CRISPR-Cas9 is a revolutionary gene-editing tool, but it’s not without its downsides. Now, scientists at Harvard have demonstrated an alternative genetic engineering system called Retron Library Recombineering (RLR), which works without cutting DNA and can be quickly applied to huge populations of cells.
CRISPR works like a pair of genetic scissors, able to make precise cut-and-paste edits to the genome of living cells. The system can seek out a particular DNA sequence, then uses an enzyme, most commonly Cas9, to make a cut there. As the cell performs its DNA repair procedures, CRISPR instructs it to use a different sequence instead of the original one, thereby editing the genome.
This system is already proving invaluable in a range of applications, from treating diseases like cancer, HIV and muscular dystrophy, to pest control, improving crops, and building biological computers out of bacteria.
There are, however, potential problems. Cutting DNA could cause some unintended side effects, and concerns have been raised that CRISPR can make edits in the wrong section of the genome. It can also be a little tricky to scale up to make larger amounts of edits at once, and to track which mutants are having which effect in lab tests.
The new gene editing tech, from researchers at Harvard Medical School and the Wyss Institute, attempts to solve these issues. RLR’s main point of difference is that it doesn’t cut the DNA at all – instead, it introduces the new DNA segment while a cell is replicating its genome before dividing.
It does so using retrons, which are segments of bacterial DNA that produce pieces of single-stranded DNA (ssDNA). This, it turns out, was originally a self-defense mechanism that bacteria use to check if they’ve been infected with a virus.
By adding both the desired DNA segment along with a single-stranded annealing protein (SSAP), the RLR system makes sure that the intended DNA segment ends up in the genome of the daughter cell, after the original cell divides.
“We figured that retrons should give us the ability to produce ssDNA within the cells we want to edit rather than trying to force them into the cell from the outside, and without damaging the native DNA, which were both very compelling qualities,” says Daniel Goodman, co-first author of the study.
The new system has a few other advantages too. It scales well, allowing millions of mutations to be produced at once, and the proportion of edited cells actually increases over time as cells replicate. The retron sequence can also be tracked like a “barcode,” allowing scientists to easily check which cells received which edit, when trying to study the effects.
To test the system out, the researchers put it to work editing populations of E. coli. They used the retrons to introduce antibiotic resistance genes to the bacteria, and after making a few other tweaks to the bugs to stop them repairing the DNA “errors,” they found that over 90 percent of the population incorporated the desired sequence after 20 generations. And thanks to the barcode nature of the retrons, the team was able to easily track which edits had transferred the desired genes into the bacterial genome.
While there’s still plenty more work to be done, the team says that the new RLR tool could have a range of applications. In the shorter term, it could be a powerful new tool to study bacterial genomes and mutations, potentially helping create new beneficial strains or uncover treatment options for problems like antibiotic resistance. Longer term, it may lead to a safer alternative to CRISPR in other organisms, even humans.
“Being able to analyze pooled, barcoded mutant libraries with RLR enables millions of experiments to be performed simultaneously, allowing us to observe the effects of mutations across the genome, as well as how those mutations might interact with each other,” says George Church, senior author of the study. “This work helps establish a road map toward using RLR in other genetic systems, which opens up many exciting possibilities for future genetic research.”
The research was published in the journal PNAS.
Source: Wyss Institute
