Exogenous DNA can be a template to precisely edit a cell’s genome. However, the delivery of in vitro -produced DNA to target cells can be inefficient, and low abundance of template DNA may underlie the low rate of precise editing. One potential tool to produce template DNA inside cells is a retron, a bacterial retroelement involved in phage defense. However, little effort has been directed at optimizing retrons to produce designed sequences. Here, we identify modifications to the retron non-coding RNA that result in more abundant reverse transcribed DNA. By testing architectures of the retron operon that enable efficient reverse transcription, we find that gains in DNA production are portable from prokaryotic to eukaryotic cells and result in more efficient genome editing. Finally, we show that retron RT-DNA can be used to precisely edit cultured human cells. These experiments provide a general framework to produce DNA using retrons for genome modification.

A structure–activity–distribution relationship study of antimycin-type depsipeptides is demonstrated with bioorthogonal Raman microscopy.

No 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.

Retrons are bacterial retroelements that produce single-stranded, reverse-transcribed DNA (RT-DNA) that is a critical part of a newly discovered phage defense system. Short retron RT-DNAs are produced from larger, structured RNAs via a unique 2′-5′ initiation and a mechanism for precise termination that is not yet understood. Interestingly, retron reverse transcriptases (RTs) typically lack an RNase H domain and, therefore, depend on endogenous RNase H1 to remove RNA templates from RT-DNA. We find evidence for an expanded role of RNase H1 in the mechanism of RT-DNA termination, beyond the mere removal of RNA from RT-DNA:RNA hybrids. We show that endogenous RNase H1 determines the termination point of the retron RT-DNA, with differing effects across retron subtypes, and that these effects can be recapitulated using a reduced, in vitro system. We exclude mechanisms of termination that rely on steric effects of RNase H1 or RNA secondary structure and, instead, propose a model in which the tertiary structure of the single-stranded RT-DNA and remaining RNA template results in termination. Finally, we show that this mechanism affects cellular function, as retron-based phage defense is weaker in the absence of RNase H1.

Biological processes depend on the differential expression of genes over time, but methods to make true physical recordings of these processes are limited. Here we report a strategy for making time-ordered recordings of transcriptional events into living genomes. We do this via engineered RNA barcodes, based on prokaryotic retrons, which are reverse-transcribed into DNA and integrated into the genome using the CRISPR-Cas system. This approach enables the targeted recording of time-ordered transcriptional events in cells. The unidirectional integration of barcodes by CRISPR integrases enables reconstruction of transcriptional event timing based on a physical record via simple, logical rules rather than relying on pre-trained classifiers or post-hoc inferential methods.

Small-molecule natural products have been an essential source of pharmaceuticals to treat human diseases, but very little is known about their behavior inside dynamic, living human cells. Here, we demonstrate the first structure-activity-distribution study of complex natural products, the anti-cancer antimycin-type depsipeptides, using the emerging bioorthogonal Stimulated Raman Scattering (SRS) Microscopy. Our results show that the intracellular enrichment and distribution of these compounds are driven by their potency and specific protein targets, as well as the lipophilic nature of compounds.

The cumbersome encoding of digital data to cellular DNA hinders the use of cells as living hard drives. A new approach transfers digital information directly into cellular DNA by converting electrical signals into stable and interpretable changes in the genomes of bacterial populations.