Researchers at the Wyss Institute for Biologically Inspired Engineering at Harvard have developed a new gene editing technique called Retron Library Recombineering (RLR), which could potentially serve as an alternative to CRISPR. Further, the technique achieves better editing rates and is safer.
CRISPR, of which CRISPR-Cas9 is the most widely used, is a gene editing tool that can be programmed to make specific cuts to double stranded DNA and used to trick the cell into using an alternate piece of DNA when the break is repaired. While the system allows accurate cuts to be made, there is potential for off-target effects that can prove toxic to cells. It can also be difficult to deliver CRISPR-Cas9 to cells in sufficient numbers and achieve a sufficiently high editing rate.
RLR uses fragments of bacterial DNA known as retrons, which can be used to produce fragments of single-stranded DNA in a process called reverse transcription. Retrons have been known to science for more than 30 years, but the function of the single stranded DNA was not understood until recently. Scientists have now determined that single stranded DNA forms part of the bacterial immune system. It is used to determine whether bacteria have been infected with a virus.
When the segments of single stranded DNA are introduced into replicating cells, the strands are then incorporated into daughter cells, without any cuts being made to double stranded DNA.
RLR can be used to generate millions of mutations simultaneously and can be used to barcode mutant bacterial cells, which allows an entire pool of cells to be screened at once. Retrons allow researchers to produce single stranded DNA within the cells they want to edit, rather than CRISPR which needs to be forced in from outside. RLR allow allows edits to be made without damaging DNA and risking off-target effects. The ability to barcode cells allows individuals in a pool of bacteria to be identified when they have received the retron sequence, which results in much faster pooled screens of precisely created mutant strains.
To test the effectiveness of the tool for use in gene editing, the researchers created circular plasmids of bacterial DNA that included antibiotic resistance genes inserted with retron sequences, along with a single-stranded annealing protein (SSAP) gene that enabled integration of the retron sequence into the bacterial genome. The retron plasmids were inserted into E. coli bacteria; however, after 20 generations, only 0.1% of the E. coli had antibiotic resistance.
The researchers suspected that the changes were being corrected prior to being passed on, so they inactivated the natural repair machinery which may have prevented the changes from being passed on and inactivated two genes involved in coding for exonucleases which may have been destroying free floating single stranded DNA in the cells. They then were able to get the retron sequence incorporated into the genome of 90% of cells after 20 generations.
The researchers also showed that they were able to sequence the barcodes of the retrons rather than individual mutants to identify antibiotic resistance much more quickly that was previously possible, to the extent that millions of experiments can be performed simultaneously to see the effects of the mutations across the genome and how those mutations interact with others.
“RLR enabled us to do something that’s impossible to do with CRISPR: we randomly chopped up a bacterial genome, turned those genetic fragments into single-stranded DNA in situ, and used them to screen millions of sequences simultaneously,” said Max Schubert, Ph.D., a postdoc in the lab of Wyss Core Faculty member George Church, Ph.D. and co-first author of the study. “RLR is a simpler, more flexible gene editing tool that can be used for highly multiplexed experiments, which eliminates the toxicity often observed with CRISPR and improves researchers’ ability to explore mutations at the genome level.”
Another advantage of RLR over CRISPR is the proportion of bacteria that have the retron sequence incorporated increases over time as the cells reproduce, whereas CRISPR is a one-shot treatment that will either succeed or fail. It may also be possible to combine RLR with CRISPR to improve editing efficiency.
The researchers say that much more work is required on RLR to standardize the editing rate but that this could be an invaluable tool in genetic research. “This new synthetic biology tool brings genome engineering to an even higher levels of throughput, which will undoubtedly lead to new, exciting, and unexpected innovations,” said Don Ingber, M.D., Ph.D., the Wyss Institute’s Founding Director.
You can read more about the study in the paper –High-throughput functional variant screens via in vivo production of single-stranded DNA – which was recently published in PNAS . DOI: 10.1073/pnas.2018181118