Researchers at the Gladstone Institutes say they found a way to edit the human genome one letter at a time. They add that this finding will not only boost the ability to model human disease, but will also pave the way for improved gene therapy approaches.

Writing in Nature Methods, the team described hey how to efficiently and accurately capture rare genetic mutations that cause disease—as well as how to fix them.

“Advances in human genetics have led to the discovery of hundreds of genetic changes linked to disease, but until now we’ve lacked an efficient means of studying them,” explained Gladstone’s Bruce Conklin, M.D. “We must have the capability to engineer the human genome, one letter at a time, with tools that are efficient, robust, and accurate. And the method that we outline in our study does just that.”

One of the major challenges preventing researchers from efficiently generating and studying these genetic diseases is that they can exist at frequencies as low as 1%, making the task of finding and studying them labor-intensive.

“For our method to work, we needed to find a way to identify a single mutation among hundreds of normal, healthy cells,” added Gladstone research scientist Yuichiro Miyaoka, Ph.D., the paper’s lead author. “So we designed a special fluorescent probe that would distinguish the mutated sequence from the original sequences. We were then able to sort through both sets of sequences and detect mutant cells—even when they made up as little one in every thousand cells. This is a level of sensitivity more than one hundred times greater than traditional methods.”

“Genome engineering is faced with a logistical challenge: high-fidelity precise mutagenesis results in rarer mutagenic events, but isolating a rare mutant cell without antibiotic resistance is exceedingly difficult,” wrote the investigators in their article (“Isolation of single-base genome-edited human iPScells without antibiotic selection”). “To help solve this problem, we developed a method that allows efficient detection of a mutation, sib-selection (originally a yeast cloning method) and isolation of rare, scarless clones with the desired mutation. We used the recently developed droplet digital PCR (ddPCR) and adapted iPS cell growth conditions so that rare mutant clones can be isolated with unprecedented efficiency. We used our method to introduce disease-associated point mutations into five genes (PHOX2B, PKP2, RBM20, PRKAG2 and BAG3). For each gene, we designed a pair of TALE nucleases (TALENs) or a guide RNA (gRNA) to target sequences close to the desired mutation site and a 60-nucleotide (nt) single-stranded oligonucleotide DNA donor containing the mutation.”

“Our method provides a novel way to capture and amplify specific mutations that are normally exceedingly rare,” continued Dr. Conklin. “Our high-efficiency, high-fidelity method could very well be the basis for the next phase of human genetics research.”

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