Since Drs. Jennifer Doudna and Emmanuelle Charpentier co-invented using CRISPR-Cas9 for gene editing nearly 15 years ago, research in CRISPR (clustered regularly interspaced short palindromic repeats) technologies for use in treating disease has rapidly progressed. While these innovations were occurring, the use of RNA in and as a target for therapeutics also advanced. Unsurprisingly, these two fields have intersected, with several major breakthroughs in the past few years.  

In this eBlog, we will review the current status of RNA-targeting CRISPR and its role in RNA therapeutics. 

How CRISPR works 

For traditional, DNA-targeting CRISPR, the mechanism requires a Cas protein (traditionally a Cas9) and a guide RNA (gRNA), which is a short RNA designed to be complementary to the DNA sequence that researchers want to edit. The gRNA binds to the Cas protein, and the complex scans the DNA to locate a PAM (protospacer adjacent motif), which is a short sequence that allows the complex to bind to the DNA. Once the complex locates the PAM, the Cas protein unwinds the DNA and compares it to the gRNA. If the DNA's bases are complementary to the gRNA’s, the Cas protein cuts both strands of the DNA, leaving behind double-stranded blunt ends on the cut DNA. 

These blunt ends are then repaired through either non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ joins DNA fragments together without exogenous homologous DNA. This mechanism is more commonly used by cells, but it can result in errors from small random insertions or deletions. HDR is more precise and uses a homologous DNA template to insert a specific sequence into the DNA. For gene editing, NHEJ is useful to knock out a gene, and HDR is useful for changing mutations. 

Why target RNA with CRISPR 

In recent years, researchers have explored using CRISPR to edit RNA instead of DNA. Editing the transcriptome rather than the genome offers greater flexibility and safety from the effects of CRISPR editing. Since RNA inherently degrades over time in cells, any editing performed on RNA ultimately disappears. This means there is no permanent change to cells undergoing CRISPR, removing the risk of permanent accidental deletions, insertions, or other editing issues. RNA editing with CRISPR is also easier to dose than when editing DNA, so researchers can better fine tune how much expression the editing drives or reduces. 

There are several mechanisms involved in RNA-targeted CRISPR, each with their own considerations and uses.  

RNA knockdown 

Often, researchers edit RNA with CRISPR using Cas13 systems rather than Cas9. Cas13 is often used for RNA knockdown, which causes therapeutic effects by reducing or silencing a gene associated with a specific mRNA. To perform gene knockdown, Cas13 processes pre-crRNA into mature crRNA and forms a crRNA-Cas13 complex. This complex then identifies the targeted mRNA sequence, usually by locating a protospacer flanking sequence (PFS), which activates the complex to cleave the RNA, disrupting gene expression. 

While this mechanism is effective, Cas13 systems are known to also cleave non-targeted RNA nearby the targeted sequencing, causing “collateral damage.” Another consideration of Cas13 systems is that Cas13 proteins are large molecules that can be difficult to deliver to cells. Cas proteins are typically delivered to patients via adeno-associated virus (AAV) vectors, which can only deliver a small amount of material. 

Fortunately, researchers are identifying and developing Cas13 systems that address these challenges. Cas13d subtypes are the smallest Cas13 proteins, making them more suitable for AAV vectors. In addition, unlike many other Cas13 proteins, RfxCas13d does not require a PFS and performs efficient knockdown with less collateral damage when delivered at lower levels of expression. The protein still reduces the targeted RNA expression, but it keeps nuclease levels in check to reduce unintended cleavage. 

Worth noting, a recent journal article by Watkins et al. suggests measuring knockdown efficiency using RT-qPCR, a common measurement method for this data, overestimates efficiency in CRISPR systems. Future research will explore this topic further. 

RNA base editing 

Beyond gene knockdown, CRISPR can swap individual nucleotide bases in an RNA sequence. This method uses deactivated Cas13, called dCas13, that can bind to RNA without cleaving it. For this process, CRISPR-dCas13 RNA fuses with deaminase to create a base editor, which binds to the target RNA, and identifies the mismatched base between the target RNA and the crRNA. Once the editor locates the mismatch, deaminase swaps the base for the correct one. 

There are a few base editors that each work best for different uses and conditions. Two common base editors are REPAIR and RESCUE, both of which use a dCas13b ortholog fused to an adenosine deaminase acting on RNA2 (ADAR2). REPAIR swaps adenosine for inosine (RNA editing for programmable A-to-I replacement), and RESCUE swaps cytidine to uridine (RNA editing for specific C-to-U exchange). Over time, researchers have developed new versions of REPAIR and RESCUE that improve editing efficiency, reduce off-target effects, and are more compact for delivery. 

Additional base editors are made up of different molecules, such as the C-to-U editor CURE, which is made up of dCasRx and hAPOBEC3A. An additional recently reported base editor fused ADAR2 to IscB, an evolutionary ancestor of Cas9, instead of Cas13 for A-to-I editing. 

RNA Splicing  

RNA-targeting CRISPR can also correct aberrant splicing, which, with further research, could treat or prevent many different diseases. dCas13 can be engineered to induce exon skipping or exon inclusion. When bound to exons or sites required for splicing, dCas13 prevents splicing and forces exon skipping. When the Cas complex binds to a downstream intron, it promotes alternative splicing and corrects the original aberrant splicing.  

Researchers are also developing CRISPR artificial splicing factors (CASFx) to better influence exon skipping and inclusion as desired. Additional splicing platforms work on more than one RNA molecule at once, such as the CRISPR-assisted mRNA fragment trans-splicing (CRAFT) system, which trans-splices exogenous RNA into endogenous pre-mRNA. 

The future of RNA-targeting CRISPR 

RNA-targeting CRISPR has research and therapeutic uses beyond knockdown, base editing, and splicing. Researchers are fusing dCas13 with methyltransferases or demethylases to modify N6-methyladenosine, as well as using a dCasRx-SINEB2 complex to improve translation efficiency. Additional researchers are using green fluorescent protein in CRISPR complexes to gather live-cell RNA images. Finally, AI models are constantly providing data on gRNA sequence design and CRISPR predictions. 

Clearly, the use of CRISPR to edit RNA is a rapidly advancing field. However, to edit RNA with any of these mechanisms, researchers require information of the RNA’s transcriptome. Eclipsebio’s eSTRAND portfolio can provide that analysis with full-length, nanopore-based, direct RNA sequencing data. eSTRAND Transcriptome offers insights into the entire length of RNA in a single experiment, and eSTRAND RNA QC provides the quality control insights needed to make informed decisions on how to use CRISPR in RNA therapeutics. 

Ready to gain the transcriptome insights you need to best use CRISPR? Contact Eclipsebio today. 

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