High-throughput functional variant screens via in vivo production of single-stranded DNA

This study presents a new genome editing method called “Retron Library Recombineering” (RLR). The new method uses so-called retrons to induce targeted changes in the genome of bacteria.

Basics about retrons

Retrons occur naturally in many different bacterial strains and are part of the bacterial immune system: they protect bacteria from certain viruses, so-called bacteriophages. Activation of retrons causes bacteria that have been infected with bacteriophages to destroy themselves. This prevents the bacteriophages from replicating in the infected bacteria and subsequently infecting other bacteria in the population. A retron is a genetic element that can be found in the genome of bacteria and consists of DNA which encodes an enzyme called reverse transcriptase (RT) and a short piece of RNA. After the RT is formed, it translates the short piece of RNA into the respective DNA. The RNA and DNA then form an RNA/DNA hybrid that remains linked to the RT. The retron is activated as soon as a bacteriophage infects a bacterium, causing an effector protein that is toxic to the bacterial cell to destroy the cell wall. The bacterium dies before it infects other bacteria.

Retrons as a new genome editing technique

In the study, which was, amongst others, published by the scientist George Church, retrons were adapted so that they could be used by researchers to make targeted changes in the genome of bacteria. George Church was one of the developers of the CRISPR/Cas genome editing technique in human cells in 2013. CRISPR/Cas gene scissors also occur naturally in bacterial cells and are part of the bacterial immune response.

The retrons are used in a process called Retron Library Recombineering (RLR), in which RT is used to form short single-stranded DNA (ssDNA) from an RNA template in bacteria. The ssDNA is homologous to specific regions of the bacterial genome and is inserted at these sites during replication (i.e. duplication of the genome). Scientists can use the ssDNAs to introduce small targeted changes in the genome. This new method has an advantage over gene scissors applications since it does not involve cutting the DNA, and there are no restrictions on targeting DNA, i.e. it works without PAM sequences in the vicinity of the target sequence(s).

So far, the RLR method has been used in bacterial strains that carry specific gene knockouts to increase the efficiency of the method. For example, it requires turning off a repair mechanism called DNA mismatch repair (MMR) in advance that normally detects mismatches (e.g. base-base mispairing) during replication. MMR ordinarily detects such structural deviations in the DNA and repairs them. The MMR mechanism is switched off by mutations from previous experiments and has now been used for the RLR method. This means that the RLR method can be used to introduce changes into the genome much more efficiently, as the MMR can no longer recognise and repair structural deviations. So far, RLR has only been applied in bacteria and has not been transferred to mammalian cells. However, the scientists believe it has great potential for use in other organisms.

Schubert, M.G.; Goodman, D.B.; Wannier, T.M.; Kaur, D.; Farzadfard, F.; Lu, T.K.; Shipman, S.L.; Church, G.M. High-throughput functional variant screens via in vivo production of single-stranded DNA. Proceedings of the National Academy of Sciences 2021, 118, e2018181118, doi:10.1073/pnas.2018181118.