Advances in DNA Editing Are Changing the Field
Dr. David Liu had to think outside the box. Facing limitations from traditional genome- editing techniques, he pursued a multidisciplinary approach that’s revolutionizing the field.
Fusing biology, chemistry and technology, Liu created a more efficient, more precise method called base editing, which has the potential to target and treat genetic diseases.
“He’s doing organic chemistry inside the cell,” said NIH director Dr. Francis Collins, introducing Liu’s recent Dr. Marshall Nirenberg Lecture. “Liu’s research has taken us to the next level in gene editing, not where you cut the DNA and hope something good happens after that, but where you very precisely go in and do an edit of one base to the one you wanted it to be.”
There are more than 60,000 genetic variants associated with disease, most of which are point mutations that could be repaired by a single base change. Base editors can directly, permanently convert one base pair to another without the double-stranded DNA breaks, deletions and rearrangements that often occur with standard genome-editing methods.
“We imagined: what if you could do chemistry directly on a target nucleotide within the genome of a living cell and directly catalyze the conversion of [the bases]?” said Liu, Richard Merkin professor and vice-chair of the faculty at the Broad Institute of Harvard and MIT.
Using protein engineering, Liu and his lab have developed and continue to optimize two classes of base editors that can efficiently make four types of base-to-base changes (C to T, T to C, A to G and G to A) in DNA in many cell types and organisms. “Collectively, these four transitions account for about 62 percent of pathogenic [variants],” said Liu.
One of the early challenges was figuring out how to correct a point mutation with precision, since most catalysts would operate on many DNA bases near the target site. The solution involved a bit of insight and chemical surgery. The Cas-9 gene editor unwinds the DNA’s double helix, so Liu began testing various enzymes that only accepted single-stranded DNA.
“Since the vast majority of your genome is double stranded, we wouldn’t expect that activity would promiscuously spread to dozens or hundreds of bases nearby,” said Liu. “This simple idea was really exciting to us because we thought maybe we could programmably direct this chemical transformation on DNA.”
It’s been 2 years since Liu published his first papers on base editing and already researchers around the world are using these tools widely, developing their own variants across different organisms, from bacteria, yeast and wheat to plants, fish, insects, even mammals. One scientist tested the editor on goats toward increasing the cashmere yield. Another used base editing to correct the Marfan syndrome mutation in human embryos.
The first-generation base editor Liu developed performed well in a test tube but needed modifications to potentially work in humans. If you alter a DNA base in a mammalian cell, “the cell will freak out,” he explained. “It will interpret that change as damage and initiate a highly evolved set of systems of DNA repair that will respond to, and probably try to undo, the change.”
To solve this challenge, Liu’s lab engineered a method to trick the human mismatch repair system. They’re also chemically engineering ways to target stubborn, uncooperative cells, which could have applications in such areas as pain management.
“We imagine that locally administering a base editor might be able to permanently shut down chronic pain in certain tissues,” he said.
Liu’s newer base editor can tackle previously difficult-to-edit sites, including mediating the conversion of AT to GC base pairs, which could fix some of the most common point mutations in human disease. Unveiled only a year ago, this base editor already has been tested by numerous labs in plants and mammals, and recently was used to correct muscular dystrophy in adult mice.
And now, Liu can evolve base-editor variants much faster. Liu’s lab recently developed PACE (phage-assisted continuous evolution), a system that automates the many steps of protein evolution. “It’s enabled us to perform lots and lots of rounds of evolution that would otherwise take more than a Ph.D. lifetime to do,” he said.
Using PACE, Liu’s lab developed xCas9, an enzyme Liu calls “a picky eater” due to its editing specificity. Liu is working to further improve editing efficiency and develop editors that can mediate additional base conversions.
Liu also used the opportunity of speaking at NIH to describe new research on a disease long studied by Collins, using a mouse model the NIH director previously created. Progeria, a syndrome that causes premature aging and early death, stems from a single point mutation, a mutant of the lamin A protein. Using traditional gene editors in these cases isn’t optimal because of the need to correct the mutation, and because they may inadvertently create other splicing defects.
After applying base-editing techniques in the mouse model, Liu’s team observed correction of the lamin A mutation from T back to a C. The treated cells exhibited correction at the DNA, RNA and protein levels.
“We were pretty stunned to find this extent of editing,” said Liu. “It provides us with an exciting glimpse of how one might use an integrated, engineered base editor...to address a mouse model of a serious human genetic disease.”