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Crispr Techniques Devised for Editing RNA

Salk Institute team

Salk Institute team, from left: Peter Lotfy, Silvana Konermann, Patrick Hsu, Jennifer Oki, and Nicholas Brideau (Salk Institute for Biological Studies)

16 March 2018. Geneticists and bioengineers developed a technology for applying genomic editing techniques known as Crispr to ribonucleic acid, or RNA, transcribed from the genetic code in a person’s DNA. A team from the Salk Institute in La Jolla, California describe the techniques in yesterday’s issue of the journal Cell (paid subscription required), where they applied the technology to correcting damaging proteins in nerve cells from a person with dementia.

Researchers in the Salk Institute lab led by Patrick Hsu are seeking to expand the use of Crispr to disorders that result from disrupted or erroneous RNA transcriptions. Crispr — short for clustered regularly interspaced short palindromic repeats — is a process based on bacterial defense mechanisms that use RNA to identify and monitor precise locations in DNA. The actual editing of genomes with Crispr employs enzymes that cleave DNA strands at the desired points, with Crispr-associated protein 9, or Cas9, being the enzyme used most often.

“Crispr has revolutionized genome engineering,” says Hsu in a Salk Institute statement, “and we wanted to expand the toolbox from DNA to RNA.” The Salk team explored splicing properties of RNA that enable production of some proteins, but can also have toxic effects if splicing goes wrong. Among the disorders that can result from mis-spliced RNA are spinal muscular atrophy and frontotemporal dementia. The researchers examined if other bacterial defenses against viruses could be exploited with Crispr to fix these damaging RNA errors.

A search of bacterial DNA databases revealed genomic patterns suggesting Crispr-type effects, and in the process uncovered a set of enzymes with properties indicating an ability to edit RNA. The researchers then needed to find a version of these enzymes in humans, and discovered the enzymes in a gut microbe known as Ruminococcus flavefaciens XPD3002. The team calls this human-friendly type of enzyme CasRx.

Hsu and colleagues tested the techniques with RNA splicing errors that generate accumulations of a protein called tau associated with frontotemporal dementia, as well as Alzheimer’s disease. The team engineered CasRx to target and reverse protein instructions that result in producing an overabundance of tau. And the researchers discovered that its small molecular size made it possible to efficiently deliver the enzyme with adeno-associated viruses, benign, naturally occurring microbes that can infect cells, but do not integrate with the cell’s genome or cause disease, and generate a mild immune response.

Tests were conducted on nerve cells from an individual with frontotemporal dementia, grown from the person’s stem cells. The researchers say the Crispr-edited RNA succeeded in rebalancing tau proteins in the cells to healthy levels, about 80 percent of the time. The team believes the findings show their techniques are feasible and can be programmed to treat a range of disorders that result from disrupted or erroneous RNA.

Hsu and first author Silvana Konermann tell more about their techniques in the following video.

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