A lack of techniques to image multiple genomic loci in living cells has limited our ability to investigate chromosome dynamics. Here we describe CRISPRainbow, a system for labeling DNA in living cells based on nuclease-dead (d) Cas9 combined with engineered single guide RNA (sgRNA) scaffolds that bind sets of fluorescent proteins. We demonstrate simultaneous imaging of up to six chromosomal loci in individual live cells and document large differences in the dynamic properties of different chromosomal loci.

The intranuclear location of genomic loci and the dynamics of these loci are important parameters for understanding the spatial and temporal regulation of gene expression. Recently it has proven possible to visualize endogenous genomic loci in live cells by the use of transcription activator-like effectors (TALEs), as well as modified versions of the bacterial immunity clustered regularly interspersed short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system. Here we report the design of multicolor versions of CRISPR using catalytically inactive Cas9 endonuclease (dCas9) from three bacterial orthologs. Each pair of dCas9-fluorescent proteins and cognate single-guide RNAs (sgRNAs) efficiently labeled several target loci in live human cells. Using pairs of differently colored dCas9-sgRNAs, it was possible to determine the intranuclear distance between loci on different chromosomes. In addition, the fluorescence spatial resolution between two loci on the same chromosome could be determined and related to the linear distance between them on the chromosome's physical map, thereby permitting assessment of the DNA compaction of such regions in a live cell.4D nucleome | telomeres | pericentromeric DNA | chromosomes

A major challenge for effective application of CRISPR systems is to accurately predict the single guide RNA (sgRNA) on-target knockout efficacy and off-target profile, which would facilitate the optimized design of sgRNAs with high sensitivity and specificity. Here we present DeepCRISPR, a comprehensive computational platform to unify sgRNA on-target and off-target site prediction into one framework with deep learning, surpassing available state-of-the-art in silico tools. In addition, DeepCRISPR fully automates the identification of sequence and epigenetic features that may affect sgRNA knockout efficacy in a data-driven manner. DeepCRISPR is available at http://www.deepcrispr.net/.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1459-4) contains supplementary material, which is available to authorized users.

How CRISPR Cas9–guide RNA complexes navigate the nucleus and interrogate the genome is not well understood. Ma et al. track these complexes in live cells and find that mutations in the guide seed region significantly reduced the complex’s target residence time, with a commensurate impairment of cleavage.

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Nucleostemin (NS) is a nucleolar protein expressed in adult and embryo-derived stem cells, transformed cell lines, and tumors. NS decreases when proliferating cells exit the cell cycle, but it is unknown how NS is controlled, and how it participates in cell growth regulation. Here, we show that NS is down-regulated by the tumor suppressor p14 ARF and that NS knockdown elevates the level of tumor suppressor p53. NS knockdown led to G1 cell cycle arrest in p53-positive cells but not in cells in which p53 was genetically deficient or depleted by small interfering RNA knockdown. These results demonstrate that, in the cells investigated, the level of NS is regulated by p14 ARF and the control of the G1/S transition by NS operates in a p53-dependent manner. INTRODUCTIONDestabilization or inactivation of the tumor suppressor p53 is typically required to maintain cell proliferation (Vogelstein et al., 2000). However, it is unclear which of many potential upstream regulators of p53 is responsible for cell cycle progression or exit. Nucleostemin (NS) is a nucleolar protein discovered in adult rat brain stem cells, and it is also preferentially expressed in embryo-derived stem cells, transformed cell lines, and human tumors (Tsai and McKay, 2002;Baddoo et al., 2003;Liu et al., 2004;Politz et al., 2005). NS dynamically shuttles between the nucleolus and nucleoplasm, and this exchange is based on its state of GTP binding (Tsai and McKay, 2005). Pull-down and coimmunoselection experiments reveal that NS interacts with p53 (Tsai and McKay, 2002), but whether NS controls cell proliferation in a p53-dependent manner remains unclear.In a previous investigation, we found that the NS is concentrated in regions of the nucleolus that are relatively devoid of nascent ribosomes (Politz et al., 2005). We subsequently asked whether any other cell cycle progression-related proteins might occupy this same intranucleolar territory, and we found that the tumor suppressor p14 ARF (alternate reading frame ͓ARF͔) precisely colocalizes with NS in these ribosome-sparse nucleolar regions (Supplemental Figure 1). Like NS, ARF is linked to p53, and yet NS and ARF play opposite roles in cell proliferation. These initial findings, therefore, motivated us to investigate whether ARF might regulate NS, and we found that it does. In turn, this led to the finding that NS regulates cell cycle progression via the p53 pathway. MATERIALS AND METHODS Cell Culture and TransfectionU2OS, Saos-2, and HeLa cells were cultured at 37°C in DMEM supplemented with 10% fetal bovine serum (FBS). The NARF6 cell line (Stott et al., 1998) was maintained in DMEM containing 10% FBS, 150 g/ml hygromycin, and 300 g/ml Geneticin (G-418; Invitrogen, Carlsbad, CA). For transient transfections, cells were plated in 35-mm dishes, and plasmid DNA was introduced using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocols. ARF expression in NARF6 cells was induced by the addition of isopropyl-␤-d-thiogalactopyranoside at 1 mM for 48 h. For analysis of cell cycle progr...

The authors note that, due to a printer's error, references 41-50 appeared incorrectly. The corrected references follow. The authors note: "Our paper unfortunately missed reference to an earlier suggestion of the T6 structure (43). This work entitled 'A hypothetical dense 3,4-connected carbon net and related B 2 C and CN 2 nets built from 1,4-cyclohexadienoid units' by M. J. Bucknum and R. Hoffmann was published in J Am Chem Soc 116: 11456-11464 (1994), where the electronic structure of a hypothetical 3,4-connected tetragonal allotrope of carbon is discussed. The results in this article are consistent with what we find. The same group had also suggested a metallic carbon structure (44) that was published in J Am Chem Soc 105: 4831-4832 (1983), which we also missed to cite. We thank Prof. Hoffmann for bringing these papers to our attention."The complete references appear below. www.pnas.org/cgi

The recently developed single-cell CRISPR screening techniques, independently termed Perturb-Seq, CRISP-seq, or CROP-seq, combine pooled CRISPR screening with single-cell RNA-seq to investigate functional CRISPR screening in a single-cell granularity. Here, we present MUSIC, an integrated pipeline for model-based understanding of single-cell CRISPR screening data. Comprehensive tests applied to all the publicly available data revealed that MUSIC accurately quantifies and prioritizes the individual gene perturbation effect on cell phenotypes with tolerance for the substantial noise that exists in such data analysis. MUSIC facilitates the single-cell CRISPR screening from three perspectives, i.e., prioritizing the gene perturbation effect as an overall perturbation effect, in a functional topic-specific way, and quantifying the relationships between different perturbations. In summary, MUSIC provides an effective and applicable solution to elucidate perturbation function and biologic circuits by a model-based quantitative analysis of single-cell-based CRISPR screening data.