CRISPR-Cas13 in RNA editing (Part 57- CRISPR in Gene Editing and Beyond)
Welcome to the 57th part of the multi-part series on applications of CRISPR in gene editing and beyond.
CRISPR-Cas13 is a revolutionary gene-editing technology that has gained attention for its potential in RNA editing. While CRISPR-Cas9 and CRISPR-Cas12 are well-known for their ability to edit DNA, CRISPR-Cas13 is uniquely suited for RNA manipulation.
Introduction to CRISPR-Cas13
Cas13 is a family of CRISPR-associated proteins that have been repurposed for RNA editing. Unlike Cas9 and Cas12, which target DNA, Cas13 targets RNA.
Components of CRISPR-Cas13: CRISPR-Cas13 systems consist of two main components: a CRISPR array containing guide RNAs (gRNAs) and the Cas13 protein. The gRNA is a synthetic RNA molecule designed to be complementary to the target RNA sequence, guiding the Cas13 protein to the desired RNA.
Targeting RNA: The gRNA is designed to recognize a specific RNA sequence by base-pairing with it. Once the gRNA binds to the target RNA, it undergoes a conformational change that activates its RNA cleavage activity. Cas13 effector protein contains two higher eukaryotes’ and prokaryotes’ nucleotide-binding domains (HEPN) that provide RNase activity. The Cas13 protein, when associated with gRNA, forms an RNA-guided RNA-targeting complex to recognize and cleave ssRNA targets. This cleavage can be used to knock down or degrade the target RNA.
But Cas13 also show collateral cleavage activity, which means the unintended cleavage of non-targeted RNA molecules. It represents a challenge in using CRISPR-Cas13 for RNA editing. To minimize it, different variants of Cas13 exhibit reduced collateral cleavage, are used, making them more suitable for applications requiring high specificity.
It is important to note that PAM sites are not required. RNA editing with CRISPR-Cas13 is guided solely by the complementary base pairing between the gRNA and the target RNA sequence, allowing for precise and specific RNA manipulation without the need for PAM sequences. Thus making them more flexible than Cas9 enzymes.
Applications in RNA Editing
CRISPR-Cas13 can be used for various RNA editing purposes:
(i) RNA Knockdown: By cleaving and degrading specific RNA molecules, Cas13 can reduce the levels of a particular RNA, effectively silencing the gene it encodes.
When introduced inside the cell, the Cas13 protein searches for the target RNA using the guidance of the gRNA. When the Cas13 protein encounters the specific RNA sequence that matches the gRNA, it binds to the target RNA with high specificity due to complementary base pairing. Activated Cas13 then functions as an RNA “scissor” and cleaves the target RNA at the site specified by the gRNA. This cleavage disrupts the integrity of the RNA molecule. As the target RNA is degraded, the gene it encodes is effectively silenced. This gene silencing can lead to a decrease in the production of the corresponding protein, which in turn can impact cellular functions.
Additionally, editing the sequence of RNA can, therefore, temporarily edit gene expression without the serious risks associated with permanent changes to the genome. This could be used in the treatment of acute diseases and temporary reduction in inflammation during organ transplantation.
(ii) Base Editing: In addition to cleavage, some variants of Cas13 can perform base editing, allowing for precise changes in individual RNA bases without causing extensive RNA degradation. While the primary function of Cas13 is to cleave RNA, a modified version called “dCas13” (dead Cas13) has been developed for RNA editing. dCas13 lacks RNA cleavage activity but retains its RNA-binding capability. When fused with an adenosine deaminase enzyme, such as ADAR (adenosine deaminase acting on RNA), it enables RNA base editing. dCas13-ADAR fusions can convert adenosine (A) bases to inosine (I) in the target RNA sequence. Inosine is read as guanosine (G) by the cell’s translational machinery.
This method allows for the precise editing of single RNA bases without causing extensive RNA degradation. The targeted A-to-I conversion provides a specific and controlled change that can be used for a variety of purposes, including correcting RNA mutations responsible for genetic diseases, studying RNA transcripts’ functions, introducing a premature stop codon to render an RNA nonfunctional, or even developing RNA-based therapies.
(iii) RNA Interference against Viruses: RNA interference (RNAi) is an innate antiviral immunity mechanism that has been successfully used to combat various plant viruses. However, the availability of such antiviral strategies remains constrained to specific virus groups. Many viruses readily mutate and have developed different counter-defense mechanisms, necessitating the continuous development of antiviral approaches. The recently introduced CRISPR/Cas13 tool holds great promise for engineering plant immunity against a broad range of RNA viruses that constitute a majority of plant viruses.
Specificity and Off-Target Effects
Like other CRISPR systems, specificity is a concern in CRISPR-Cas13. Ensuring that the gRNA is highly specific to the target RNA sequence is crucial to avoid unintended off-target effects.
Ongoing research focuses on improving the specificity of CRISPR-Cas13 for safe and precise RNA editing by introducing mutations in the Cas13 enzyme, ADAR, and gRNA.
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