CRISPR-Cas9 is revolutionary gene-editing tool, but it’s not without its downsides. Now, scientists at Harvard have demonstrated an alternate genetic-engineering system called Retron Library Recombineering (RLR), which works without cutting DNA and may be quickly applied to large populations of cells.
CRISPR works like a pair of genetic scissors, ready to make precise cut-and-paste edits to the genome of living cells. The system can hunt down a specific DNA sequence, then uses an enzyme, most ordinarily Cas9, to mak a cut there. because cell performs its DNA repair procedures, CRISPR instructs it to use a special sequence rather than the original one, thereby editing the genome.
This system is already proving invaluable during 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 are raised that CRISPR can make edits within the wrong section of the genome. It also can be a touch tricky to scale up to form larger amounts of edits at once , and to trace which mutants are having which effect in lab tests.
The new gene editing tech, from researchers at Harvard Medical School & the Wyss Institute, attempts to unravel 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 seems , was originally a self-defense mechanism that bacteria use to see if they’ve been infected with virus .
By adding both the specified DNA segment along side a single-stranded annealing protein (SSAP), the RLR system makes sure that the intended DNA segment ends up in genome of the cell , after original cell divides.
“We figured that retrons should give us the power to produce ssDNA within the cells we would like to edit instead of 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 few other advantages too. It scales well, allowing many mutations to be produced directly at once , and therefore the proportion of edited cells actually increases over time as cells replicate. The retron sequence also can be tracked like a “barcode,” allowing scientists to simply check which cells received which edit, when trying to review 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 couple of other tweaks to the bugs to prevent them repairing the DNA “errors,” they found that over 90% of the population incorporated the specified sequence after 20 generations. And because of the barcode nature of the retrons, the team was ready to easily track which edits had transferred the specified 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 variety of applications. within the shorter term, it might be powerful new tool to review bacterial genomes and mutations, potentially helping create new beneficial strains or uncover treatment options for problems like antibiotic resistance. Long term, it’s going to cause a safer alternative to CRISPR in other organisms, even humans.
“Being ready to analyze pooled, barcoded mutant libraries with RLR enables many experiments to be performed simultaneously, allowing us to watch the effects of mutations across the genome, also as how those mutations might interact with one another ,” says George Church, senior author of the study. “This work helps establish a road map toward using RLR in other genetic systems, which exposes many exciting possibilities for future genetic research.”
The research was published in the journal PNAS.