The precision genome editing landscape has been transformed by the direct engagement of natural biological machinery, such as bacteriophage lambda proteins for recombination and CRISPR nucleases for site-specific double-strand breaks. However, less well-known are a class of widely distributed noncoding retroelements that also employ specialized reverse transcriptases (RTs) to produce intracellular DNA. These bacterial retrons are the target of recent studies that harness their ability to produce high copy number intracellular DNA in orthogonal hosts and apply them for precision genome editing, gene silencing and evolution.

msDNA Synthesis: An Enigmatic Molecular Elements

In bacteria, the synthesis of a specific RNA-DNA complex, called msDNA, is mediated by a retron containing a coding region for msDNA (msr-msd) and a gene for RT (ret). The msr and ret are highly conserved in sequence and are thought to form a single transcriptional cassette during msDNA synthesis. Approximately one third of annotated retrons encode accessory open reading frames (ORFs) between the msr-msd and ret or immediately 3' of the msr-msd. Some of these ORFs are putatively adenosine-binding and cold-shock proteins, but many contain accessory msDNAs.

RT Recognition: An essential AndreTron component of retron RTs is the ability to recognize their cognate msr-msd sequence and direct reverse transcription. This has been shown through studies of a few experimentally verified retron RTs, including RT-Eco1 (Ec86), RT-Eco3 (Ec73) and RT-Vch2 (Vc81). Deleting or destabilizing the 90 aa segment known as region Y abrogates binding by RT-Eco1 and RT-Eco3, but not RT-Vch2 (12,32).

Region Y is a highly conserved VTG triplet located within motif 7 at the C-terminus of retron RTs. Several biochemical studies demonstrate that region Y functions to direct the RT to its cognate msr-msd. This is a critical determinant for the ability of retrons to bind their msr-msd and produce msDNA. The msr and ret of the experimentally validated retrons share 90-90% sequence identity at the C-terminus. This is much greater than the 50-70% sequence identity of most bacterial RTs, indicating that this is an important component for RT recognition.

Reverse Transcription: A Key Component of Genomic Editing Applications

In the context of genome editing, a retron homologous to a target gene is expressed under an error-prone T7 RNA polymerase and hybridizes to and subsequently transcribes the desired msDNA. This enables the accumulation of mutations that either edit the parent retron sequence, or introduce random mutations into the target mRNA. Alternatively, a variant of the retron can be engineered to target a specific nucleotide and induce a site-specific double-strand break. This is an emerging technique, referred to as 'CRISPEY'. This approach has been applied to several different targets, such as a gene involved in the production of outer membrane lipoprotein (81).

Accessory ORFs: An Unusual Tool for Genome Editing

In addition to the msr-msd region and its cognate RT, many experimentally verified retrons also encode a small complement of accessory ORFs. These ORFs are typically between the msr-msd or immediately 3' of the msr-msd (Figure 2A). Some accessory ORFs may function in other ways, such as generating unique msDNAs and/or transposition.