PT  - JOURNAL ARTICLE
AU  - Santiago C. Lopez
AU  - Kate D. Crawford
AU  - Santi Bhattarai-Kline
AU  - Seth L. Shipman
TI  - Improved architectures for flexible DNA production using retrons across kingdoms of life
AID  - 10.1101/2021.03.26.437017
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.03.26.437017
4099  - http://biorxiv.org/content/early/2021/03/26/2021.03.26.437017.short
4100  - http://biorxiv.org/content/early/2021/03/26/2021.03.26.437017.full
AB  - Exogenous DNA is a critical molecular tool for biology. This is particularly true for gene editing, where exogenous DNA can be used as a template to introduce precise changes to the sequence of a cell’s genome. This DNA is typically synthesized or assembled in vitro and then delivered to target cells. However, delivery can be inefficient, and low abundance of template DNA may be one reason that precise editing typically occurs at a low rate. It has recently been shown that producing DNA inside cells can using reverse transcriptases can increase the efficiency of genome editing. One tool to produce that DNA is a retron, a bacterial retroelement that has an endogenous role in phage defense. However, little effort has been directed at optimizing the retron for production of designed sequences when used as a component of biotechnology. Here, we identify modifications to the retron non-coding RNA that result in more abundant reverse transcribed DNA. We also test architectures of the retron operon that enable efficient reverse transcription across kingdoms of life from bacteria, to yeast, to cultured human cells. We find that gains in DNA production using modified retrons are portable from prokaryotic to eukaryotic cells. Finally, we demonstrate that increased production of RT-DNA results in more efficient genome editing in both prokaryotic and eukaryotic cells. These experiments provide a general framework for production of DNA using a retron for biotechnological applications.Competing Interest StatementS.L.S. is a named inventor on a patent application related to the technologies described in this work.