Progress Reported in Development of Gene Editing Tool

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.

Since 2013, the CRISPR/Cas9 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 enzyme 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 that shepherd the Cas9 protein to the target gene on a DNA strand. In 2016, investigators at the University of California, San Diego (USA) reported the design of an RNA-targeting Cas9 (RCas9).

Microsatellite repeat expansions in DNA produce pathogenic RNA species that cause dominantly inherited diseases such as myotonic dystrophy type 1 and 2 (DM1/2), Huntington’s disease, and C9orf72-linked amyotrophic lateral sclerosis (C9-ALS). Means to target these repetitive RNAs are required for diagnostic and therapeutic purposes.

In a study published in the August 10, 2017, online edition of the journal Cell, the University of California, San Diego investigators described the development of a programmable CRISPR system capable of specifically visualizing and eliminating these toxic RNAs. RCas9 worked similarly to the regular CRISP/Cas9 editing tool, but the guide RNA directed the Cas9 enzyme to an RNA molecule instead of DNA.

The investigators reported that RCas9 eliminated 95% or more of the RNA foci linked to myotonic dystrophy type 1 and type 2, C9-ALS, and Huntington's disease. The approach also eliminated 95% of the aberrant repeat RNAs in myotonic dystrophy patient cells cultures.

In another step forward, the investigators produced a truncated version of Cas9 that could be packaged into an adeno-associated virus transport vehicle. This smaller version of Cas9 was created by deleting regions of the protein that were necessary for DNA cleavage, but dispensable for binding RNA.

"This is exciting because we are not only targeting the root cause of diseases for which there are no current therapies to delay progression, but we have re-engineered the CRISPR/Cas9 system in a way that is feasible to deliver it to specific tissues via a viral vector," said senior author Dr. Gene Yeo, professor of cellular and molecular medicine at the University of California, San Diego. "The main thing we do not know yet is whether or not the viral vectors that deliver RCas9 to cells would elicit an immune response. Before this could be tested in humans, we would need to test it in animal models, determine potential toxicities and evaluate long-term exposure."

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University of California, San Diego