RNA targeting of Cas9
Technology of using CRISPR/Cas9 for the treatment of RNA-associated diseases
Anna Kerman, XX2 century, based on materials Phys.org: New version of DNA editing system corrects underlying defects in RNA-based diseases
Until recently, the CRISPR/Cas9 gene editing technique could only be used for manipulating DNA. In a study conducted in 2016, scientists from the School of Medicine at the University of California San Diego repurposed the method of tracking RNA in living cells in a method called RNA targeting Cas9 (RCas9).
In a new study published in the journal Cell, the team takes RCas9 one step further. Now scientists have used this method to correct molecular errors that lead to diseases with repeated expansion of the microsatellite, for example, myotonic dystrophy of the first and second types and Huntington's disease.
"This is interesting because we are not only targeting the root cause of diseases for which there are currently no treatments to slow the progression. We also redesigned the CRISPR/Cas9 system in such a way that it was possible to deliver it to certain tissues using a viral vector," said senior author Gene Ye, PhD, professor of cellular and molecular medicine (Gene Yeo, PhD, professor of cellular and molecular medicine) at the California School of Medicine San Diego.
If DNA is like an architect's design for a cell, RNA is an engineer's interpretation of the design. Life on Earth is based on the following mechanism: genes encoded in DNA in the nucleus are transcribed into RNA, and RNA carries the message to the cytoplasm, where they are translated to create proteins.
Diseases of expansion of non-coding (microsatellite) repeats occur due to erroneous repeats in RNA sequences. They are toxic to the cell, partly because they interfere with the formation of essential proteins. These repetitive RNAs accumulate in the nucleus or cytoplasm of cells, forming dense nodes called foci.
In this study, which proved the concept, Ye's team used RCas9 to eliminate problematic RNAs associated with disease expansion of non-coding repeats in patients' cells in cellular disease models in the laboratory.
Typically, CRISPR/Cas9 works as follows: researchers develop a "guide" RNA to match the sequence of a specific target gene. The RNA then directs the Cas9 enzyme to the right place in the genome, where it "cuts" DNA. The cell inaccurately repairs the DNA break, thus deactivating the gene. Otherwise, the researchers replace the area adjacent to the incision with an adjusted version of the gene. RCas9 works similarly, but as part of this technology, RNA directs Cas9 to the RNA molecule instead of DNA.
The researchers tested the new RCas9 system in the laboratory. RCas9 eliminated 95% or more of the RNA foci associated with type 1 and type 2 myotonic dystrophy, one type of amyotrophic lateral sclerosis and Huntington's disease. This approach also eliminated 95% of aberrant repetitive RNAs in the cells of a patient with myotonic dystrophy cultured in the laboratory.
Muscle cells of a patient with type 1 myodystrophy before (left) and after (right) treatment. The repeating RNA is highlighted in red, the cell nucleus is blue.
Another discovery made during the study was related to MBNL1. It is a protein that normally binds RNA, but is separated from its RNA targets by RNA foci in type 1 myotonic dystrophy. When the researchers applied RCas9, they reversed 93% of these dysfunctional RNA targets in the patient's muscle cells, and the cells eventually began to resemble healthy control cells.
"Although this study provides the first evidence that the approach works in the laboratory, there is a long way to go before RCas9 can be tested in patients," Ye explained.
"The main thing we don't know yet is whether the viral vectors that deliver RCas9 to cells will be deprived of an immune response," he said. "Before it can be tested in humans, we need to test it in animal models, determine potential toxicity and assess long–term effects."
To do this, Ye and his colleagues launched a specialized company called Locana. It is designed to handle the preclinical steps required to move RCas9 from the laboratory to the clinic for the treatment of diseases associated with RNA disorders.
"We are very excited about this work because we have not only identified a new potential therapeutic mechanism for CRISPR-Cas9, but also demonstrated how it can be used to treat a whole class of diseases for which there are no successful treatment options," said David Nelles, co–author of the first study of the study with Ranjan Batra, another researcher from Ye's lab.
"There are more than 20 genetic diseases caused by microsatellite expansions in different places of the genome,– Batra said. –The ability to program the RCas9 system to target different repeats combined with a low risk of side effects is the main strength [of our development]."
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04.09.2017