Synthetic RNA Improves the CRISPR/Cas 9 Genome Editing Tool

By replacing natural CRISPR guide RNA with a selectively synthesized version, molecular biologists have developed a modified gene editing technique that gives researchers more precise control in correcting and inactivating genes.

CRISPRs (clustered regularly interspaced short palindromic repeats) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to a bacterial virus or plasmid. CRISPRs are found in approximately 40% of sequenced bacteria genomes and 90% of sequenced archaea. CRISPRs are often associated with cas genes that code for proteins related to CRISPRs. The CRISPR/Cas complex comprises a prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. Since 2013, the CRISPR/Cas system has been used in research for gene editing (adding, disrupting, or changing the sequence of specific genes) and gene regulation. By delivering the Cas9 protein and appropriate guide RNAs into a cell, the organism's genome can be cut at any desired location. The conventional CRISPR-Cas9 system is composed of two parts: the Cas9 enzyme, which cleaves the DNA molecule and specific RNA guides (CRISPRs) that shepherd the Cas9 protein to the target gene on a DNA strand.

Investigators at the University of California, San Diego (USA) and Ionis (previously Isis) Pharmaceuticals (Carlsbad, CA, USA) have described an improvement to the classical CRISPR/Cas 9 gene editing technique. They developed drug that was in essence a chemically modified, 29-nucleotide synthetic CRISPR RNA (scrRNA). They showed that this molecule could functionally replace the natural CRISPR crRNA, producing enhanced cleavage activity at a target DNA site with apparently reduced off-target cleavage. Incorporation of rational chemical modifications known to protect against nuclease digestion and stabilize RNA–RNA interactions in crRNA yielded a scrRNA with enhanced activity compared with the unmodified crRNA and comparable gene disruption activity to the previously published single guide RNA.

“The RNA-based drugs we developed in this study provide many advantages over the current CRISPR/Cas9 system, such as increased editing efficiency and potential selectivity,” said senior author Dr. Don Cleveland, professor of cellular and molecular medicine at the University of California, San Diego. “In addition, they can be synthesized efficiently, on an industrial scale and in a commercially feasible manner today. These findings provide a platform for multiple therapeutic applications, especially for nervous system diseases, using successive application of designer CRISPR RNA drugs.”

Related Links:
University of California, San Diego
Ionis (previously Isis) Pharmaceuticals