CRISPR-Cas13: how does the technology compare to its famous Cas9 cousin?

As the first Cas protein that scientists repurposed for gene editing, CRISPR-Cas9 has been a revolutionary tool, making gene editing easier and faster than ever before. But it is not the only CRISPR-based tool that scientists are putting to good use. Its lesser-known cousin, CRISPR-Cas13, is also making headway in a number of applications, both as a diagnostic tool and as a gene therapy. In this article, we explore how CRISPR-Cas13 differs from CRISPR-Cas9, its advantages and disadvantages, and what the technology can be used for.

CRISPR-Cas13 vs CRISPR/Cas9: What is the difference? 

Although both are CRISPR-Cas systems, Cas9 and Cas13 are actually quite different in terms of how they work. This is because CRISPR-Cas9 uses a single guide RNA (sgRNA) to target and cleave DNA, whereas CRISPR-Cas13, despite also being an RNA-guided nuclease, targets RNA instead of DNA. 

CRISPR-Cas13 also indiscriminately cleaves single-stranded RNA (ssRNA) when engaged at its RNA target sequence. This RNA-targeting ability makes it perfect for targeting mRNA and is useful as a mechanism of transient (non-permanent) knockdown of gene expression – unlike CRISPR-cas9, whose edits on DNA are permanent.

Because of its mechanism of action, CRISPR-Cas13 has stirred up interest in its potential as a viral therapeutic that could specifically degrade target viral RNA. 

Applications of CRISPR-Cas13

In terms of applications, CRISPR-Cas13 is perhaps best known for its ability to detect and inhibit viruses. It can also be used to diagnose bacteria, other pathogens, and even cancer. While it has the potential to act as a gene therapy treatment for indications such as muscular dystrophy and certain cancer types, its role in disease treatment is still being researched and much remains to be discovered about its potential uses in this area. 

Fighting viruses

It has been known for a while now that CRISPR-Cas13 can effectively detect viruses in people’s systems. But the fact that scientists say it could also be used to fight viruses, due to its ability to target RNA and cleave ssRNA, is an extremely welcome proposition. RNA viruses account for many human diseases and pandemic events but are often not targetable by traditional therapeutic modalities. Here are a few examples of studies showing how CRISPR-Cas13 has the potential to treat certain viruses:

• In a 2023 study, researchers showed that adeno-associated virus (AAV)-delivered CRISPR-Cas13 directly targeted and eliminated human enterovirus – a genus of positive-sense single-stranded RNA viruses associated with several human and mammalian diseases – in cells and infected mice. Here, the new antiviral AAV-CRISPR-Cas13 modality was seen to represent an effective direct-acting prophylactic and therapeutic agent against lethal RNA viral infections.

• Not only can CRISPR-Cas13 be used for better detecting COVID-19, but it turns out that it could also help to fight against the virus. In 2020, a team of bioengineers at Stanford University, who had already been working on a system to fight the flu when the COVID-19 pandemic emerged, reported that they had developed a method of inhibiting 90% of coronaviruses, including SARS-CoV-2. The technology, called PAC-MAN, combines a guide RNA with Cas13, directing Cas13 to destroy certain nucleotide sequences in the SARS-CoV-2 genome and effectively neutralizing it. The technology was also found to lower the viral load in human lung epithelial cells infected with the H1N1 strain of the flu.

• A 2020 study reported the use of the CRISPR-Cas13a system to inhibit human immunodeficiency virus type 1 (HIV-1) infection. This was done by targeting HIV-1 RNA and reducing viral gene expression. A strong inhibitory effect of the CRISPR-Cas13a system on HIV-1 infection in human cells was observed experimentally, and the technology was found not only to reduce the levels of newly synthesized viral RNA, but also target and destroy viral RNA entering cells within the viral capsid, resulting in a strong inhibitory effect on HIV-1 infection. 

• In a study conducted last year, scientists detailed the three-dimensional structure of one of the smallest known CRISPR-Cas13 systems, CRISPR-Cas13bt3, used for RNA modification, which operates differently from other proteins in the same family. This discovery allowed them to enhance the tool’s precision, enabling better access and delivery to target editing sites. This means that this particular CRISPR-Cas13 system holds extreme promise in targeting RNA and shredding viruses.

Killing drug-resistant bacteria 

Drug-resistant bacteria are an ever-growing threat in today’s world and there is a significant need to come up with effective alternatives to antibiotics. 

Luckily, CRISPR technology could hold promise in this area, as it makes it possible to design CRISPR antimicrobials with specific bacteria-killing effects. In a study conducted in 2020, researchers used the CRISPR-Cas13a system to enable crRNA to recognize the promiscuous RNA cutting ability (promiscuous means binding to many RNAs without the requirement for an obvious or well-defined protein-binding pattern) of the target RNA, resulting in host cell death and also resulting in a new class of bacterial antimicrobial agents. 

Additionally, in terms of killing efficiency, CRISPR-Cas13a is thought to be better than CRISPR-Cas9, which has also been studied to defeat bacteria and can be used with antibiotic-resistant bacteria, such as beta-lactam or vancomycin-resistant staphylococcus. In contrast to CRISPR-Cas9-based antimicrobials, which lack bactericidal ability when the target gene is located on the plasmid, CRISPR-Cas13a shows strong bacteria-killing activity when recognizing the target gene. 

The study reported the development of an antimicrobial nucleocapsid based on CRISPR-Cas13a, called CapsidCas13a(s). It can specifically kill carbapenem-resistant E.coli and methicillin-resistant Staphylococcus aureus by identifying the corresponding sequence of antimicrobial resistance genes.

Treating cancer

CRISPR-Cas13a has been used to detect tumor markers, such as the aberrant expression of microRNAs (miRNAs), which is known to be associated with various tumors and has been confirmed as a biomarker for initial cancer diagnosis. And, as well as being used for diagnosis, the CRISPR-Cas13a system has been studied as a cancer therapy.

An oncogene is a kind of gene that is related to the transformation of tumor cells, and its abnormal expression often leads to the occurrence of tumors. The CRISPR-Cas13a system can inhibit tumor growth by specifically recognizing and reducing the mRNA expression of oncogenes in cancer cells, inducing programmed cell death of these cells. 

While further investigation needs to be done into how CRISPR-Cas13 could treat cancer, it is being studied as a potential therapy for cancers such as glioma, pancreatic cancer, cervical cancer, and bladder cancer. 

Treating genetic diseases

Although CRISPR-Cas9 is the first choice when it comes to the treatment of genetic diseases, CRISPR-Cas13 could also potentially be used as a gene-editing tool in this manner. 

In a study conducted in 2022, the University of Illinois Urbana-Champaign researchers used a targeted CRISPR-Cas13 technique in the central nervous systems of mice to turn off the production of mutant proteins that can cause amyotrophic lateral sclerosis (ALS) and Huntington’s disease. Here, the team developed Cas13 systems to target and cut RNAs that code for mutant proteins that trigger ALS and Huntington’s disease, effectively silencing the mutant genes without disturbing the cell’s DNA.

Meanwhile, another study conducted last year showed that mini-dCas13X–mediated RNA editing restored dystrophin expression in a humanized mouse model of Duchenne muscular dystrophy. 

Targeting RNA with CRISPR-Cas13: a promising future?

As revolutionary as CRISPR-Cas9 gene-editing tools have been, they face unique safety and efficacy limitations due to the permanent nature of DNA editing. In fact, CRISPR-Cas13 was investigated in a study at Stanford University to circumvent the limitations of CRISPR-Cas9. Scientists developed an RNA editing tool called multiplexed effector guide arrays (MEGA) and used Cas13 and a pooled array of guide RNAs to simultaneously edit multiple gene transcripts in primary human T cells without targeting or cutting genomic DNA. This multi-targeting method addressed an unmet need in cell therapy optimization by allowing the researchers to dynamically regulate several pathways per T cell, rather than add or ablate individual genes completely, one at a time. 

So, as RNA editing has recently made its way into the clinic, with more and more companies choosing to focus on this field, the ability of CRISPR-Cas13 to act on RNA instead of DNA could make it a very promising and valuable tool.

New treatments related to CRISPR-Cas13

• RNA-Programmed NMD Activation (RP-NMD) System – University of Rochester