Next-generation CRISPR tools and therapies improved by modified RNA guides

By Angela Betsaida B. Laguipo, BSNAug 3 2021

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), a gene-editing tool, is a promising way to treat diseases by removing, adding, or altering sections of deoxyribonucleic acid (DNA) sequences. It is currently the most straightforward, most flexible, and accurate method of gene manipulation.

A new type of gene-editing tool, CRISPR-Cas13, has shown promise in RNA targeting. Previous studies have shown the tool as a potential treatment method for viral diseases, including coronavirus disease (COVID-19).

Researchers at the lab of Neville Sanjana at New York University (NYU) and the New York Genome Center (NYGC) have developed chemically modified guide RNAs for a CRISPR system that targets RNA instead of DNA.

The research, published in the journal Cell Chemical Biology, demonstrated that co-delivery of modified CRISPR RNA (crRNA) and recombinant Cas13 enzyme in ribonucleoprotein (RNP) complexes can alter gene expression in primary CD4+ and CD8+ T cells, representing an effective and potent method to modulate transcripts without gene manipulation.

Study: Chemically modified guide RNAs enhance CRISPR-Cas13 knockdown in human cells. Image Credit: Vchal / Shutterstock

What is CRISPR-Cas13?

CRISPR-Cas13 is an RNA editing tool that can alter or edit protein sequences without modifying the cell’s genome. Recent advances in CRISPR-Cas13 technology show that it can now be utilized to locate and reduce cancer-linked gene expression.

Apart from this, it has been used in treating acute diseases and reduce inflammation during organ transplantation. It sheds light on RNA processing in disease, specifically RNA editing and alternative splicing. Since there are few changes to gene levels, scientists can explore the possible gene knockdowns to help find cute to certain diseases.

The method has also shown promise in helping scientists explore the use of CRISPR technology on viral diseases, including COVID-19, which has now caused a worldwide pandemic.

Chemical modification of CRISPR-Cas13

In the current study, the researchers aimed to explore different RNA modifications, detailing how the modified guides enhance efficiencies of CRISPR activity from two to five-fold compared with unmodified guides.

To arrive at the study findings, the team compared the several chemical RNA modifications at various positions to determine synthetic crRNAs that enhance RNA targeting efficiency and half-life in human cells. They tested if chemically modified synthetic crRNAs can be delivered into human cells to improve Cas-13-mediated transcript knockdown.

The study findings showed that Cas13 crRNA chemical modifications improve transcript knockdown. The team also found that adding three bases with a different type of chemical bond linking them together, called phosphorothioate modification, extended RNA target knockdown ability by a couple of days in human cells. In primary T cells, the modification caused a 60 to 65-percent knockdown of CD46 expression, a receptor essential for immune system regulation, compared with attaining a 40 to 45-percent knockdown when using an unmodified guide.

Further, the team developed effective Cas13 RNP complexes, giving a platform for non-viral delivery of Cas13. They showed the use of transcriptome engineering in various applications, including targeting endogenous human transcripts, antiviral defense against the pathogen against COVID-19, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and in human primary cells where CRISPR editing has been complex.

“Our study highlights the utility of optimized, chemically modified crRNAs for efficient transcriptome engineering. We anticipate that chemically modified crRNAs for RNA-targeting CRISPRs will be useful for both in vitro RNA diagnostics and in vivo where DNA editing is not feasible or desirable,” the team concluded in the study.

With the development of Cas13-based research tools, diagnostics, and therapeutics, chemically modified crRNAs can enhance CRISPR-Cas13 RNA editing for many applications in both medicine and biotechnology. Using the tool, for example, can help develop better treatments and vaccines against COVID-19, the pandemic that has infected nearly 200 million individuals around the world.