Targeting Activity Time of CRISPR RNA Improved with Modified CRISPR-Cas13 Guides

Targeting Activity Time of CRISPR RNA Improved with Modified CRISPR-Cas13 Guides

The CRISPR gene editing system is most commonly used for making cuts to double stranded DNA to remove or replace faulty genes, but that is not the only way that CRISPR can been used. Recently, a new type of CRISPR technology has been developed that targets single stranded RNA rather DNA to modulate gene expression outcomes. This is a preferred method of using CRISPR, as genetic expression is modulated without making permanent changes to the underlying DNA.

While this relatively new method of using CRISPR has great potential, it is not without its challenges. When CRISPR RNA (crRNA) is delivered into human cells there is rapid guide degradation, which severely limits the CRISPR-targeting activity time, which is only about 48 hours.

A team of researchers at the New York Genome Center (NYGC) have been studying how chemical RNA modifications at various positions of the guide RNA can improve the efficiency of CRISPR activity. Their studies have shown some of these modifications can extend the CRISPR targeting activity from 48 hours to 4 days.

“We were inspired by the guide modifications developed for other DNA-targeting CRISPRs and wanted to test if chemically modified guides could improve knockdown time for RNA-targeting CRISPR-Cas13 in human cells,” said Alejandro Méndez-Mancilla, PhD, a New York Genome Center (NYGC) post-doc and co-first author of the study.

Source: Mendez-Mancilla et al.

The researchers had previously conducted a study, published in Nature Biotechnology in March 2020, which outlined the principles for optimal Cas13 guide design. Following on from the findings of that study, chemical changes were made to the guide to test whether it was possible to improve efficiency and increase half-life. The researchers found that adding three bases linked together with a phosphorothioate modification significantly increased the half-life in human cell lines.

The researchers targeted cell surface receptors in human T cells from healthy donors and showed that their modification resulted in a 60-65% knockdown of expression of the immune system regulation receptor CD46, compared to a 40-45% knockdown when using an unmodified guide. In addition, they found that it was possible to improve Cas13 activity with certain methylation and inverted terminator modifications; however, those modifications needed to be carefully placed, as in some cases, modifications halted all Cas13 activity. The researchers demonstrated the knockdown of cell surface receptors that are part of a universal segment of the genetic sequence that is shared by all currently known variants of SARS-COV-2.

By targeting this sequence on subgenomic RNAs used by the virus when making essential proteins for replication and the infection of other cells, RNAcr could potentially be used as a treatment to protect cells against SARS-CoV-2 infection.

“These modified guides further expand the toolbox for genome and transcriptome engineering. For non-coding elements in the human genome, targeting DNA may not be effective, and other organisms, such as RNA viruses like coronavirus or flu, cannot be targeted at all,” said NYGC core faculty member, Neville Sanjana, PhD.

 

 

You can read more about the study in the paper – Chemically modified guide RNAs enhance CRISPR-Cas13 knockdown in human cells– which was recently published in Cell Chemical Biology. DOI: 10.1016/j.chembiol.2021.07.011