The exciting discovery of an adaptive “immune system” of bacteria against infection by bacteriophages has captivated the imagination of scientists for the last few years. The harnessing of this system to introduce precise breaks in almost any desired sequence of DNA has potential applications in many fields of fundamental biology, but also agricultural research, disease modelling and medicine (Hsu et al. 2014).
Nevertheless, new upgrades and modifications to the system keep being reported, making this tool even more versatile, powerful and precise. The latest breakthrough was published in Cell, with Ranjan Batra and colleagues describing the use of a modified Cas9 capable of binding and cutting target microsatellite repeat expansion (MRE) RNAs (Batra et al. 2017). This strategy showed remarkable efficiency in ameliorating the molecular hallmarks in cellular models of MRE disorders, including Myotonic dystrophy type I and II, Huntington’s disease and C9ORF72-ALS/FTD.
MREs can be located in either exonic or intronic regions of protein-coding genes, originating toxic RNA species that can trigger the development of several neurological and neuromuscular diseases. Among these are Myotonic dystrophy type I, caused by expanded repetitions of the ‘CTG’ trinucleotide, Myotonic dystrophy type II, caused by ‘CTTG’ expansions, C9ORF72-ALS/FTD (‘GGGGCC’) and Huntington’s disease ‘CUG’ expansions.
Currently approved treatments, when available, are incapable of delaying the disease progression while conventional gene therapy strategies such as RNA-interference and antisense-oligonucleotides (ASOs) are generally inefficient in silencing repeat expanded RNAs. Considering this, the authors harnessed the potential of the CRISPR-Cas9 tool and repurposed it to target RNA molecules, instead of DNA, bypassing in this way the ethical and safety concerns related to genome editing.
Indeed, the authors fused a non-specific RNA endonuclease to a catalytically dead Cas9 (incapable of cutting DNA sequences), creating a tool that conserves the high specificity of Cas9 while eliminating the danger of off-target genome cutting. The authors then tested this technology in in vitro models of MRE diseases, including cells from patients carrying the causative mutations, and observed a reversal of disease-associated defects. Finally, the paper also reports a truncated version of this modified Cas9, which retains its RNA-cutting properties but is small enough to be packaged into an adeno-associated virus (AAV).
MRE disorders represent one of the most challenging topics in gene therapy due to the highly complex and unstable nature of nucleotide expansion regions, for which reason the implications of these findings are notorious. In addition to eliminating these expanded RNA species with high efficiency, the designed vector can be packaged in the most promising viral delivery system for the central nervous system: AAVs, with an impeccable safety profile, proven by dozens of clinical trials around the world (Murlidharan et al. 2014). We look forward to seeing this tool tested in vivo, from which eventual positive results would bring a new hope for the treatment of these devastating disorders.
This BSGCT Blog! article was co-authored by Professor Mimoun Azzouz and João Miguel da Conceição Aves-Cruzeiro.
Batra, R., et al. (2017). “Elimination of Toxic Microsatellite Repeat Expansion RNA by RNA-Targeting Cas9.” Cell 170(5): 899-912 e810.
Hsu, P. D., et al. (2014). “Development and applications of CRISPR-Cas9 for genome engineering.” Cell 157(6): 1262-1278.
Murlidharan, G., et al. (2014). “Biology of adeno-associated viral vectors in the central nervous system.” Front Mol Neurosci 7: 76.