Improved architectures for flexible DNA production using retrons across kingdoms of life

Abstract

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.

High-efficiency retron-mediated single-stranded DNA production in plants

“…Here, we selected the retron Ec86 system to produce ssDNA in N. benthamiana . ssDNA production is dependent on the retron ncRNA structure and the retron RT [9,31]. Instead of using WT Ec86 ncRNA, we replaced the WT msd template with our DRT sequences (100 bp) at the msd loop region as suggested by Sharon et al [25].…”

“…Because retron-mediated ssDNA has been used for HDR in yeast and mammalian cells [9,25], and because we successfully produced ssDNA with our retron designs, we hypothesized that retron-meditated ssDNA overproduction might facilitate HDR in N. benthamiana cells (Supplementary Figure 6). To test this, we extracted total genomic DNA from the infiltrated leaf samples (two independent biological replicates for each target), amplified the target DNA by PCR, and used the PCR amplicons for deep sequencing.…”

“…Considering the potential of retrons as promising tools in molecular biology, two important improvements, enhancing ssDNA production by retrons and adapting retrons to work in more host organisms are essential. Lopez et al [9] demonstrated different strategies to improve ssDNA production in mammalian cells. Remarkably, the extension of the a1/a2 region of the retron ncRNA facilitated higher ssDNA production in mammalian cells [9].…”

“…Lopez et al [9] demonstrated different strategies to improve ssDNA production in mammalian cells. Remarkably, the extension of the a1/a2 region of the retron ncRNA facilitated higher ssDNA production in mammalian cells [9]. Viswanathan et al [10] downregulated the expression of the endogenous exonuclease and showed efficient retron-mediated genome editing in Escherichia coli .…”

“…Researchers have repurposed retrons as powerful tools for genome engineering in several organisms [6,7,23]. For example, retrons have been coupled with CRISPR-Cas9 tools to enrich ssDNA templates at the targeted editing site for HDR in yeast, bacteria, and mammalian cells [9,24,25].…”