Ordinarily, CRISPR-Cas9 gene editing systems cut all the way across double-stranded DNA. But these systems can be modified so that they inflict nothing more drastic than a nick. The advantage of a nick, as opposed to a double-strand break, is a certain degree of refinement, one that can lead to safer therapeutic applications. Consider the nick-making CRISPR-Cas9 systems known as base editors. In a recent study, a base editor was used to correct the genetic mutations that cause cystic fibrosis.
The new study, led by by Hans Clevers, PhD, group leader at the Hubrecht Institute for Developmental Biology and Stem Cell Research, and Jeffrey Beekman, PhD, professor, cellular disease models, UMC Utrecht, relied on an adenine base editor (ABE), which enables enzymatic conversion from A-T to G-C base pairs. The ABE enabled efficient repair of faulty genes in cystic fibrosis organoids, three-dimensional cultures of stem cells derived from cystic fibrosis patients.
Detailed results from the study appeared February 20 in Cell Stem Cell, in an article entitled, “CRISPR-Based Adenine Editors Correct Nonsense Mutations in a Cystic Fibrosis Organoid Biobank.” Because the ABE used in the study didn’t inflict any double-strand breaks on DNA, nothing invoked the usual mistake-prone DNA repair mechanisms. Also, there was no need to supply donor DNA.
The operation of the ABE was described by study co-author Maarten Geurts, PhD student, Hubrecht Institute as follows: “Instead of creating a cut and replacing the faulty DNA, the mutation is directly repaired on site, making this a more effective genome editing tool.” Besides introducing nicks where needed, the ABE avoided adding them where the weren’t needed. In fact, the study’s authors reported that the ABE caused no detectable off-target effects.
“Here, we describe a cystic fibrosis (CF) intestinal organoid biobank, representing 664 patients, of which ~20% can theoretically be repaired by ABE,” the article’s authors wrote. “We apply SpCas9-ABE (PAM recognition sequence: NGG) and xCas9-ABE (PAM recognition sequence: NGN) on four selected CF organoid samples. Genetic and functional repair was obtained in all four cases, while whole-genome sequencing (WGS) of corrected lines of two patients did not detect off-target mutations.”
The Hubrecht Organoid Technology Foundation and the UMC Utrecht have generated a biobank consisting of intestinal organoids, tiny versions of the gut, derived from the stem cells of cystic fibrosis patients. The biobank was set up together with many cystic fibrosis centers across Europe and the Dutch Cystic Fibrosis Foundation.
The organoids have been developed to serve as disease models and advance the development of new therapies. In the current study, organoids from the biobank were used to test whether the new base editing technique can be applied in human stem cells.
“Cystic fibrosis is caused by a mistake, a mutation, in the CFTR gene leading to malfunctioning of the gene,” noted Geurts. “As a consequence, the mucus in many organs, including the lungs, is less hydrated, resulting in mucus build-up and organ failure. With the new base editing technique, the mutation in the CFTR gene can be detected and repaired without creating further damage in the genome.”
Even though this research shows that this novel CRISPR tool is effective in the lab, this does not mean that patients can already benefit from it. “This research represents a big step toward genetic repair of diseases in patients,” said Eyleen de Poel, PhD student, UMC Utrecht and another study co-author. “However, a big question that remains is how to deliver the CRISPR enzyme to the appropriate organs in the patient.”
Until delivery difficulties are resolved, CRISPR tools may be of limited utility against diseases such as cystic fibrosis, which affects multiple organs. For the time being, then, CRISPR gene editing should focus on diseases that affect a single organ or tissue such as sickle-cell anemia. Indeed, it is with such diseases that CRISPR gene editing is beginning to show clinical potential.
The researchers responsible for the current study noted that further research is needed before CRISPR base editing can be used for clinical application. However, they added that their work is contributing to progress, and that optimism is justified: “These observations exemplify the value of large, patient-derived organoid biobanks representing hereditary disease and indicate that ABE may be safely applied in human cells.”