Biology subjects

Collins, S. P.

Publications and source records attributed to Collins, S. P..

5 recordsLinked to original sources

bioRxiv2024

Targeted DNA ADP-ribosylation triggers templated repair in bacteria and base mutagenesis in eukaryotes

Base editors create precise genomic edits by directing nucleobase deamination or removal without inducing double-stranded DNA breaks. However, a vast chemical space of other DNA modifications remains to be explored for genome editing. Here, we harness the bacterial anti-phage toxin DarT2 to append ADP-ribosyl moieties to DNA, unlocking distinct editing outcomes in bacteria versus eukaryotes. Fusing an attenuated DarT2 to a Cas9 nickase, we program site-specific ADP-ribosylation of thymines within a target DNA sequence. In tested bacteria, targeting drives efficient homologous recombination in tested bacteria, offering flexible and scar-free genome editing without base replacement nor counterselection. In tested eukaryotes including yeast, plants and human cells, targeting drives substitution of the modified thymine to adenine or a mixture of adenine and cytosine with limited insertions or deletions, offering edits inaccessible to current base editors. Altogether, our approach, called append editing, leverages the addition of a chemical moiety to DNA to expand current modalities for precision gene editing.

synthetic biology↗

bioRxiv2022

Discovery of an alternate mechanism for the inhibition of particulate guanylyl cyclases by the natriuretic peptide 'clearance' receptor

The cardiac natriuretic peptides (NPs) control pivotal physiological actions such as fluid and electrolyte balance, cardiovascular homeostasis, and adipose tissue metabolism by activating their receptor enzymes (NPRA and NPRB). These receptors are homodimers that generate intracellular cyclic guanosine monophosphate (cGMP). The NP receptor NPRC, nicknamed the clearance receptor, lacks a guanylyl cyclase domain; instead, it can bind the NPs to internalize and degrade them. The conventional paradigm is that by competing for and internalizing NPs, NPRC blunts the ability of NPs to signal through NPRA and NPRB. Here we show another previously unknown mechanism by which NPRC can interfere with the cGMP signaling function of the NP receptors. By forming a heterodimer with monomeric NPRA or NPRB, NPRC can prevent the formation of a functional guanylyl cyclase domain and thereby suppress cGMP production in a cell-autonomous manner. Significance StatementNatriuretic peptides (NP) are hormones that are established regulators of vascular and cardiac function, in part through their regulation of fluid and electrolyte balance. NPs signal through particulate guanylyl cyclases (NPRA and NPRB), which are homodimeric membrane-bound receptor enzymes that generate cGMP upon NP binding. Additionally, a silent NP receptor (NPRC) lacks the guanylyl cyclase domain and is a negative regulator of NP signaling. It has been demonstrated that NPRC undergoes internalization and recycling and thus removes NPs, thereby blunting activation of the guanylyl cyclase-containing receptors. Here we show an additional mechanism by which NPRC inhibits NP signaling. Our results show that NPRC can directly interact with NPRA and NPRB, forming non-functional receptor heterodimers with NPRA and NPRB, thereby abrogating NP-evoked cGMP production. This finding establishes another novel mechanistic role for NPRC.

biochemistry↗

bioRxiv2021

A target expression threshold dictates invader defense and autoimmunity by CRISPR-Cas13

Immune systems must recognize and clear foreign invaders without eliciting autoimmunity. CRISPR-Cas immune systems in prokaryotes manage this task by following two criteria: extensive guide:target complementarity and a defined target-flanking motif. Here we report an additional requirement for RNA-targeting CRISPR-Cas13 systems: expression of the target transcript exceeding a threshold. This finding is based on targeting endogenous non-essential transcripts, which rarely elicited dormancy through collateral RNA degradation. Instead, eliciting dormancy required over-expressing targeted transcripts above a threshold. A genome-wide screen confirmed target expression levels as the principal determinant of cytotoxic autoimmunity and revealed that the threshold shifts with the guide:target pair. This expression threshold ensured defense against a lytic bacteriophage yet allowed tolerance of a targeted beneficial gene expressed from an invading plasmid. These findings establish target expression levels as a third criterion for immune activation by RNA-targeting CRISPR-Cas systems, buffering against autoimmunity and distinguishing pathogenic and benign invaders. HIGHLIGHTSO_LICas13-induced dormancy requires RNA target levels to exceed an expression threshold C_LIO_LIThe expression threshold can prevent cytotoxic self-targeting for endogenous transcripts C_LIO_LIThe threshold shifts depending on the CRISPR RNA guide:target pair C_LIO_LIThe threshold allows cells to distinguish pathogenic and benign infections C_LI

molecular biology↗

bioRxiv2020

Establishing Probiotic Saccharomyces boulardii as a Model Organism for Synthesis and Delivery of Biomolecules

Saccharomyces boulardii is a widely used yeast probiotic which can counteract various gastrointestinal disorders1. As a relative of Saccharomyces cerevisiae, S. boulardii exhibits rapid growth and is easy to transform2 and thus represents a promising chassis for the engineered secretion of biomolecules. To establish S. boulardii as a platform for delivery of biomolecules to the mammalian gut, we measured the amount and variance in protein expression enabled by promoters, terminators, selective markers, and copy number control elements in this organism. These genetic elements were characterized in plasmidic and genomic contexts, revealing strategies for tunable control of gene expression and CRISPR-mediated genome editing in this strain. We then leveraged this set of genetic parts to combinatorially assemble pathways enabling a wide range of drug and vitamin titers. Finally, we measured S. boulardiis residence time in the gastrointestinal tracts of germ-free and antibiotic-treated mice, revealing the relationships between dosing strategy and colonization level. This work establishes S. boulardii as a genetically tractable commensal fungus and provides a set of strategies for engineering S. boulardii to synthesize and deliver biomolecules during gut colonization.

synthetic biology↗

bioRxiv2017

Rapid and scalable characterization of CRISPR technologies using an E. coli cell-free transcription-translation system

CRISPR-Cas systems have offered versatile technologies for genome engineering, yet their implementation has been outpaced by the ongoing discovery of new Cas nucleases and anti-CRISPR proteins. Here, we present the use of E. coli cell-free transcription-translation systems (TXTL) to vastly improve the speed and scalability of CRISPR characterization and validation. Unlike prior approaches that require protein purification or live cells, TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression. To demonstrate the applicability of TXTL, we rapidly measure guide RNA-dependent DNA cleavage and gene repression for single- and multi-effector CRISPR-Cas systems, accurately predict the strength of gene repression in E. coli, quantify the inhibitory activity of anti-CRISPR proteins, and develop a fast and scalable high-throughput screen for protospacer-adjacent motifs. These examples underscore the potential of TXTL to facilitate the characterization and application of CRISPR technologies across their many uses.

synthetic biology↗