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Abstract The CRISPR integrases Cas1-Cas2 create immunological memories of viral infection by storing phage-derived DNA in CRISPR arrays, a process known as CRISPR adaptation. A number of host factors have been shown to influence adaptation, but the full pathway from infection to a fully integrated, phage-derived sequences in the array remains incomplete. Here, we deploy a new CRISPRi-based screen to identify putative host factors that participate in CRISPR adaptation in the Escherichia coli Type I-E system. Our screen and subsequent mechanistic characterization reveal that SspA, through its role as a global transcriptional regulator of cellular stress, is required for functional CRISPR adaptation. One target of SspA is H-NS, a known repressor of CRISPR interference proteins, but we find that the role of SspA on adaptation is not H-NS-dependent. We propose a new model of CRISPR-Cas defense that includes independent cellular control of adaptation and interference by SspA. 

Abstract The bacterial retron reverse transcriptase system has served as an intracellular factory for single-stranded DNA in many biotechnological applications. In these technologies, a natural retron non-coding RNA (ncRNA) is modified to encode a template for the production of custom DNA sequences by reverse transcription. The efficiency of reverse transcription is a major limiting step for retron technologies, but we lack systematic knowledge of how to improve or maintain reverse transcription efficiency while changing the retron sequence for custom DNA production. Here, we test thousands of different modifications to the Retron-Eco1 ncRNA and measure DNA production in pooled variant library experiments, identifying regions of the ncRNA that are tolerant and intolerant to modification. We apply this new information to a specific application: the use of the retron to produce a precise genome editing donor in combination with a CRISPR-Cas9 RNA-guided nuclease (an editron). We use high-throughput libraries in Saccharomyces cerevisiae to additionally define design rules for editrons. We extend our new knowledge of retron DNA production and editron design rules to human genome editing to achieve the highest efficiency Retron-Eco1 editrons to date. 

Tumor vasculature plays a crucial role in tumor progression, affecting nutrition and oxygen transportation as well as the efficiency of drug delivery. While targeting pro-angiogenic growth factors has been a significant focus for treating tumor angiogenesis, recent studies indicate that metabolism also plays a role in regulating endothelial cell behavior. Like cancer cells, tumor endothelial cells undergo metabolic changes that regulate rearrangement for tip cell position during angiogenesis. Our previous studies have shown that altered mechanical properties of the collagen matrix regulate angiogenesis and can promote a tumor vasculature phenotype. Here, we examine the effect of collagen density on endothelial cell tip–stalk cell rearrangement and cellular energetics during angiogenic sprouting. We find that increased collagen density leads to an elevated energy state and an increased rate of tip–stalk cell switching, which is correlated with the energy state of the cells. Tip cells exhibit higher glucose uptake than stalk cells, and inhibition of glucose uptake revealed that invading sprouts rely on glucose to meet elevated energy requirements for invasion in dense matrices. This work helps to elucidate the complex interplay between the mechanical microenvironment and the endothelial cell metabolic status during angiogenesis, which could have important implications for developing new anti-cancer therapies. 

Vinculin is a protein found in both focal adhesions (FAs) and adherens junctions (AJs) which regulates actin connectivity to these structures. Many studies have demonstrated that mechanical perturbations of cells result in enhanced recruitment of vinculin to FAs and/or AJs. Likewise, many other studies have shown “cross-talk” between FAs and AJs. Vinculin itself has been suggested to be a probable regulator of this adhesion cross-talk. In this study we used MDCK as a model system of epithelia, developing cell lines in which vinculin recruitment was reduced or enhanced at AJs. Careful analysis of these cells revealed that perturbing vinculin recruitment to AJs resulted in a reduction of detectable FAs. Interestingly the cross-talk between these two structures was not due to a limited pool of vinculin, as increasing expression of vinculin did not rescue FA formation. Instead, we demonstrate that vinculin translocation between AJs and FAs is necessary for actin cytoskeleton rearrangements that occur during cell migration, which is necessary for large, well-formed FAs. Last, we show using a wound assay that collective cell migration is similarly hindered when vinculin recruitment is reduced or enhanced at AJs, highlighting that vinculin translocation between each compartment is necessary for efficient collective migration.