A new genome editing system, developed in Drosophila, performs efficient somatic repair of both double strand breaks (DSBs) and single-strand breaks (SSBs) using intact sequences from the homologous chromosome. The authors refer to the process, which uses a nickase, as homologous chromosome-templated repair (HTR).

Repair of DSBs in somatic cells, which is generally accomplished by error-prone non homologous end joining. The repair phenotypes elicited by the nickase (versus Cas9) are described to be differentiated by both developmental timing (late versus early stages, respectively) and the production of undesired mutagenic events (rare versus frequent).

The nickase-mediated HTR, the authors note, represents an “efficient and unanticipated mechanism for allelic correction, with far-reaching potential applications in the field of gene editing.”

This work is published in Science Advances in the article “Cas9/Nickase-induced allelic conversion by homologous chromosome-templated repair in Drosophila somatic cells.

In many cases, those suffering from genetic disorders carry distinct mutations in the two copies of genes. Therefore, a mutation on one chromosome will have a functional sequence on the other chromosome. The researchers employed CRISPR genetic editing tools to exploit this fact.

“The healthy variant can be used by the cell’s repair machinery to correct the defective mutation after cutting the mutant DNA,” said Annabel Guichard, PhD, a project scientist in the lab of Ethan Bier at the University of California at Santa Cruz.  “Remarkably, this can be achieved even more efficiently by a simple harmless nick.”

The researchers used eye color mutants to visualize homologous chromosome-templated repair, or HTR. Such mutants initially featured entirely white eyes. But when the same flies expressed a guide RNA plus Cas9, they displayed large red patches across their eyes, a sign that the cell’s DNA repair machinery had succeeded in reversing the mutation using the functional DNA from the other chromosome.

They then tested their new system with nickases that nick one strand of DNA instead of both. Nicks also gave rise to high-level restoration of red eye color nearly on par with wild type flies. They found that the HTR-mediated allelic conversion at the white locus was more efficient (40–65%) in response to SSBs induced by Cas9-derived nickases D10A or H840A than to DSBs induced by fully active Cas9 (20–30%).

graphic abstract
Restorative gene editing using sequences from the counterpart chromosome: The standard CRISPR enzyme Cas9 offers the ability to make repairs but also potentially results in unintended mutations at the targeted site and possibly elsewhere in the genome (left). In contrast, the nickase enzyme results in more efficient gene correction and no mutagenic events (right). [Guichard/Bier]
“I could not believe how well the nickase worked—it was completely unanticipated,” said Sitara Roy, PhD, a post-doc in the Bier lab at UCSD. The versatility of the new system could serve as a model for fixing genetic mutations in mammals, the researchers noted.

“We don’t know yet how this process will translate to human cells and if we can apply it to any gene,” said Guichard. “Some adjustment may be needed to obtain efficient HTR for disease-causing mutations carried by human chromosomes.”

The new research extends the group’s previous achievements in precision-editing with “allelic-drives,” which expand on principles of gene-drives with a guide RNA that directs the CRISPR system to cut undesired variants of a gene and replace them with a preferred version of the gene.

A key feature of the team’s research is that their nickase-based system causes far fewer on- and off-target mutations than traditional Cas9-based CRISPR edits. They also note that a slow, continuous delivery of nickase components across several days may prove more beneficial than one-time deliveries.

“Another notable advantage of this approach is its simplicity,” said Ethan Bier, PhD, professor in the section of cell and developmental biology at UCSD. “It relies on very few components and DNA nicks are ‘soft,’ unlike Cas9, which produces full DNA breaks often accompanied by mutations.”

“If the frequency of such events could be increased either by promoting interhomolog pairing or by optimizing nick-specific repair processes,” notes Roy, “such strategies could be harnessed to correct numerous dominant or trans-heterozygous disease-causing mutations.”