Special Focus on CRISPR: CRISPR on the moveSeptember 2017by Jeffrey Bouley  |  Email the authorEDIT CONNECTSHARING OPTIONS:

Special Focus: CRISPR Gene EditingCRISPR on the moveGene-editing technology continues to evolve In the relatively short time since gene editing involving clustered regularly interspaced short palindromic repeats (CRISPR) arrived on the life-sciences scene—most particularly in the handful of years since we got CRISPR/Cas9 and a much more simplified editing process—the technology has seen its ups and downs with regard to how safe, specific, efficient and reliable it is. But there is no doubt the technology continues to advance and almost certainly will hold a key position in the genomics arena for a long time to come. Germany’s Merck KGaA (not to be confused with U.S.-based Merck & Co.), for example, recently developed an alternative CRISPR genome editing method that it says makes CRISPR “more efficient, flexible and specific, giving researchers more experimental options and faster results that can accelerate drug development and access to new therapies.” Merck KGaA calls the new technique proxy-CRISPR and maintains that it provides access to previously unreachable areas of the genome. Most natural CRISPR systems, found in bacteria, cannot work in human cells without significant re-engineering, the company notes; however, proxy-CRISPR is said to provide a simpler and quicker method to increase their usability without the need to re-engineer native CRISPR proteins. “With more flexible and easy-to-use genome-editing technologies, there is greater potential in research, bioprocessing and novel treatment modalities,” said Udit Batra, a member of the company’s executive board and CEO of its Life Science unit. “As a leader in genome editing, Merck’s new technology is just one example of our commitment to solving challenges in the genome editing field, and we will continue to make CRISPR research a priority.” The company has filed several patent applications on the proxy-CRISPR technology, just one of several CRISPR patent application filings made by the company since 2012. Merck’s research on proxy-CRISPR, “Targeted Activation of Diverse CRISPR-Cas Systems for Mammalian Genome Editing via Proximal CRISPR Targeting,” was published in the April 7, 2017, edition of Nature Communications. The new technology is a follow-on to Merck’s existing CRISPR applications, and the company’s next suite of genome-editing tools for the research community—planned for launch later this year—is expected to include novel and modified versions of Cas and Cas-like proteins. More progress on the RNA front Researchers in the medical school at the University of California, San Diego (UC San Diego) in a 2016 study repurposed the CRISPR/Cas9 technique to track RNA in live cells in a method called RNA-targeting Cas9 (RCas9). In a new study, published Aug. 10 in Cell, the team took RCas9 a step further, using 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 amyotrophic lateral sclerosis and Huntington’s disease. “This is exciting because we’re not only targeting the root cause of diseases for which there are no current therapies to delay progression, but we’ve re-engineered the CRISPR/Cas9 system in a way that’s feasible to deliver it to specific tissues via a viral vector,” said senior author Dr. Gene Yeo, professor of cellular and molecular medicine at UC San Diego School of Medicine. 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. There is still a ways to go before RCas9 could be tested in patients, though, Yeo acknowledged. One bottleneck is efficient delivery of RCas9 to patient cells, as the non-infectious adeno-associated viruses that are commonly used in gene therapy are typically too small to hold Cas9 to target DNA. Yeo’s team made a smaller version of Cas9 by deleting regions of the protein that were necessary for DNA cleavage, but dispensable for binding RNA. “The main thing we don’t know yet is whether or not the viral vectors that deliver RCas9 to cells would illicit an immune response,” he said. “Before this could be tested in humans, we would need to test it in animal models, determine potential toxicities and evaluate long-term exposure.”