Refining tools for gene editing in human cells

Refining tools for gene editing in human cells

In a span of a few years, the bacterial clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated (Cas) system, which evolved as a defense against bacteriophages, has been developed into a powerful tool for editing genomic DNA within eukaryotic cells.

In one version of this method, a DNA sequence-specific CRISPR RNA (crRNA) is delivered to cells expressing the Cas9 protein and a transactivating RNA (tracrRNA). The three macromolecules form a complex that generates a double-stranded break at the target site.

To improve the therapeutic potential of CRISPR-Cas researchers in the journal PNAS sought to generate a synthetic CRISPR RNA (scrRNA) with enhanced function.

Replacing the phosphate backbone of a 42-nucleotide crRNA with phosphothioate (PS), which is more resistant to nucleases, the authors obtained a scrRNA with enhanced activity in cell culture.

To stabilize the interactions of scrRNA with both DNA and tracrRNA, the authors generated substitutions at the 2′ hydroxyl group of select ribose groups while experimenting with shorter lengths of scrRNA. A 29-nucleotide scrRNA containing a complete PS backbone and two types of 2′ modifications, located within the DNA and tracrRNA recognition domains, demonstrated activity similar to that obtained with a 102-nucleotide single guide RNA.

The ease of synthesis and stability of scrRNA, along with its potential for transient therapeutic delivery, might aid the development of improved strategies for gene editing, according to the authors.