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We present GuideScan software for the design of CRISPR guide RNA libraries that can be used to edit coding and noncoding genomic regions. GuideScan produces high-density sets of gRNAs for single- and paired-gRNA genome-wide screens. We also show that by using a trie data structure GuideScan designs gRNAs that are more specific than those designed by existing tools.

SUMMARY Inflammatory bowel disease (IBD) results from a dysregulated interaction between the microbiota and a genetically susceptible host. Genetic studies have linked TNFSF15 polymorphisms and its protein TNF-like ligand 1A (TL1A) with IBD, but the functional role of TL1A is not known. Here, we found that adherent IBD-associated microbiota induced TL1A release from CX3CR1+ mononuclear phagocytes (MNPs). Using cell- specific genetic deletion models, we identified an essential role for CX3CR1+MNP- derived TL1A in driving group 3 innate lymphoid cell (ILC3) production of interleukin 22 and mucosal healing during acute colitis. In contrast to this protective role in acute colitis, TL1A-dependent expression of co-stimulatory molecule OX40L in MHCII+ ILC3s during colitis led to co-stimulation of antigen-specific T cells that was required for chronic T cell colitis. These results identify a role for ILC3s in activating intestinal T cells and reveal a central role for TL1A in promoting ILC3 barrier immunity during colitis.

The epithelial cell adhesion molecule (EpCAM) is an integral transmembrane protein that is frequently overexpressed in embryonic stem cells, tissue progenitors, carcinomas and cancer-initiating cells. In cancer cells, expression of EpCAM is associated with enhanced proliferation and upregulation of target genes including c-myc. However, the exact molecular mechanisms underlying the observed EpCAM-dependent cell proliferation remained unexplored. Here, we show that EpCAM directly affects cell cycle progression via its capacity to regulate the expression of cyclin D1 at the transcriptional level and depending on the direct interaction partner FHL2 (four-and-a-half LIM domains protein 2). As a result, downstream events such as phosphorylation of the retinoblastoma protein (Rb) and expression of cyclins E and A are similarly affected. In vivo, EpCAM expression strength and pattern are both positively correlated with the proliferation marker Ki67, high expression and nuclear localisation of cyclin D1, and Rb phosphorylation. Thus, EpCAM enhances cell cycle progression via the classical cyclin-regulated pathway.

In order to better characterize the new rapid staining method for flow cytofluorometry proposed by Krishan, we have tested its stability and several other properties, and have carried out a quantitative comparison of the fluorescence histograms obtained using propidium iodide or the acriflavine-Feulgen staining procedure.Using a human hematopoietic cell line in the logarithmic phase of growth, and analyzing the data by means of a mathematical method we have devised, we found that the fluorescence intensity of cells stained with propidium iodide remains stable for at least 48 h; it is insensitive to dye concentration between 0.025 and 0.10 mg/ml (37-150 tzM); it is not affected by incubation with ribonuclease before staining; propidium iodide in 0.1% sodium citrate remains stable for at least 20 days; and quantitative estimates of the fractions of cells in the different phases of the cell cycle are in good agreement with those obtained from acriflavine-Feulgen staining and from autoradiography after pulse labeling with tritiated thymidine. We conclude that this method is useful for the measurement of relative DNA content by flow cytofluorometry, although modifications in the technique are necessary for some cell types which grow in monolayers.Because it can be used to measure selected properties of large numbers of individual cells in a population rapidly and conveniently, flow cytofluorometry has become increasingly popular as a research tool, and its potential for clinical application is being intensively explored. In principle, the intercellular distribution of any cellular component can be measured provided an appropriate fluorescent dye can be used which binds stoichiometrically to the component in sufficient quantity to be detectable by the available instrumentation. Most applications of the method have until now been concerned with the determination of the relative distribution of DNA content among the cells of a population. A method that permits one to carry out rapid determinations of relative DNA content has important implications not only as a more convenient and potentially more accurate means of cell cycle analysis of populations in culture, but also for monitoring the status and kinetic response to therapy of patients with leukemia or other neoplasms (8,9,12,13).Until very recently, the fluorescent staining methods used by most investigators were the acri-

The rapid hypotonic staining procedure developed by Krishan for DNA determina-This investigation was supported by grants CA-16757 and CA-19117 awarded by the National Cancer Institute, and by grant ACS-CH-6G awarded by the American Cancer Society. MATERIALS AND METHODS Cell Lines and Growth Conditions HeLa S3(I): These cells were obtained from the American Type Culture Collection, Rockville, Md., as CCL 2.2. They are grown without FLOW CYTOFLUOROMETRY OF MONOLAYER CELLS

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Ionizing radiation (IR) can cause gastrointestinal syndrome (GIS), a lethal disorder, by means of unknown mechanisms. We show that high-dose irradiation increases unconventional prefoldin RPB5 interactor (URI) levels in mouse intestinal crypt, but organ regeneration correlates with URI reductions. URI overexpression in intestine protects mice from radiation-induced GIS, whereas halving URI expression sensitizes mice to IR. URI specifically inhibits β-catenin in stem cell–like label-retaining (LR) cells, which are essential for organ regeneration after IR. URI reduction activates β-catenin–induced c-MYC expression, causing proliferation of and DNA damage to LR cells, rendering them radiosensitive. Therefore, URI labels LR cells which promote tissue regeneration in response to high-dose irradiation, and c-MYC inhibitors could be countermeasures for humans at risk of developing GIS.