Genome-wide Cas9-mediated screening of essential non-coding regulatory elements via libraries of paired single-guide RNAs.

The functions of non-coding regulatory elements (NCREs), which constitute a major fraction of the human genome, have not been systematically studied. Here we report a method involving libraries of paired single-guide RNAs targeting both ends of an NCRE as a screening system for the Cas9-mediated deletion of thousands of NCREs genome-wide to study their functions in distinct biological contexts. By using K562 and 293T cell lines and human embryonic stem cells, we show that NCREs can have redundant functions, and that many ultra-conserved elements have silencer activity and play essential roles in cell growth and in cellular responses to drugs (notably, the ultra-conserved element PAX6_Tarzan may be critical for heart development, as removing it from human embryonic stem cells led to defects in cardiomyocyte differentiation). The high-throughput screen, which is compatible with single-cell sequencing, may allow for the identification of druggable NCREs.

Systemic coagulopathy promotes host lethality in a new Drosophila tumor model.

Malignant tumors trigger a complex network of inflammatory and wound repair responses, prompting Dvorak's characterization of tumors as "wounds that never heal."1 Some of these responses lead to profound defects in blood clotting, such as disseminated intravascular coagulopathy (DIC), which correlate with poor prognoses.2,3,4 Here, we demonstrate that a new tumor model in Drosophila provokes phenotypes that resemble coagulopathies observed in patients. Fly ovarian tumors overproduce multiple secreted components of the clotting cascade and trigger hypercoagulation of fly blood (hemolymph). Hypercoagulation occurs shortly after tumor induction and is transient; it is followed by a hypocoagulative state that is defective in wound healing. Cellular clotting regulators accumulate on the tumor over time and are depleted from the body, suggesting that hypocoagulation is caused by exhaustion of host clotting components. We show that rescuing coagulopathy by depleting a tumor-produced clotting factor improves survival of tumor-bearing flies, despite the fact that flies have an open (non-vascular) circulatory system. As clinical studies suggest that lethality in patients with high serum levels of clotting components can be independent of thrombotic events,5,6 our work establishes a platform for identifying alternative mechanisms by which tumor-driven coagulopathy triggers early mortality. Moreover, it opens up exploration of other conserved mechanisms of host responses to chronic wounds.

The mIAA7 degron improves auxin-mediated degradation in Caenorhabditiselegans.

Auxin-inducible degradation is a powerful tool for the targeted degradation of proteins with spatiotemporal control. One limitation of the auxin-inducible degradation system is that not all proteins are degraded efficiently. Here, we demonstrate that an alternative degron sequence, termed mIAA7, improves the efficiency of degradation in Caenorhabditiselegans, as previously reported in human cells. We tested the depletion of a series of proteins with various subcellular localizations in different tissue types and found that the use of the mIAA7 degron resulted in faster depletion kinetics for 5 out of 6 proteins tested. The exception was the nuclear protein HIS-72, which was depleted with similar efficiency as with the conventional AID* degron sequence. The mIAA7 degron also increased the leaky degradation for 2 of the tested proteins. To overcome this problem, we combined the mIAA7 degron with the C. elegans AID2 system, which resulted in complete protein depletion without detectable leaky degradation. Finally, we show that the degradation of ERM-1, a highly stable protein that is challenging to deplete, could be improved further by using multiple mIAA7 degrons. Taken together, the mIAA7 degron further increases the power and applicability of the auxin-inducible degradation system. To facilitate the generation of mIAA7-tagged proteins using CRISPR/Cas9 genome engineering, we generated a toolkit of plasmids for the generation of dsDNA repair templates by PCR.

ERM-1 Phosphorylation and NRFL-1 Redundantly Control Lumen Formation in the C. elegans Intestine.

Reorganization of the plasma membrane and underlying actin cytoskeleton into specialized domains is essential for the functioning of most polarized cells in animals. Proteins of the ezrin-radixin-moesin (ERM) and Na+/H+ exchanger 3 regulating factor (NHERF) family are conserved regulators of cortical specialization. ERM proteins function as membrane-actin linkers and as molecular scaffolds that organize the distribution of proteins at the membrane. NHERF proteins are PDZ-domain containing adapters that can bind to ERM proteins and extend their scaffolding capability. Here, we investigate how ERM and NHERF proteins function in regulating intestinal lumen formation in the nematode Caenorhabditis elegans. C. elegans has single ERM and NHERF family proteins, termed ERM-1 and NRFL-1, and ERM-1 was previously shown to be critical for intestinal lumen formation. Using CRISPR/Cas9-generated nrfl-1 alleles we demonstrate that NRFL-1 localizes at the intestinal microvilli, and that this localization is depended on an interaction with ERM-1. However, nrfl-1 loss of function mutants are viable and do not show defects in intestinal development. Interestingly, combining nrfl-1 loss with erm-1 mutants that either block or mimic phosphorylation of a regulatory C-terminal threonine causes severe defects in intestinal lumen formation. These defects are not observed in the phosphorylation mutants alone, and resemble the effects of strong erm-1 loss of function. The loss of NRFL-1 did not affect the localization or activity of ERM-1. Together, these data indicate that ERM-1 and NRFL-1 function together in intestinal lumen formation in C. elegans. We postulate that the functioning of ERM-1 in this tissue involves actin-binding activities that are regulated by the C-terminal threonine residue and the organization of apical domain composition through NRFL-1.

C-terminal phosphorylation modulates ERM-1 localization and dynamics to control cortical actin organization and support lumen formation during Caenorhabditiselegans development.

ERM proteins are conserved regulators of cortical membrane specialization that function as membrane-actin linkers and molecular hubs. The activity of ERM proteins requires a conformational switch from an inactive cytoplasmic form into an active membrane- and actin-bound form, which is thought to be mediated by sequential PIP2 binding and phosphorylation of a conserved C-terminal threonine residue. Here, we use the single Caenorhabditiselegans ERM ortholog, ERM-1, to study the contribution of these regulatory events to ERM activity and tissue formation in vivo Using CRISPR/Cas9-generated erm-1 mutant alleles, we demonstrate that a PIP2-binding site is crucially required for ERM-1 function. By contrast, dynamic regulation of C-terminal T544 phosphorylation is not essential but modulates ERM-1 apical localization and dynamics in a tissue-specific manner, to control cortical actin organization and support lumen formation in epithelial tubes. Our work highlights the dynamic nature of ERM protein regulation during tissue morphogenesis and the importance of C-terminal phosphorylation in fine-tuning ERM activity in a tissue-specific context.