three Cas13 protein families have been identified to date: Cas13a (C2c2), Cas13b, and Cas13c. Interestingly, the biochemical PFS was not required for RNA interference with Cas13a. Like dCas9, the modified dCas13 retains its speciﬁc RNA-binding programmability. The binding of dCas13-crRNA complexes can hinder viral replication and direct speciﬁc modiﬁcations to different RNAs. For example, dCas13 can be fused to a RNA adenosine deaminase (ADAR) that can convert adenosine to inosine (inosine is read as guanosine) to recover functional proteins, block virus replication, or increase plant transcriptome plasticity to improve stress tolerance through customized alternative splicing. The programmable nature of Cas13 enzymes makes them an attractive starting point to develop tools for RNA binding and perturbation applications.
Recently, the newly identiﬁed class II type VI CRISPR-Cas13 systems were found to be amenable for use in RNA manipulation in eukaryotic cells. Cas13 enzymes have two Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) endo RNase domains that mediate precise RNA cleavage with a preference for targets with protospacer flanking site (PFS) motifs observed biochemically and in bacteria. The CRISPR-Cas13 system functions as an 'adaptive' immune system in bacteria and archaea to fend off invading RNA elements, such as RNA viruses, by recognizing the invading RNAs and mediating their subsequent degradation. CRISPR-Cas13 system holds great promise as a robust, precise, scalable RNA-targeting platform for RNA manipulations and RNA virus targeting in many fields.
On the ﬁrst encounter with RNA phages, CRISPR-Cas13 systems acquire a short (28-30 nt) spacer sequence from the phage genome and incorporate it into their CRISPR array through an unknown mechanism. Bacterial acquired immunity occurs via three steps: (i) expression, where both the CRISPR array and Cas13 are transcribed and Cas13 protein is produced; (ii) processing, where pre-crRNA is processed by Cas13 enzyme to form individual, short single crRNAs (28-30nt spacers and a 28-36nt Cas13-binding scaffold); and (iii) targeting or viral interference, where the invading phage RNA is cleaved by the crRNA-Cas13 complex. Upon binding to its target, Cas13 promiscuously degrades cellular RNA, leading to programmed cell death (PCD).
Fig 1. The bacterial CRISPR-Cas13 system against RNA phages.
RNA editing has multiple advantages over more traditional DNA editing systems. For example, RNA editing doesn't require homology-directed repair (HDR) machinery, and could thus be used in non-dividing cells. Cas13 enzymes also don't require a PAM sequence at the target locus, making them more flexible than Cas9/Cpf1. Some Cas13 enzymes prefer targets with a given single base protospacer flanking site (PFS) sequence, but orthologs like LwaCas13a do not require a specific PFS. In addition, by editing RNA rather than DNA, it might be possible to confer temporary, reversible genetic edits, rather than the CRISPR's permanent genome edits. This would allow the potential for temporal control over editing outcomes, as well as avoid the ethical issues that have arisen around genome editing.
Cas13 enzymes do not contain the RuvC and HNH nuclease domains responsible for DNA cleavage, so they can't directly edit the genome. A Cas13-based CRISPR RNA editing system would also avoid genomic off-targets or indels introduced through non-homologous end joining (NHEJ). Now that these REPAIR systems have been shared with the scientific community, we look forward to learning more about their function across the entire transcriptome, and whether they can efficiently edit both low- and high-copy RNA species. We are also curious if REPAIR can function in a multiplexed manner to edit multiple RNAs, or multiple mutations in a single RNA. It's clear that CRISPR RNA editors will open new doors for scientific research.
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