Retrons: Progressing From Enigma to Engineering Tool

Retrons are attractive because their sequences may be used as barcodes to identify which individuals in a pool of bacteria have received each retron sequence, allowing for much faster-pooled screening of precisely generated mutant strains.

FREMONT CA: Researchers have created a new gene-editing tool named Retron Library Recombineering (RLR) that can produce up to millions of mutations concurrently and 'barcodes' mutant bacterial cells to screen the whole pool at once. It can be employed in situations when CRISPR is toxic or impossible to use, and it improves editing rates.

Retrons are single-stranded DNA fragments produced by reverse transcription of bacterial DNA segments (ssDNA). Retrons have been known for decades, but the role of the ssDNA they make has baffled scientists since the 1980s, when a team eventually discovered out that retron ssDNA discovers whether a virus has infected the cell, developing part of the bacterial immune system.

Retrons were often thought to be little more than a curious bacterial oddity, but in recent years, researchers have been more interested in them because, like CRISPR, they could be utilized for precise and flexible gene editing in yeast, bacteria, and even human cells.

The integration of ssDNA with the desired mutation into an organism's DNA is needed for recombination-based gene editing approaches, which can be accomplished in one of two ways.Double-stranded DNA can be physically cut (for instance, with CRISPR-Cas9) to cause the cell to combine the mutant sequence into its genome during the repair process,

or the mutant DNA strand and a Single-Stranded Annealing Protein (SSAP) can be imported into a replicating cell so that the SSAP combines the mutant strand into the daughter cells' DNA.

Retrons are also attractive because their sequences may be used as barcodes to identify which individuals in a pool of bacteria have received each retron sequence, allowing for much faster-pooled screening of precisely generated mutant strains. To determine if retrons might be used to accomplish successful recombineering, one researcher and his colleagues first constructed circular plasmids of bacterial DNA containing antibiotic resistance genes within retron sequences, as well as an SSAP gene to allow retron sequence integration into the bacterial genome. After 20 generations of cell replication, they put these retron plasmids into E. coli bacteria to check if the genes were successfully incorporated into their genomes. Initially, the required mutation was found in less than 0.1 percent of E. coli carrying the retron recombineering system.

The team made many genetic changes to the bacteria to boost its initial performance. They first turned off the cells' normal mismatch repair mechanism, which corrects DNA replication errors and may have been "fixing" the intended mutations before they could be transmitted down to the next generation. They also turned off two bacterial genes that code for exonucleases, which are enzymes that break down free-floating ssDNA. The proportion of bacteria that integrated the retron sequence expanded rapidly as a result of these alterations, reaching more than 90 percent of the population.