CRISPR technology could be classified as a form a gene therapy, but differs from classical forms of gene therapy discussed previously in this forum (Issue #49, April 2015). In classical forms of gene therapy a healthy form of a gene, oftentimes on a modified viral vector, is introduced into patient cells harboring mutant copies of the gene. This newly introduced gene can be inserted anywhere in the genome, thus both healthy and mutated genes typically persist in treated cells at distinct locations. In genome editing using CRISPR, the ultimate goal is to replace disease-causing mutations in a gene's native location within our chromosomes with healthy sequence without any other alterations to the genome. If this sounds a bit like science fiction or a someday hoped for future, let me simply say that the future may be closer than you think. A recent paper published in Human Gene Therapy describes the use of CRISPR technology to correct a disease causing mutation in skin cells derived from a patient with Fanconi anemia[2]. While these corrected cells are a proof of principle and have not been reintroduced back into the patient for use therapeutically, it would seem to be only a matter of time before similar experiments in more therapeutically relevant cells will extend this technology to the clinic.

  

Before discussing current limitations of CRISPR technology, let me first provide a brief overview of what is involved. The basic idea behind genome editing by CRISPR is relatively straightforward and involves introducing a double stranded break in DNA near a site to be edited and then using an exogenous fragment of DNA to repair the double-stranded break. Because the sequences of the exogenous DNA are used as a template to replace DNA that is found before, through and after the break, the original DNA in the cleaved strand is replaced by homologous sequences derived from the exogenously supplied DNA. If the original chromosomal DNA near the double-stranded cut harbors a disease causing mutation and the exogenous DNA used to repair the cut has a sequence found in healthy individuals, the net effect of the repair process is to replace the mutant DNA with healthy DNA. This form of DNA repair is referred to as homology-directed repair.

  

The key to CRISPR technology is therefore the ability to direct a double-stranded cut in DNA to a region of a chromosome near a disease-causing mutation. The enzyme that carries out the double-stranded cut is a nuclease referred to as Cas9. Cas9 forms a complex with a guide RNA that directs it to specific sequences in DNA. The guide RNA does this through its ability to form Watson/Crick base pairs with a target DNA.   Fortunately, the sequences of the guide RNA can be manipulated without dramatically affecting its ability to interact productively with Cas9, thereby allowing investigators to target virtually any DNA sequence for cleavage. Once Cas9, a synthetic guide RNA, and a exogenous DNA template are introduced into a cell, the homology-directed DNA repair machinery present in most cells uses the exogenous template to repair the guide RNA-directed double-stranded cut and when all is said and done, the mutated DNA is replaced by healthy DNA. This can all be done through the transient expression of CRISPR components in cells, such that once the genome has been edited all exogenous components used in editing are lost and the edited sequence is the only lasting change present in the modified cells. Optimal methods for delivering these various components to cells and biasing repair toward homology-directed repair as opposed to more error-prone mechanisms of DNA repair are currently being worked out to enhance the efficiency of genome editing.