Biology subjects

Shipman, S. L.

Publications and source records attributed to Shipman, S. L..

9 recordsLinked to original sources

bioRxiv2026

High-throughput variant libraries and machine learning enable robust target-site selection for toehold-switch design in Detectrons

Programmable RNA sensors enable sequence-specific detection of RNA inputs for diagnostics, genetic circuit control, and multiplexed molecular readouts. Although readily programmable, their performance varies substantially with the target site selected within a transcript, making target-site selection a major challenge. Here, we report DeSTAR (Detectron Sensor TARget-site ranker), a machine-learning-assisted target-site ranking model for programmable RNA sensors. To train this model, we generated transcript-wide, single-nucleotide-resolution tiling sensor libraries across six diverse RNA targets, enabling generalization to be tested across transcripts rather than within a single RNA. In leave-one-transcript-out evaluation, DeSTAR's top-ranked sites were enriched for high-performing sensors in held-out transcripts, driven predominantly by interpretable sequence grammar in the sensor stem rather than transcript-specific patterns. Prospectively, on an unseen SARS-CoV-2 transcript, the model's top-ranked sites yielded working sensors roughly five-fold more often than random selection. These results support DeSTAR as a generalizable strategy for target-site selection in RNA sensors across transcripts.

synthetic biology↗

bioRxiv2025

Reverse transcribed ssDNA derepresses translation of a retron antiviral protein

Retrons are bacterial immune systems that prevent the spread of phages by initiating a toxic response within infected hosts. All previously characterized retrons produce high levels of multicopy single-stranded DNA (msDNA) in the cell by reverse transcription, which acts as an antitoxin in the absence of phage infection. However, we describe here a non-canonical mechanism for Type VI retrons, which do not produce detectable msDNA in the absence of phage, yet still provide phage defense. Focusing primarily on Retron-Vpa2, a Type VI retron from Vibrio parahaemolyticus, we show broad defense against phages and identify triggers of the system within phage recombination systems. Within the Retron-Vpa2 operon, we find a highly enriched, structured transcript that we term a hybrid RNA (hyRNA), which contains both the retrons reverse transcription template and a translationally repressed toxic effector coding sequence. We find that phage infection induces the accumulation of high levels of msDNA and that this msDNA is necessary for derepressing translation of the antiviral toxin. These findings present key biological and mechanistic insights into a distinct group of retrons while highlighting the diversity of systems that participate in bacterial immunity.

microbiology↗

bioRxiv2024

An experimental census of retrons for DNA production and genome editing

Retrons are bacterial immune systems that use reverse transcribed DNA as a detector of phage infection. They are also increasingly deployed as a component of biotechnology. For genome editing, for instance, retrons are modified so that the reverse transcribed DNA (RT-DNA) encodes an editing donor. Retrons are commonly found in bacterial genomes; thousands of unique retrons have now been predicted bioinformatically. However, only a small number have been characterized experimentally. Here, we add substantially to the corpus of experimentally studied retrons. We synthesized >100 previously untested retrons to identify the natural sequence of RT-DNA they produce, quantify their RT-DNA production, and test the relative efficacy of editing using retron-derived donors to edit bacterial genomes, phage genomes, and human genomes. We add 62 new empirically determined, natural RT-DNAs, which are not predictable from the retron sequence alone. We report a large diversity in RT-DNA production and editing rates across retrons, finding that top performing editors outperform those used in previous studies, and are drawn from a subset of the retron phylogeny.

bioengineering↗

bioRxiv2023

Simultaneous multi-site editing of individual genomes using retron arrays

Our understanding of genomics is limited by the scale of our genomic technologies. While libraries of genomic manipulations scaffolded on CRISPR gRNAs have been transformative, these existing approaches are typically multiplexed across genomes. Yet much of the complexity of real genomes is encoded within a genome across sites. Unfortunately, building cells with multiple, non-adjacent precise mutations remains a laborious cycle of editing, isolating an edited cell, and editing again. Here, we describe a technology for precisely modifying multiple sites on a single genome simultaneously. This technology - termed a multitron - is built from a heavily modified retron, in which multiple donor-encoding msds are produced from a single transcript. The multitron architecture is compatible with both recombineering in prokaryotic cells and CRISPR editing in eukaryotic cells. We demonstrate applications for this approach in molecular recording, genetic element minimization, and metabolic engineering.

bioengineering↗

bioRxiv2023

High-throughput colocalization pipeline quantifies efficacy of mitochondrial targeting signals across different protein types

Efficient metabolic engineering and the development of mitochondrial therapeutics often rely upon the specific and strong import of foreign proteins into mitochondria. Fusing a protein to a mitochondria-bound signal peptide is a common method to localize proteins to mitochondria, but this strategy is not universally effective with particular proteins empirically failing to localize. To help overcome this barrier, this work develops a generalizable and open-source framework to design proteins for mitochondrial import and quantify their specific localization. By using a Python-based pipeline to quantitatively assess the colocalization of different proteins previously used for precise genome editing in a high-throughput manner, we reveal signal peptide-protein combinations that localize well in mitochondria and, more broadly, general trends about the overall reliability of commonly used mitochondrial targeting signals.

synthetic biology↗

bioRxiv2023

Continuous Multiplexed Phage Genome Editing Using Recombitrons

Bacteriophages, which naturally shape bacterial communities, can be co-opted as a biological technology to help eliminate pathogenic bacteria from our bodies and food supply1. Phage genome editing is a critical tool to engineer more effective phage technologies. However, editing phage genomes has traditionally been a low efficiency process that requires laborious screening, counter selection, or in vitro construction of modified genomes2. These requirements impose limitations on the type and throughput of phage modifications, which in turn limit our knowledge and potential for innovation. Here, we present a scalable approach for engineering phage genomes using recombitrons: modified bacterial retrons3 that generate recombineering donor DNA paired with single stranded binding and annealing proteins to integrate those donors into phage genomes. This system can efficiently create genome modifications in multiple phages without the need for counterselection. Moreover, the process is continuous, with edits accumulating in the phage genome the longer the phage is cultured with the host, and multiplexable, with different editing hosts contributing distinct mutations along the genome of a phage in a mixed culture. In lambda phage, as an example, recombitrons yield single-base substitutions at up to 99% efficiency and up to 5 distinct mutations installed on a single phage genome, all without counterselection and only a few hours of hands-on time.

bioengineering↗

bioRxiv2021

Reconstructing transcriptional histories by CRISPR acquisition of retron-based genetic barcodes

Biological processes depend on the differential expression of genes over time, but methods to make physical recordings of these processes are limited. Here we report a molecular system for making time-ordered recordings of transcriptional events into living genomes. We do this via engineered RNA barcodes, based on prokaryotic retrons1, which are reverse-transcribed into DNA and integrated into the genome using the CRISPR-Cas system2. 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. For disambiguation in the field, we will refer to this system as a Retro-Cascorder.

synthetic biology↗

bioRxiv2021

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

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 cells 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.

synthetic biology↗

bioRxiv2020

High throughput functional variant screens via in-vivo production of single-stranded DNA

Tremendous genetic variation exists in nature, but our ability to create and characterize individual genetic variants remains far more limited in scale. Likewise, engineering proteins and phenotypes requires the introduction of synthetic variants, but design of variants outpaces experimental measurement of variant effect. Here, we optimize efficient and continuous generation of precise genomic edits in Escherichia coli, via in-vivo production of single-stranded DNA by the targeted reverse-transcription activity of retrons. Greater than 90% editing efficiency can be obtained using this method, enabling multiplexed applications. We introduce Retron Library Recombineering (RLR), a system for high-throughput screens of variants, wherein the association of introduced edits with their retron elements enables a targeted deep sequencing phenotypic output. We use RLR for pooled, quantitative phenotyping of synthesized variants, characterizing antibiotic resistance alleles. We also perform RLR using sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for antibiotic resistance variants. In doing so, we demonstrate that RLR is uniquely suited to utilize non-designed sources of variation. Pooled experiments using ssDNA produced in vivo thus present new avenues for exploring variation, both designed and not, across the entire genome.

synthetic biology↗