Skip to main content
Premium Trial:

Request an Annual Quote

New CRISPR/Cas9-based Tech Edits Single Nucleotides Without Breaking DNA


NEW YORK (GenomeWeb) – By fusing a nucleotide-flipping enzyme to Cas9, scientists from Harvard University have developed a new way to edit point mutations without making a double-stranded break. Though presently limited in which base conversions it can make, the technique could reduce the effect of unwanted indels in gene editing and could be applied to correct mutations associated with genetic diseases.

Led by first author Alexis Komor and senior author David Liu, the researchers published their results today in Nature.

"Most known disease-associated human genetic variations are point mutations," Liu told GenomeWeb. "But current genome-editing mutations are better at introducing indels and disrupting genomes and are not particularly good at point mutations."

"The efficiency and cleanliness of correcting point mutations under conditions that are therapeutically relevant is modest," he said.

The new study shows the potential of making more precise single-nucleotide edits in cells. Fusing a cytidine deaminase to a nuclease-null Cas9 (dCas9), the researchers were able to change cystosine to uracil, essentially effecting a "C" to "T" edit. Then, by taking advantage of the cell's DNA repair response, they were able to also change guanine to adenine.

The technology is far from advanced. While it limits indels and favors point mutations, its efficiency at correcting actual mutations stood in the single digits, percentage-wise, and the scientists were only able to demonstrate two of the 12 possible base substitutions. Still, Liu said it provides an opening for research on human diseases.

"Even if base editors only can do one sixth of the possible changes, there are still many disease-associated mutations that fit that scope," he said.

The protein fusion is the latest attempt to wrest more control over the gene editing process from the whims of the cell. While major advances have been made in reducing off-target effects of the Cas9 enzyme, gene editing has almost entirely relied on cutting the DNA open.

Often, the cell's primary response is the quick and dirty non-homologous end joining repair, which incorporates random indels to create knockouts. There's another repair pathway that can incorporate a genomic template of one's choosing, homology directed repair (HDR), "but only rarely will the donor template be incorporated," Liu said.

Scientists are finding ways to raise the efficiency of HDR, but it's far from a guarantee that the insert one wants to see will be incorporated where it's supposed to go. "This situation is what leads my colleague [Harvard Medical School professor] George Church to note 'much of what passes for genome editing should really be called genome vandalism,'" Liu said.

To gain more precision in editing, Liu's lab looked for a way to avoid a double-stranded break and directly catalyze the conversion of one base to another.

Early on, the scientists hit upon the idea of tethering a cytidine deaminase to a dCas9 to catalyze the conversion of cytosine to uracil, but they also predicted two complications.

The first was that it would lose the specificity that makes CRISPR/Cas9 so useful. "If you simply tether the enzymes, that fusion can operate on dozens of bases of DNA near the Cas9 binding site," Liu said. He pointed to a recently published paper that illustrated the problem, where DNA methyltransferases tethered to dCas9 would methylate bases in a 35 base-pair window.

To overcome this, Liu's lab looked at cytidine deaminases that would only work on single-stranded DNA. When the Cas9-guide RNA complex binds to its genomic target, it unzips a portion of the DNA. While much of the non-target strand gets fixed to the Cas9 enzyme, there's a small section on the non-target strand near the PAM recognition site that doesn't, which is approximately 11 base pairs long.

Because it's the only single-stranded section of DNA available to the protein fusion, the enzyme's effects are limited to that portion. "Our idea was to perform direct chemical surgery within that bubble of single-stranded DNA," Liu said. "That way it doesn't just edit dozens of DNA bases, which would make for a not-so-useful editor."

Liu's team hypothesized that the cytidine deaminase could work in that window. By tinkering with the length of the enzyme linker, they were able to restrict the window of the cytosine deaminase to about three to six bases from the Cas9 binding site.

The other concern about the approach was that it would be inefficient. Moving the dCas9-cytidine deaminase fusion into cells resulted in a tenfold decrease in efficiency, Liu said. "The DNA repair response will fight the change you just made," specifically a uracil-guanine mismatch.

Liu's lab also used Cas9 fusions to manipulate the cell's base excision repair and mismatch repair pathways, turning that into a bonus. For this, they fused a uracil glycosylase inhibitor to a Cas9 nickase targeting the non-edited strand, which helped the repair mechanism favor excising the guanine instead and replace it with an adenine.

C to T and G to A are only two of the possible changes, but they open a world of possibility for people interested in studying the diseases. "With just those two [base changes], we can reverse disease-associated mutations in cells and study them, helping to pave the way to potential future human therapeutics," Liu said.

In the paper the researchers reversed a mutation in APOE4 that is associated with Alzheimer's disease and cardiovascular disease, as well as a mutation in p53, which is strongly associated with several cancers.

"There are hundreds to thousands, depending on the criteria of the genetic disease, for which C to T or G to A can undo the mutation," Liu said.

The next step for Liu is to find ways to induce the other base substitutions. His lab is exploring both enzymatic and chemical catalysts. So far, Liu's been pleased with his progress. 

"We are working on the development of other base editors, but it's too early to describe them at this point," he said.