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Christiana Care Scientists Adapt Single-Nucleotide Editing Strategy for Use With CRISPR/Cas9


NEW YORK (GenomeWeb) – CRISPR/Cas9 can help improve the efficiency of a method of single-nucleotide editing directed by single-stranded DNA oligos (ssODNs), according to studies published by scientists at Christiana Care Health System's Gene Editing Institute. While the method could help edit point mutations that cause human diseases, it is prone to leaving behind indels at the Cas9 cut site.

If it sounds like gene knock-in using the homology-directed repair pathway, think again, Eric Kmiec of the Gene Editing Institute told GenomeWeb. "Oligos can fix point mutations without having to be totally inserted into the DNA," he said. "Our method is directed by homology, but calling it homology-directed repair is misleading because it implies that the entire oligo gets inserted."

Instead, the method known as targeted gene alteration leverages the cell's mismatch repair mechanisms to change a single base. By inserting an ssODN that hybridizes perfectly to the target sequence at all but one nucleotide, a mismatch is created, which should be repaired by the cell.

"We're interested in point mutation repair," Kmiec said, "not editing large segments of the genome. We think this is a slightly different pathway. It's more of a surgical approach to fixing a point mutation."

In that sense, it's a bit like nuclease-null Cas9 (dCas9) fused to a cytidine deaminase, as described by Harvard University's David Liu. But unlike that strategy, where there is no cutting activity, Kmiec's method works best when a double-strand break is placed close to the DNA target, within 35 to 50 bases.

That's one of the findings from studies led by Kmiec in the past several years. While Kmiec and colleagues have showed Cas9 is a good choice for making that DSB, it's not the only way to do so. They've also used transcription activator-like effector nucleases (TALENs), and the idea of adding a DSB to aid ssODN-directed editing goes back to at least 2006, according to a study led by University Hospital Ulm scientist Klaus Schwarz and published that year in Molecular Therapy.

And the history of targeted gene alteration goes back even further than that, making it an attractive option for editing point mutations in gene therapy. "This stuff has been known for 20 years," Kmiec said. The fact that his method shares many similarities with antisense ssODN therapies is also promising, since the US Food and Drug Administration has already approved them for use in humans.

In a study published in PLoS One in June 2015, a team led by Kmiec found that Cas9 cleavage could elevate rates of ssODN-directed repair, even when compared to a similar TALEN-aided strategy. But that's not all they found. The flexibility and low cost of Cas9 helped them extract lots of information about the repair mechanism: ssODNs hybridizing to the non-transcribed strand led to more mutation repair than those targeting the transcribed strand; cleavage must be close — within 50 bases — to the gene target; and a DSB is better than a single-stranded nick.


Not only has Kmiec been looking at the point correction, he's also been looking at the Cas9 cleavage site. In a study published earlier this month in Scientific Reports, Kmiec and his colleagues described further analysis of ssODN-directed point mutation repair in a human gene, hemoglobin beta (HBB).

"CRISPR/Cas9 has an interesting ability to do things [at the cut site]," causing a variety of mutations, he said, but the addition of the ssODNs makes CRISPR/Cas9 less mutagenic at its target site.

This has two implications. First, Cas9 could introduce its own mutations at its cut site, which will shock nobody in the field. In addition, and somewhat more surprising, oligos change the dynamics of DNA repair at the Cas9 cut site.

The oligo helped make a cleaner cut, "like using a Band-Aid to hold the ends together," and preventing NHEJ from inserting or deleting nucleotides, Kmeic said. "This was a side-bar observation. But we followed up on it, and found that the combination of the CRISPR and the oligonucleotide was doing something that is much more widely applicable for anyone doing this kind of work."

If it holds up, it would be just one of several interesting DNA repair-related findings to pop out of CRISPR/Cas9 experiments. A report from University of California, Berkeley scientist Jacob Corn suggests something akin to an opposite-side-of-the-coin finding, that the presence of short, synthetic oligos with no homology actually helps mutagenesis, leading to more efficient knock-outs when using Cas9.

Kmiec's team made the oligo-Cas9 cut site connection while investigating edits to HBB, a gene of interest for many in the gene editing field. While they were in several instances able to make a point edit without a mutation at the Cas9 cut site, he found "a whole population of indels," in other cases. It's an important reminder that those looking to use CRISPR/Cas9 in clinical editing should be careful.

"CRISPR/Cas9 is fast and vicious," he said. "You better look at what the collateral damage is at the cut site."

Nevertheless, Kmiec said he was "extremely cautiously optimistic" that he could continue pushing the oligo-Cas9 combination towards clinical use.

"[Cas9-induced mutations are] not a negative to the technology, it just has to be discussed," he said. Especially compared to the early, dark days of gene therapy, there's enough good news coming from his lab and others that he feels he's not throwing a wet blanket on his method of gene editing. "Smart people will be able to find their ways around it," he said.

Already he's working on follow-up studies and is seeing a healthy number of perfectly corrected point mutations, with no collateral damage.