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Abstract The bacterial retron reverse transcriptase system has served as an intracellular factory for single-stranded DNA in many biotechnological applications. In these technologies, a natural retron non-coding RNA (ncRNA) is modified to encode a template for the production of custom DNA sequences by reverse transcription. The efficiency of reverse transcription is a major limiting step for retron technologies, but we lack systematic knowledge of how to improve or maintain reverse transcription efficiency while changing the retron sequence for custom DNA production. Here, we test thousands of different modifications to the Retron-Eco1 ncRNA and measure DNA production in pooled variant library experiments, identifying regions of the ncRNA that are tolerant and intolerant to modification. We apply this new information to a specific application: the use of the retron to produce a precise genome editing donor in combination with a CRISPR-Cas9 RNA-guided nuclease (an editron). We use high-throughput libraries in Saccharomyces cerevisiae to additionally define design rules for editrons. We extend our new knowledge of retron DNA production and editron design rules to human genome editing to achieve the highest efficiency Retron-Eco1 editrons to date.
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This content will become publicly available on January 29, 2026
Phage defense and genome editing using novel retrons sourced from isolated environmental bacteria
ABSTRACT Retrons are bacterial immune systems that protect a bacterial population against phages by killing infected hosts. Retrons typically comprise a reverse transcriptase, a template noncoding RNA that is partially reverse transcribed into RT-DNA, and a toxic effector. The reverse transcriptase, noncoding RNA, and RT-DNA complex sequester the toxic effector until triggered by phage infection, at which point the toxin is released to induce cell death. Due to their ability to produce single-stranded DNA in vivo, retrons have also been engineered to produce donor templates for genome editing in both prokaryotes and eukaryotes. However, the current repertoire of experimentally characterized retrons is limited, with most retrons sourced from clinical and laboratory strains of bacteria. To better understand retron biology and natural diversity, and to expand the current toolbox of retron-based genome editors, we developed a pipeline to isolate retrons and their bacterial hosts from a variety of environmental samples. Here, we present six of these novel retrons, each isolated from a different host bacterium. We characterize the full operon of these retrons and test their ability to defend against a panel ofE. coliphages. For two of these retrons, we further unravel their mechanism of defense by identifying the phage genes responsible for triggering abortive infection. Finally, we engineer these retrons for genome editing inE. coli, demonstrating their potential use in a biotechnological application.
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- Award ID(s):
- 2137692
- PAR ID:
- 10620634
- Publisher / Repository:
- bioRxiv
- Date Published:
- Format(s):
- Medium: X
- Institution:
- bioRxiv
- Sponsoring Org:
- National Science Foundation
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