Scientist Uses New Version of DNA Editing System to Correct Molecular Mistakes

Muscle cells from a patient with myotonic dystrophy type I, untreated (left) and treated with the RNA-targeting Cas9 system (right). The MBNL1 protein is in green, repetitive RNA in red and the cell's nucleus in blue. MBNL1 is an important RNA-binding protein and its normal function is disrupted when it binds repetitive RNA. In the treated cells on the right, MBNL1 is released from the repetitive RNA. Credit: UC San Diego Health

Until recently, the CRISPR-Cas9 gene editing technique could only be used to manipulate DNA. In a 2016 study,[..] researchers repurposed the technique to track RNA in live cells in a method called RNA-targeting Cas9 (RCas9).[..] they use the technique to correct molecular mistakes that lead to microsatellite repeat expansion diseases, which include myotonic dystrophy types 1 and 2, the most common form of hereditary ALS, and Huntington's disease.

Microsatellite repeat expansion diseases arise because there are errant repeats in RNA sequences that are toxic to the cell, in part because they prevent production of crucial proteins. These repetitive RNAs accumulate in the nucleus or cytoplasm of cells, forming dense knots, called foci.

In this proof-of-concept study, Yeo's team used RCas9 to eliminate the problem-causing RNAs associated with microsatellite repeat expansion diseases in patient-derived cells and cellular models of the diseases in the laboratory.

Normally, CRISPR-Cas9 works like this: researchers design a "guide" RNA to match the sequence of a specific target gene. The RNA directs the Cas9 enzyme to the desired spot in the genome, where it cuts DNA. The cell repairs the DNA break imprecisely, thus inactivating the gene, or researchers replace the section adjacent to the cut with a corrected version of the gene. RCas9 works similarly but the guide RNA directs Cas9 to an RNA molecule instead of DNA.

The researchers tested the new RCas9 system on microsatellite repeat expansion disease RNAs in the laboratory. RCas9 eliminated 95 percent or more of the RNA foci linked to myotonic dystrophy type 1 and type 2, one type of ALS and Huntington's disease. The approach also eliminated 95 percent of the aberrant repeat RNAs in myotonic dystrophy patient cells cultured in the laboratory.

Another measure of success centered on MBNL1, a protein that normally binds RNA, but is sequestered away from hundreds of its natural RNA targets by the RNA foci in myotonic dystrophy type 1. When the researchers applied RCas9, they reversed 93 percent of these dysfunctional RNA targets in patient muscle cells, and the cells ultimately resembled healthy control cells.

News Reference New version of DNA editing system corrects underlying defects in RNA-based diseases

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