Novel Gene-Editing Tool Specifically Modifies RNA

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. 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 (sgRNAs) 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.

Investigators at the Salk Institute for Biological Research (La Jolla, CA, USA) analyzed prokaryotic genome and metagenome sequences to identify uncharacterized families of RNA-guided, RNA-targeting CRISPR systems. Biochemical characterization and protein engineering of seven distinct orthologs generated a ribonuclease effector (CasRx) derived from the gut bacterium Ruminococcus flavefaciens XPD3002 with robust activity in human cells. CasRx-mediated knockdown exhibited high efficiency and specificity relative to RNA interference across diverse endogenous transcripts. In addition to high efficiency and specificity, CasRx was one of the most compact single-effector Cas enzymes and could be flexibly packaged into an adeno-associated virus for in in vivo delivery.

To demonstrate the potential clinical value of the CasRx system, the investigators used a viral vector to insert it into neuronal stem cells derived from a patient with the neurodegenerative disorder frontotemporal dementia (FTD). This disorder is characterized by an imbalance in the ratio of two forms of the tau protein. The inserted CasRx had been genetically engineered to target RNA sequences for the version of the tau protein that was in excess.

Results published in the March 15, 2018, online edition of the journal Cell revealed that CasRx was 80% effective in rebalancing the levels of tau protein to healthy levels.

"Bioengineers are like nature's detectives, searching for clues in patterns of DNA to help solve the mysteries of genetic diseases," said senior author Dr. Patrick Hsu, a Helmsley-Salk Fellow at the Salk Institute for Biological Research. "CRISPR has revolutionized genome engineering, and we wanted to expand the toolbox from DNA to RNA."

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Salk Institute for Biological Research