Duke University researcher Charles Gersbach and colleagues point to the promise of gene editing to treat people with neuromuscular conditions such as Duchenne muscular dystrophy3. An edit correcting a mutation in the gene encoding dystrophin leads to expression of the protein, whereas with the mutation it’s prematurely terminated and thus missing in the patients’ muscle cells. An important methods challenge is how to best deliver gene-editing constructs. Viral vectors such as adeno-associated viruses are most often used and have been introduced into mouse embryos in utero to edit mutations that lead to congenital disorders4. Concerns include these viruses’ relatively low load-carrying capacity and immunogenicity risks. Labs thus explore alternatives such as non-viral direct injection of plasmid DNA or oligonucleotides. Harvard Medical School researcher George Church and colleagues note that many variables shape successful gene silencing in somatic cells: guide RNAs might not perform as well as assumed; local chromatin effects can decrease edit efficiency; Cas9 and endogenous transcriptional regulators compete for binding; epigenetic marks can interfere with gene editing5. In the work from the Mitalipov lab, the two alleles differed by four base pairs. Many genetic conditions involve only single-nucleotide variants (SNVs). Another issue is that after a spot in the genome is cut and repaired, CRISPR does not immediately cease cutting the genome. That can mean, for example, that labs might need to introduce adjacent silent mutations in the donor DNA to avoid re-cleavage by Cas96.

In gene-editing experiments, the founder’s cells can have differing genotypes. This animal is mosaic. Credit: M. Wiles/Genetic Engineering Technology, JAX

Wiles sees the promise gene editing holds for somatic cell-based therapies, and keeps them in mind as he develops techniques and validates new methods at Jackson Lab. As experimental models, mice are a “reasonable surrogate” for people and help labs verify science, he says. If a gene-editing technique generates a mistake, experiments in mice reveal the challenges for researchers to address before considering applications in people. On a daily basis, Wiles and his team inject CRISPR reagents into mouse zygotes. “Each CRISPR reagent, each guide, behaves very slightly differently,” says Wiles. A designed guide RNA that seemed perfect can end up cutting poorly, yet a guide designed to cut just ten bases adjacent to that first location might do a better job. Micro-injecting embryos takes great skill, and even with people who do this daily, there is some variability, he says. “The micro-injectors are not robots, they are people,” he says. Experiments targeting a similar genomic region with the same guide RNAs and same genetic background can deliver different knockout efficiencies at a targeted site. One experiment might lead to 80% gene-editing efficiency, the next can drop to 20%, “and we don’t quite know why one of them changed,” he says.

Wiles began doing gene-editing experiments with zinc-finger nucleases and then transcription activator-like effector nucleases (TALENs), which he and colleagues applied to a retinal gene mutation that causes developmental blindness in mice7. “We were very happy, because we repaired the gene,” he says. He switched to CRISPR, and when he generated the first gene-edited mouse with the desired genomic deletion at a pre-designated location, the Jackson Lab colleagues he did this for were dumbfounded, he says. Gene editing is now commonplace at The Jackson Laboratory. The majority of Wiles’s work still involves using “fairly crude Cas9s,” he says. He reminds himself that CRISPR was not born a lab tool but rather is a bacterial defense system for combating phage infection. When it fails, bacteria can die. “The idea that it would have 100% specificity may not be the best idea evolutionarily,” he says. That is why optimization is an integrated part of work with CRISPR. Labs are engineering what they need, such as molecular scissors with a 100% requirement for a specific genomic 20-base target. Massive “intellectual horsepower” is being brought to bear to make CRISPR–Cas a versatile, precise and efficient tool, he says. Wiles is testing some of the many engineered Cas9s such as eSpCas9, HypaCas9 and Cas-9-HF-1, which appear to have the same efficiency as Cas9 but greater specificity. “We’re still using spCas9 for most of our work here,” he says, “although we are switching over to base editors for some things.” Around half of human genetic variants associated with disease are single-base changes, which may be more amenable to base editing, he says.