RT Journal Article
SR Electronic
T1 Improved architectures for flexible DNA production using retrons across kingdoms of life
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.03.26.437017
DO 10.1101/2021.03.26.437017
A1 Santiago C. Lopez
A1 Kate D. Crawford
A1 Santi Bhattarai-Kline
A1 Seth L. Shipman
YR 2021
UL http://biorxiv.org/content/early/2021/03/26/2021.03.26.437017.abstract
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.