The gene editing toolkit expanded this week, as two groups of researchers announced techniques that enable scientists to make targeted modifications to DNA and RNA. Instead of the original CRISPR gene editing system – a relatively imprecise and unexpected type of molecular scissors that cut large segments of DNA – the new systems rewrite individual letters or genetic bases. The ability to modify single bases means researchers can now try to correct more than half of human genetic diseases.
Expanding Precision Editing Tools for Single Bases
These tools were developed by separate teams at the Broad Institute of Technology and Harvard University in Cambridge, Massachusetts. While most previous attempts to use CRISPR-based methods to correct individual bases were crude – akin to using a dull knife to remove a wart – the new techniques more resemble “precise chemical surgery,” according to David Liu, a biochemical scientist at the Broad Institute who led one of the studies.
Editing Single Bases in DNA
Last year, his team reported the first “base editing” method to convert a targeted DNA letter into another without necessitating the cleavage of the DNA double helix. It has been used worldwide to correct genes in fungi, plants, fish, mice, and even in human embryos carrying a defective gene that could cause a blood disorder. However, this base editor can achieve only two types of chemical conversions: converting cytosine (C) to thymine (T) or guanine (G) to adenine (A).
Editing Single Bases in RNA
Another method, described in a study published on October 25 in Science and led by bioengineer Feng Zhang from the Broad Institute, converts adenine (A) to inosine (I), which is read as guanine (G) by the cell’s protein-building machinery. This allows for a temporary correction of the mutation causing the disease without a permanent change to the genome – a safer option when it comes to gene therapy, although the treatment would need to be administered repeatedly. It also means researchers can adjust the treatment as they gain a better understanding of the disease. “If you use RNA therapy,” says Zhang, “you can upgrade.”
His team’s RNA editor relies on a naturally occurring enzyme that rearranges atoms in adenine to resemble inosine. They then fused the enzyme with a deactivated version of the CRISPR system – including an RNA-targeting enzyme called Cas13 instead of the Cas9 that binds to DNA. With the help of a sequence-specific RNA guide molecule, they successfully corrected disease-causing mutations by 23-35%, with low rates of off-target activity.
Editing Single Bases in DNA
In the base editing method developed by Liu’s team last year, researchers developed a naturally occurring enzyme and linked it to inactive Cas9, allowing them to convert cytosine to thymine. However, there is no equivalent enzyme in nature for the reverse conversion in DNA. Therefore, the researchers started with an RNA editing enzyme similar to the one used by Zhang’s group. The team guided bacterial cell evolution through seven generations and used some lab protein engineering to produce an enzyme capable of recognizing and manipulating DNA. The enzyme was able to rearrange atoms in adenine to convert it to inosine, which the cell reads as a gene. The system then tricked the cell into inserting cytosine into the unedited DNA strand.
Editing
Individual Base Editing – A Bold Step
This method represents a heroic effort, according to Dana Carroll, a genome engineering researcher at the University of Utah in Salt Lake City, who noted that the approach relying on directed evolution was somewhat in the dark. “I wouldn’t have dared to try what they did,” says Carroll. “I respect David Liu.”
The ability to perform four types of individual base conversions – from A to G, from G to A, from C to T, and from T to C – “will be extremely valuable for therapeutic editing and precise agriculture,” according to Caixia Gao, a plant geneticist at the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences in Beijing. It may also be useful for drug discovery and DNA-based data storage, according to Marcello Maresca, a gene editing researcher at AstraZeneca in Gothenburg, Sweden.
Developing any other base editors will require enzymes not present in nature, even for the conversions in RNA. But this type of hurdle has not stopped Liu before. “We will keep trying until the community develops all possible base editors,” he says.
Source: https://www.nature.com/news/crispr-hacks-enable-pinpoint-repairs-to-genome-1.22884
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