A race to bring CRISPR to the clinic

It has nowbeen two years sincewe first wrote about the therapeutic potential of CRISPR-Cas9 in ourMay 2015 Editorial “CRISPR–Cas9 Based Therapeutics: Not So Fast”. At the time, we shared serious concerns resonating within the community that the potential for undesirable off-target mutations was significant, and that the technology was not ready for introduction into humans as a gene modification strategy. Since then, the field has moved at lightning speed. Researchers have made great strides toward eliminating off-target effects and improving specificity. This has been achieved in part by modifying the Cas9 enzyme, using shorter guide RNAs, and by reducing the amount of Cas9 expression within the cell, for example. Furthermore, the ability to detect and monitor any potential off-target mutations has been greatly improved, with extremely sensitive detection methods such as CIRCLE-seq. This technique combines PCR amplification of regions containing Cas9 cleavage sites with next-generation sequencing to provide highly accurate detection of CRISPR-Cas9 mutation sites. Along with a substantial increase in targeting specificity, the repertoire of practical applications for CRISPR-Cas systems has also expanded—within both the basic and clinical realms. For example, in the April 28 2017 issue of Science, researchers report the development of SHERLOCK—a nucleic acid detection method combining the specificity offered by CRISPR-Cas (in this case Cas13a) with isothermal amplification to detect attomolar levels of target RNA orDNA. The authorswere able to use SHERLOCK to detect very low levels of Zika virus (ZIKV) and to distinguish ZIKV from dengue virus, which can be a challenge with current diagnostic tools, given the genetic relatedness of these two flaviviruses. To further demonstrate utility, they also applied the technique to detect antibiotic resistance genes within pathogenic bacteria, and to identify mutations in cell-free tumor DNA. One can imagine a range of potential diagnostic and research applications for this technology in the future. In addition to this exciting new tool, the CRISPR-Cas systemhas been used in countless recent basic research applications including screens to identify genes essential for tumorigenesis, identification of host factors relevant for pathogen survival, and for the creation of disease-relevant preclinical models. Cellular, organoid, and animal models have recently been created using CRISPR for awide range of diseases including cancer, atherosclerosis, and neurological diseases. CRISPR has also been used to treat disease inmammals, within amousemodel of Duchennemuscular dystrophy (DMD) where the mutated dystrophin exon was removed. Researchers delivered the Cas9 gene modifying system using an AAV9 vector—systemically in neonatal mice, and both systemically and locally in adult mice. Dystrophin expressionwas induced in differentiated muscle cells and cardiomyocytes as well as muscle cell precursors.