Improving Gene Editing by Promoting Error-free Repair of CRISPR/Cas9-cut DNA

Cystic fibrosis (CF) is a chronic, progressive genetic disease affecting the lungs, pancreas and other organs. Although current treatment options manage the symptoms, reduce complications and improve quality of life, there is no cure and for most, the condition is eventually fatal. On average, individuals with CF have a lifespan of approximately 30 years.

However, innovative new gene editing technology brings hope for a cure. Since the publication of the seminal papers in 2012 and 2013 laying out the protocol for genome editing, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) has garnered much attention and generated a lot of excitement for its ability to make precise, permanent changes to DNA in animals and plants. CRISPR has the potential to provide novel therapies for patients suffering from severe diseases caused by a single gene mutation, including CF. It is also being investigated as a treatment for sickle cell anemia, Huntington’s disease, and as a way to improve immunotherapy for cancer.

With the first clinical trial of CRISPR gene editing in progress and the controversy of the first gene-edited babies continuing to spark discussion, it is more important than ever to better understand not only the benefits but also the risks of CRISPR-based therapies.

Keeping CRISPR on target

The CRISPR/Cas9 system is able to target and edit DNA by making precise cuts in double-stranded DNA. These cuts are then repaired by one of several possible mechanisms in the cell: the two main ones being non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ tends to be more prominent in cells, despite its tendency to introduce unintended insertions and deletions (indels) into the DNA it repairs. This is a particular concern if there are any off-target cleavage sites, which can lead to undesired indels and potential off-target effects (OTEs). These OTEs could impact the safety profile of a potential CRISPR therapy and so, determine whether that therapy is approved for clinical use. For many CRISPR experiments and therapies, HDR is preferred because it is less error prone than NHEJ and can mediate a specific mutation or insertion within the genome.

To improve the chance of successfully developing CRISPR-based therapies, companies like Integrated DNA Technologies (IDT) are constantly innovating to design and provide better tools for life science researchers working with CRISPR. With such tools, researchers led by Professor Bill Skarnes at the Jackson Laboratory for Genomic Medicine (JAX) in Farmington, Connecticut, USA have developed a CRISPR protocol for improving HDR efficiency in human stem cells.

There are three key improvements in the protocol that promote HDR over NHEJ. One key improvement involves the use of IDT’s Alt-R modified HDR Donor Oligos, which are single-stranded oligo deoxynucleotides (ssODNs) specially modified with end-blocks at 5’ and 3’ ends, which increases the efficiency of the HDR. Then, the addition of IDT’s Alt-R HDR Enhancer, a small-molecule compound that promotes HDR, to the cell culture media for one to two days after the introduction of the CRISPR/Cas9 complex, further enhances the HDR. The third key improvement is that of cold shocking the cells after the entry of the CRISPR/Cas9 complex into the cells. By implementing these three improvements into their CRISPR protocol, the team at JAX have been able to shift the ratio of HDR:NHEJ from 0.5 to 3.7. At a ratio of 0.5, NHEJ predominated and occurred in twice as many cells as HDR did. With the 7-fold improved ratio of 3.7, HDR was predominant and occurred in the majority, that is 70%, of unselected human cells.

This refined method is being applied in JAX to the development of cellular models of human disease, generated through the CRISPR gene editing of induced pluripotent stem cells. To faithfully recapitulate many disease variants, the gene editing must efficiently and precisely modify the DNA. These models inform ground-breaking research to elucidate the pathological mechanisms of many diseases and to test potential therapies. The publication of this dramatically improved CRISPR/Cas9 protocol will undoubtedly facilitate more efficient and precise CRISPR genome editing and thus, hopefully serve to help scientists accelerate the development of new therapies, including a cure for CF.

IDT recently announced the release of a free, web-based Alt-R HDR Design Tool that was published using extensive wet bench testing and customer validation. The tool allows users to design and order optimized HDR donor templates and associated Cas9 guide RNAs for multiple species.

Towards a cure for cystic fibrosis

As CF is one of the most common inherited disorders in the world and is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, several researchers are investigating the potential of gene therapies to correct these mutations. One such researcher is Professor Matthew Porteus, Professor of Pediatrics (Stem Cell Transplantation) at Stanford University, who is developing a CRISPR gene-edited, stem cell-based therapy for treating chronic sinusitis due to CF. The therapy will involve first extracting airway stem cells from individual patients with CF. The CF-causing mutation in these cells will be corrected using CRISPR gene editing. For this particular therapy, this will involve rectifying the most common CF mutation (CFTR –deltaF508) and/or replace the entire CFTR gene in the stem cells. The corrected cells will then be transplanted back into the nasal cavities of the individual patients, in what is termed autologous transplantation.

Other research studies are being conducted to develop gene-edited, stem cell-based therapies for treating CF. Some of these include the use of allogenic rather than autologous transplantation. Allogenic transplants are transplants from matched donors rather than from the patient him/herself. A Phase I clinical trial (CEASE-CF trial) is already underway, with another (HAPI trial) scheduled to start this May, to evaluate the safety and tolerability of allogenic stem cell infusions in adults with CF. These clinical trials and other critical research studies will pave the way for more effective treatments for individuals with CF; thus, improving their lives as well as that of their families, while simultaneously reducing the societal cost of this devastating disease. With the ongoing and rapid advances of technologies like CRISPR, it is more a question of "when", not "if", a treatment for CF will be realized.