A new genome editing technique developed in Drosophila allows for efficient somatic repair of both double-strand breaks (DSBs) and single-strand breaks (SSBs) using intact chromosome sequences. The authors refer to the process, which involves a nickase, as homologous chromosome-templated repair (HTR).
Repair of DSBs in somatic cells is usually accomplished by error-prone non-homologous end joining. Both the development timing (late versus early stages, respectively) and the development of undesired mutations are described.
The nickase-mediated HTR, according to the authors, is an efficient and unanticipated mechanism for allelic correction, with considerable potential applications in the field of gene editing.
In the article Cas9/Nickase-induced allelic conversion by homologous chromosome-templated repair in Drosophila somatic cells, this research is published in Science Advances.
Genetic disorders are prevalent in many instances because to distinct mutations in the two copies of genes. Therefore, a mutation on one chromosome will have a functional sequence on the other. CRISPR genetic editing tools were used to exploit this fact.
Annabel Guichard, PhD, a project scientist at the University of California at Santa Cruz, believes that the healthy version might be utilized by the cells' repair machinery to correct the defective mutation after cutting the mutant DNA. This is possible even more effectively through a simple harmless nick.
Researchers used eye color mutants to visualize homologous chromosome-templated repair, or HTR. These mutants initially had completely white eyes. However, when the same flies expressed a guide RNA plus Cas9, they showed large red patches across their eyes, indicating that the cells' DNA repair machinery had successfully reversed the mutation using the functional DNA from the other chromosome.
Nicks allowed for high-level red eye color almost on par with wild type flies. In contrast to SSBs induced by fully active Cas9 (2030%), they also found that the HTR-mediated allelic conversion at the white locus was more effective (4065%).
Restorative gene editing using sequences from the opposite chromosome: Cas9, a standard CRISPR enzyme, allows for repairs, but it also allows for unintended mutations at the targeted site and elsewhere in the genome (left) ; the nickase enzyme allows for more efficient gene correction and no mutagenic events (right) ; the researchers claim the new technique would serve as a model for repairing genetic mutations in mammals.
Guichard claims that this process will not be applicable to human cells nor if we may apply it to any gene. It may take a while to obtain an efficient HTR for disease-causing mutations carried by human chromosomes.
The new study extends the group's previous achievements in precision-editing with allelic-drives, which extend on the principles of gene-drives with a guide RNA that directs the CRISPR system to remove undesired variants of a gene and replace them with a preferred version of the gene.
A key feature of the teams' research is that their nickase-based system has significantly less on-and off-target mutations than traditional Cas9-based CRISPR edits. They also note that a slow, continuous delivery of nickase components over several days may be more beneficial than one-time deliveries.
Ethan Bier, PhD, professor in the department of cell and developmental biology at UCSD, cites this approach for its simplicity. Unlike Cas9, which produces full DNA breaks often accompanied by mutations, it only requires very few components and DNA nicks are soft.
According to Roy, such measures might be utilized to correct numerous dominant or trans-heterozygous disease-causing mutations.