Recording morphogen signals reveals mechanisms underlying gastruloid symmetry breaking.

Aggregates of stem cells can break symmetry and self-organize into embryo-like structures with complex morphologies and gene expression patterns. Mechanisms including reaction-diffusion Turing patterns and cell sorting have been proposed to explain symmetry breaking but distinguishing between these candidate mechanisms of self-organization requires identifying which early asymmetries evolve into subsequent tissue patterns and cell fates. Here we use synthetic 'signal-recording' gene circuits to trace the evolution of signalling patterns in gastruloids, three-dimensional stem cell aggregates that form an anterior-posterior axis and structures resembling the mammalian primitive streak and tailbud.

Baseline unfolded protein response signaling adjusts the timing of the mammalian cell cycle.

The endoplasmic reticulum (ER) is a single-copy organelle that cannot be generated de novo, suggesting coordination between the mechanisms overseeing ER integrity and those controlling the cell cycle to maintain organelle inheritance. The Unfolded Protein Response (UPR) is a conserved signaling network that regulates ER homeostasis. Here, we show that pharmacological and genetic inhibition of the UPR sensors IRE1, ATF6, and PERK in unstressed cells delays the cell cycle, with PERK inhibition showing the most penetrant effect, which was associated with a slowdown of the G-to-S/G transition.

A benchmarked, high-efficiency prime editing platform for multiplexed dropout screening.

Prime editing installs precise edits into the genome with minimal unwanted byproducts, but low and variable editing efficiencies have complicated application of the approach to high-throughput functional genomics. Leveraging several recent advances, we assembled a prime editing platform capable of high-efficiency substitution editing across a set of engineered prime editing guide RNAs (epegRNAs) and corresponding target sequences (80% median intended editing). Then, using a custom library of 240,000 epegRNAs targeting >17,000 codons with 175 different substitution types, we benchmarked our platform for functional interrogation of small substitution variants (1-3 nucleotides) targeted to essential genes.

Improving prime editing with an endogenous small RNA-binding protein.

Prime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3' ends of CRISPR-Cas guide RNAs. To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La.

Recording morphogen signals reveals origins of gastruloid symmetry breaking.

When cultured in three dimensional spheroids, mammalian stem cells can reproducibly self-organize a single anterior-posterior axis and sequentially differentiate into structures resembling the primitive streak and tailbud. Whereas the embryo's body axes are instructed by spatially patterned extra-embryonic cues, it is unknown how these stem cell gastruloids break symmetry to reproducibly define a single anterior-posterior (A-P) axis. Here, we use synthetic gene circuits to trace how early intracellular signals predict cells' future anterior-posterior position in the gastruloid.

Comparison of Severe Acute Respiratory Syndrome Coronavirus 2 Screening Using Reverse Transcriptase-Quantitative Polymerase Chain Reaction or CRISPR-Based Assays in Asymptomatic College Students.

Importance: The reopening of colleges and universities in the US during the coronavirus disease 2019 (COVID-19) pandemic is a significant public health challenge. The development of accessible and practical approaches for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in the college population is paramount for deploying recurrent surveillance testing as an essential strategy for virus detection, containment, and mitigation.

Objective: To determine the prevalence of SARS-CoV-2 in asymptomatic participants in a university community by using CREST (Cas13-based, rugged, equitable, scalable testing), a CRISPR-based test developed for accessible and large-scale viral screening.

A Scalable, Easy-to-Deploy Protocol for Cas13-Based Detection of SARS-CoV-2 Genetic Material.

The COVID-19 pandemic has created massive demand for widespread, distributed tools for detecting SARS-CoV-2 genetic material. The hurdles to scalable testing include reagent and instrument accessibility, availability of highly trained personnel, and large upfront investment. Here, we showcase an orthogonal pipeline we call CREST (Cas13-based, rugged, equitable, scalable testing) that addresses some of these hurdles.