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Contains fulltext : 218166.pdf (Publisher’s version ) (Open Access) The recent boom in microfluidics and combinatorial indexing strategies, combined with low sequencing costs, has empowered single-cell sequencing technology. Thousands-or even millions-of cells analyzed in a single experiment amount to a data revolution in single-cell biology and pose unique data science problems. Here, we outline eleven challenges that will be central to bringing this emerging field of single-cell data science forward. For each challenge, we highlight motivating research questions, review prior work, and formulate open problems. This compendium is for established researchers, newcomers, and students alike, highlighting interesting and rewarding problems for the coming years.

CRISPR/Cas9 pooled screening permits parallel evaluation of comprehensive guide RNA libraries to systematically perturb protein coding sequences in situ and correlate with functional readouts. For the analysis and visualization of the resulting datasets, we develop CRISPRO, a computational pipeline that maps functional scores associated with guide RNAs to genomes, transcripts, and protein coordinates and structures. No currently available tool has similar functionality. The ensuing genotype-phenotype linear and three-dimensional maps raise hypotheses about structure-function relationships at discrete protein regions. Machine learning based on CRISPRO features improves prediction of guide RNA efficacy. The CRISPRO tool is freely available at gitlab.com/bauerlab/crispro. Electronic supplementary material The online version of this article (10.1186/s13059-018-1563-5) contains supplementary material, which is available to authorized users.

Genome editing technologies are rapidly evolving, and analysis of deep sequencing data from target or off-target regions is necessary for measuring editing efficiency and evaluating safety. However, no software exists to analyze base editors, perform allele-specific quantification or that incorporates biologically-informed and scalable alignment approaches. Here, we present CRISPResso2 to fill this gap and illustrate its functionality by experimentally measuring and analyzing the editing properties of six genome editing agents.

CRISPR/Cas9 pooled screening permits parallel evaluation of comprehensive guide RNA libraries to systematically perturb protein coding sequences in situ and correlate with functional readouts. For the analysis and visualization of the resulting datasets we have developed CRISPRO, a computational pipeline that maps functional scores associated with guide RNAs to genome, transcript, and protein coordinates and structure. No available tool has similar functionality. The ensuing genotype-phenotype linear and 3D maps raise hypotheses about structure-function relationships at discrete protein regions. Machine learning based on CRISPRO features improves prediction of guide RNA efficacy. The CRISPRO tool is freely available at gitlab.com/bauerlab/crispro.

CRISPR (clustered regularly interspaced short palindromic repeats) genome-editing experiments offer enormous potential for the evaluation of genomic loci using arrayed single guide RNARNARNAs (sgRNAs) or pooled sgRNA libraries. Numerous computational tools are available to help design sgRNAs with optimal on-target efficiency and minimal off-target potential. In addition, computational tools have been developed to analyze deep-sequencing data resulting from genome-editing experiments. However, these tools are typically developed in isolation and oftentimes are not readily translatable into laboratory-based experiments. Here, we present a protocol that describes in detail both the computational and benchtop implementation of an arrayed and/or pooled CRISPR genome-editing experiment. This protocol provides instructions for sgRNA design with CRISPOR (computational tool for the design, evaluation, and cloning of sgRNA sequences), experimental implementation, and analysis of the resulting high-throughput sequencing data with CRISPResso (computational tool for analysis of genome-editing outcomes from deep-sequencing data). This protocol allows for design and execution of arrayed and pooled CRISPR experiments in 4–5 weeks by non-experts, as well as computational data analysis that can be performed in 1–2 d by both computational and noncomputational biologists alike using web-based and/or command-line versions.

Single-cell transcriptomic assays have enabled the de novo reconstruction of lineage differentiation trajectories, along with the characterization of cellular heterogeneity and state transitions. Several methods have been developed for reconstructing developmental trajectories from single-cell transcriptomic data, but efforts on analyzing single-cell epigenomic data and on trajectory visualization remain limited. Here we present STREAM, an interactive pipeline capable of disentangling and visualizing complex branching trajectories from both single-cell transcriptomic and epigenomic data.

Schaefer et al. recently advanced the provocative conclusion that CRISPR-Cas9 nuclease can induce off-target alterations at genomic loci that do not resemble the intended on-target site. Using high-coverage whole genome sequencing (WGS), these authors reported finding SNPs and indels in two CRISPR-Cas9-treated mice that were not present in a single untreated control mouse. On the basis of this association, Schaefer et al. concluded that these sequence variants were caused by CRISPR-Cas9. This new proposed CRISPR-Cas9 off-target activity runs contrary to previously published work and, if the authors are correct, could have profound implications for research and therapeutic applications. Here we demonstrate that the simplest interpretation of Schaefer et al.9s data is that the two CRISPR-Cas9-treated mice are actually more closely related genetically to each other than to the control mouse. This strongly suggests that the so-called “unexpected mutations” simply represent SNPs and indels shared in common by these mice prior to nuclease treatment. In addition, given the genomic and sequence distribution profiles of these variants, we show that it is challenging to explain how CRISPR-Cas9 might be expected to induce such changes. Finally, we argue that the lack of appropriate controls in Schaefer et al.9s experimental design precludes assignment of causality to CRISPR-Cas9. Given these substantial issues, we urge Schaefer et al. to revise or re-state the original conclusions of their published work so as to avoid leaving misleading and unsupported statements to persist in the literature.

Motivation Unique molecular identifiers (UMIs) are added to DNA fragments before PCR amplification to discriminate between alleles arising from the same genomic locus and sequencing reads produced by PCR amplification. While computational methods have been developed to take into account UMI information in genome-wide and single-cell sequencing studies, they are not designed for modern amplicon-based sequencing experiments, especially in cases of high allelic diversity. Importantly, no guidelines are provided for the design of optimal UMI length for amplicon-based sequencing experiments. Results Based on the total number of DNA fragments and the distribution of allele frequencies, we present a model for the determination of the minimum UMI length required to prevent UMI collisions and reduce allelic distortion. We also introduce a user-friendly software tool called AmpUMI to assist in the design and the analysis of UMI-based amplicon sequencing studies. AmpUMI provides quality control metrics on frequency and quality of UMIs, and trims and deduplicates amplicon sequences with user specified parameters for use in downstream analysis. Availability and implementation AmpUMI is open-source and freely available at http://github.com/pinellolab/AmpUMI.

Recently described base editor (BE) technology, which uses CRISPR-Cas9 to direct cytidine deaminase enzymatic activity to specific genomic loci, enables the highly efficient introduction of precise cytidine-to-thymidine (C to T) DNA alterations in many different cell types and organisms. In contrast to genome-editing nucleases, BEs avoid the need to introduce double-strand breaks or exogenous donor DNA templates and induce lower levels of unwanted variable-length insertion/deletion mutations (indels). However, existing BEs can also efficiently create unwanted C to T alterations when more than one C is present within the five base pair "editing window" of these proteins, a lack of precision that can cause potentially deleterious bystander mutations. Mutations in the cytidine deaminase enzyme can shorten the length of the editing window and thereby partially address this limitation but these BE variants still do not discriminate among multiple cytidines within the narrowed window and also possess a more limited targeting range. Here, we describe an alternative strategy for reducing bystander mutations using a novel BE architecture that harbors an engineered human APOBEC3A (eA3A) domain, which preferentially deaminates cytidines according to a TCR>TCY>VCN (V = G, A, C, Y = C, T) hierarchy. In direct comparisons with the widely used BE3 fusion in human cells, our eA3A-BE3 fusion exhibits comparable activities on cytidines in TC motifs but greatly reduced or no significant editing on cytidines in other sequence contexts. Importantly, we show that eA3A-BE3 can correct a human beta-thalassemia promoter mutation with much higher (>40-fold) precision than BE3, substantially minimizing the creation of an undesirable bystander mutation. Surprisingly, we also found that eA3A-BE3 shows reduced mutation frequencies on known off-target sites of BE3, even when targeting promiscuous homopolymeric sites. Our results validate a general strategy to improve the precision of base editors by engineering their cytidine deaminases to possess greater sequence specificity, an important proof-of-principle that should motivate the development of a larger suite of new base editors with such properties.

CRISPR-Cas genome-editing nucleases hold substantial promise for human therapeutics1–5 but identifying unwanted off-target mutations remains an important requirement for clinical translation6, 7. For ex vivo therapeutic applications, previously published cell-based genome-wide methods provide potentially useful strategies to identify and quantify these off-target mutation sites8–12. However, a well-validated method that can reliably identify off-targets in vivo has not been described to date, leaving the question of whether and how frequently these types of mutations occur. Here we describe Verification of In Vivo Off-targets (VIVO), a highly sensitive, unbiased, and generalizable strategy that we show can robustly identify genome-wide CRISPR-Cas nuclease off-target effects in vivo. To our knowledge, these studies provide the first demonstration that CRISPR-Cas nucleases can induce substantial off-target mutations in vivo, a result we obtained using a deliberately promiscuous guide RNA (gRNA). More importantly, we used VIVO to show that appropriately designed gRNAs can direct efficient in vivo editing without inducing detectable off-target mutations. Our findings provide strong support for and should encourage further development of in vivo genome editing therapeutic strategies.

SUMMARYThe first cellular differentiation event in mouse development leads to the formation of the blastocyst consisting of the inner cell mass (ICM) and an outer functional epithelium called trophectoderm (TE). The lineage specific transcription factor CDX2 is required for proper TE specification, where it promotes expression of TE genes, and represses expression of Pou5f1 (OCT4) by inhibiting OCT4 from promoting its own expression. However its downstream network in the developing early embryo is not fully characterized. Here, we performed high-throughput single embryo qPCR analysis in Cdx2 null embryos to identify components of the CDX2-regulated network in vivo. To identify genes likely to be regulated by CDX2 directly, we performed CDX2 ChIP-Seq on trophoblast stem (TS) cells, derived from the TE. In addition, we examined the dynamics of gene expression changes using an inducible CDX2 embryonic stem (ES) cell system, so that we could predict which CDX2-bound genes are activated or repressed by CDX2 binding. By integrating these data with observations of chromatin modifications, we were able to identify novel regulatory elements that are likely to repress gene expression in a lineage-specific manner. Interestingly, we found CDX2 binding sites within regulatory elements of key pluripotent genes such as Pou5f1 and Nanog, pointing to the existence of a novel mechanism by which CDX2 maintains repression of OCT4 in trophoblast. Our study proposes a general mechanism in regulating lineage segregation during mammalian development.

To identify novel targets for acute myeloid leukemia (AML) therapy, we performed genome-wide CRISPR-Cas9 screening using AML cell lines, followed by a second screen in vivo. Here, we show that the mRNA decapping enzyme scavenger (DCPS) gene is essential for AML cell survival. The DCPS enzyme interacted with components of pre-mRNA metabolic pathways, including spliceosomes, as revealed by mass spectrometry. RG3039, a DCPS inhibitor originally developed to treat spinal muscular atrophy, exhibited anti-leukemic activity via inducing pre-mRNA mis-splicing. Humans harboring germline biallelic DCPS loss-of-function mutations do not exhibit aberrant hematologic phenotypes, indicating that DCPS is dispensable for human hematopoiesis. Our findings shed light on a pre-mRNA metabolic pathway and identify DCPS as a target for AML therapy. Yamauchi et al. perform in vitro and in vivo CRISPR-Cas9 genetic screening of p53 WT AML to identify potential therapeutic targets. They find that AML relies on the DCPS decapping enzyme, and a DCPS inhibitor shows anti-leukemia activity in tumor models without impacting normal hematopoiesis.

The transcription factor STAT3 is constitutively active in many cancers, where it mediates important biological effects, including cell proliferation, differentiation, survival, and angiogenesis. The N-terminal domain (NTD) of STAT3 performs multiple functions, such as cooperative DNA binding, nuclear translocation, and protein-protein interactions. However, it is unclear which subsets of STAT3 target genes depend on the NTD for transcriptional regulation. To identify such genes, we compared gene expression in STAT3-null mouse embryonic fibroblasts (MEFs) stably expressing wild-type STAT3 or STAT3 from which NTD was deleted. NTD deletion reduced the cytokine-induced expression of specific STAT3 target genes by decreasing STAT3 binding to their regulatory regions. To better understand the potential mechanisms of this effect, we determined the crystal structure of the STAT3 NTD and identified a dimer interface responsible for cooperative DNA binding in vitro. We also observed an Ni(2+)-mediated oligomer with an as yet unknown biological function. Mutations on both dimer and Ni(2+)-mediated interfaces affected the cytokine induction of STAT3 target genes. These studies shed light on the role of the NTD in transcriptional regulation by STAT3 and provide a structural template with which to design STAT3 NTD inhibitors with potential therapeutic value.

The first cellular differentiation event in mouse development leads to the formation of the blastocyst consisting of the inner cell mass (ICM) and trophectoderm (TE). The transcription factor CDX2 is required for proper TE specification, where it promotes expression of TE genes, and represses expression of Pou5f1 (OCT4). However its downstream network in the developing embryo is not fully characterized. Here, we performed high-throughput single embryo qPCR analysis in Cdx2 null embryos to identify CDX2-regulated targets in vivo. To identify genes likely to be regulated by CDX2 directly, we performed CDX2 ChIP-Seq on trophoblast stem (TS) cells. In addition, we examined the dynamics of gene expression changes using inducible CDX2 embryonic stem (ES) cells, so that we could predict which CDX2-bound genes are activated or repressed by CDX2 binding. By integrating these data with observations of chromatin modifications, we identify putative novel regulatory elements that repress gene expression in a lineage-specific manner. Interestingly, we found CDX2 binding sites within regulatory elements of key pluripotent genes such as Pou5f1 and Nanog, pointing to the existence of a novel mechanism by which CDX2 maintains repression of OCT4 in trophoblast. Our study proposes a general mechanism in regulating lineage segregation during mammalian development.

We present Bioconda (https://bioconda.github.io), a distribution of bioinformatics software for the lightweight, multiplatform and language-agnostic package manager Conda. Currently, Bioconda offers a collection of over 3000 software packages, which is continuously maintained, updated, and extended by a growing global community of more than 200 contributors. Bioconda improves analysis reproducibility by allowing users to define isolated environments with defined software versions, all of which are easily installed and managed without administrative privileges.

MotivationWith the increasing amount of genomic and epigenomic data in the public domain, a pressing challenge is how to integrate these data to investigate the role of epigenetic mechanisms in regulating gene expression and maintenance of cell-identity. To this end, we have implemented a computational pipeline to systematically study epigenetic variability and uncover regulatory DNA sequences that play a role in gene regulation.ResultsHaystack is a bioinformatics pipeline to characterize hotspots of epigenetic variability across different cell-types as well as cell-type specific cis-regulatory elements along with their corresponding transcription factors. Our approach is generally applicable to any epigenetic mark and provides an important tool to investigate cell-type identity and the mechanisms underlying epigenetic switches during development. Additionally, we make available a set of precomputed tracks for a number of epigenetic marks across several cell types. These precomputed results may be used as an independent resource for functional annotation of the human genome.AvailabilityThe Haystack pipeline is implemented as an open-source, multiplatform, Python package called haystack_bio available at https://github.com/pinellolab/haystack_bio.Contactlpinello@mgh.harvard.edu, gcyuan@jimmy.harvard.edu

Schaefer et al. recently advanced the provocative conclusion that CRISPR-Cas9 nuclease can induce off-target alterations at genomic loci that do not resemble the intended on-target site.1 Using high-coverage whole genome sequencing (WGS), these authors reported finding SNPs and indels in two CRISPR-Cas9-treated mice that were not present in a single untreated control mouse. On the basis of this association, Schaefer et al. concluded that these sequence variants were caused by CRISPR-Cas9. This new proposed CRISPR-Cas9 off-target activity runs contrary to previously published work2–8 and, if the authors are correct, could have profound implications for research and therapeutic applications. Here, we demonstrate that the simplest interpretation of Schaefer et al.’s data is that the two CRISPR-Cas9-treated mice are actually more closely related genetically to each other than to the control mouse. This strongly suggests that the so-called “unexpected mutations” simply represent SNPs and indels shared in common by these mice prior to nuclease treatment. In addition, given the genomic and sequence distribution profiles of these variants, we show that it is challenging to explain how CRISPR-Cas9 might be expected to induce such changes. Finally, we argue that the lack of appropriate controls in Schaefer et al.’s experimental design precludes assignment of causality to CRISPR-Cas9. Given these substantial issues, we urge Schaefer et al. to revise or re-state the original conclusions of their published work so as to avoid leaving misleading and unsupported statements to persist in the literature.

CRISPR genome editing experiments offer enormous potential for the evaluation of genomic loci using arrayed single guide RNAs (sgRNAs) or pooled sgRNA libraries. Numerous computational tools are available to help design sgRNAs with optimal on-target efficiency and minimal off-target potential. In addition, computational tools have been developed to analyze deep sequencing data resulting from genome editing experiments. However, these tools are typically developed in isolation and oftentimes not readily translatable into laboratory-based experiments. Here we present a protocol that describes in detail both the computational and benchtop implementation of an arrayed and/or pooled CRISPR genome editing experiment. This protocol provides instructions for sgRNA design with CRISPOR, experimental implementation, and analysis of the resulting high-throughput sequencing data with CRISPResso. This protocol allows for design and execution of arrayed and pooled CRISPR experiments in 4-5 weeks by non-experts as well as computational data analysis in 1-2 days that can be performed by both computational and non-computational biologists alike.

Chromatin-state analysis is widely applied in the studies of development and diseases. However, existing methods operate at a single length scale, and therefore cannot distinguish large domains from isolated elements of the same type. To overcome this limitation, we present a hierarchical hidden Markov model, diHMM, to systematically annotate chromatin states at multiple length scales. We apply diHMM to analyse a public ChIP-seq data set. diHMM not only accurately captures nucleosome-level information, but identifies domain-level states that vary in nucleosome-level state composition, spatial distribution and functionality. The domain-level states recapitulate known patterns such as super-enhancers, bivalent promoters and Polycomb repressed regions, and identify additional patterns whose biological functions are not yet characterized. By integrating chromatin-state information with gene expression and Hi-C data, we identify context-dependent functions of nucleosome-level states. Thus, diHMM provides a powerful tool for investigating the role of higher-order chromatin structure in gene regulation., Claudia Barr Award, National Institutes of Health (U.S.) (Grant R21HG006778), National Institutes of Health (U.S.) (Grant R01HL119099), National Institutes of Health (U.S.) (Grant K25HL133599)

Recent work has shown that RNA polymerase II mediated transcription at distal cis-regulatory elements serves as a mark of highly active enhancers. Production of non-coding RNAs at enhancers, termed eRNAs, correlates with higher expression of genes that the enhancer interacts with; hence eRNAs provide a new tool to model gene activity in normal and disease tissues. Moreover, this unique class of non-coding RNA has diverse roles in transcriptional regulation. Transcribed enhancers can be identified by a common signature of epigenetics marks by overlaying a series of genome-wide chromatin immunoprecipitation and RNA sequencing datasets. A computational approach to filter non-enhancer elements and other classes of non-coding RNAs is essential to not cloud downstream analysis. Here we present a protocol that combines wet and dry bench methods to accurately identify transcribed enhancers genome-wide as well as an experimental procedure to validate these datasets.

Background— Long noncoding RNAs (lncRNAs) have recently been implicated in many biological processes and diseases. Atherosclerosis is a major risk factor for cardiovascular disease. However, the functional role of lncRNAs in atherosclerosis is largely unknown. Methods and Results— We identified lincRNA-p21 as a key regulator of cell proliferation and apoptosis during atherosclerosis. The expression of lincRNA-p21 was dramatically downregulated in atherosclerotic plaques of ApoE −/− mice, an animal model for atherosclerosis. Through loss- and gain-of-function approaches, we showed that lincRNA-p21 represses cell proliferation and induces apoptosis in vascular smooth muscle cells and mouse mononuclear macrophage cells in vitro. Moreover, we found that inhibition of lincRNA-p21 results in neointimal hyperplasia in vivo in a carotid artery injury model. Genome-wide analysis revealed that lincRNA-p21 inhibition dysregulated many p53 targets. Furthermore, lincRNA-p21, a transcriptional target of p53, feeds back to enhance p53 transcriptional activity, at least in part, via binding to mouse double minute 2 (MDM2), an E3 ubiquitin-protein ligase. The association of lincRNA-p21 and MDM2 releases MDM2 repression of p53, enabling p53 to interact with p300 and to bind to the promoters/enhancers of its target genes. Finally, we show that lincRNA-p21 expression is decreased in patients with coronary artery disease. Conclusions— Our studies identify lincRNA-p21 as a novel regulator of cell proliferation and apoptosis and suggest that this lncRNA could serve as a therapeutic target to treat atherosclerosis and related cardiovascular disorders.

Tiling screens using CRISPR-Cas technologies provide a powerful approach to map regulatory elements to phenotypes of interest, but computational methods that effectively model these experimental approaches for different CRISPR technologies are not readily available. Here we present CRISPR-SURF, a deconvolution framework to identify functional regulatory regions in the genome from data generated by CRISPR-Cas nuclease, CRISPR interference (CRISPRi), or CRISPR activation (CRISPRa) tiling screens. We validated CRISPR-SURF on previously published and new data, identifying both experimentally validated and new potential regulatory elements. With CRISPR tiling screens now being increasingly used to elucidate the regulatory architecture of the non-coding genome, CRISPRSURF provides a generalizable and accessible solution for the discovery of regulatory elements.

Acute myeloid leukemia (AML) is a devastating disease with a long-term survival rate of less than 30%. While AML is a genetically heterogeneous disease, TP53 mutation is among the most powerful risk factors in AML, underscoring the critical need to devise a novel therapeutic strategy for TP53-mutated AML. To identify genes/pathways whose loss are vulnerable to TP53 deficiency in AML cells, we performed genome-wide CRISPR-Cas9 screens using Trp53-knockout (KO) and wild-type (WT) mouse AML cells. To generate AML lines with a relatively "clean" genetic background, we established mouse AML lines harboring WT Trp53 with normal karyotype (Yamauchi et al. Cancer Cell 2018). We then generated Trp53-KO AML lines using a single guide RNA (sgRNA) targeting Trp53. Genome-wide CRISPR-Cas9 dropout screens were performed using these lines to identify genes/pathways whose loss are vulnerable to TP53 deficiency. Importantly, sgRNAs targeting Trp53 were enriched only in Trp53-WT AML cells, but not in Trp53-KO cells. Furthermore, sgRNAs targeting Mdm2, which encodes a E3 ubiquitin-protein ligase for Trp53 protein, were depleted only in Trp53-WT cells, attesting to the validity of our experimental system. We identified Xpo7, a putative nuclear/cytoplasmic transporter, as a factor necessary for the survival of Trp53-KO AML cells. sgRNAs targeting Xpo7 were enriched in Trp53-WT AML cells after the 16-day culture (Yamauchi et al. Cancer Cell 2018), while they were significantly depleted in Trp53-KO cells. Trp53-KO cells were vulnerable to Xpo7 depletion, while Xpo7 functioned as a Trp53-dependent tumor suppressor in Trp53-WT AML cells. As expected, either CRISPR/Cas9-mediated Xpo7 depletion or shRNA-mediated Xpo7 knockdown significantly delayed cell cycle progression and suppressed cell growth only in Trp53-KO AML cells, but not in WT cells. These findings were also validated using the DepMap (https://depmap.org), a publicly-available CRISPR/Cas9 dropout screen dataset. Significantly, XPO7 dependency was most correlated with that of TP53 and particularly evident in TP53-WT cell lines. We next performed CRISPR-Cas9 saturation mutagenesis scan targeting all Xpo7 exons using Trp53-WT and -KO mouse AML cells. Strikingly, sgRNAs targeting Xpo7 coding regions were significantly enriched only in the Trp53-WT cells, namely in the presence of an MDM2 inhibitor, supporting the notion that Xpo7 could function as a tumor suppressor when Trp53 function is intact and evident. In a stark contrast, the same sgRNAs, which target coding exons, were mostly depleted in Trp53-KO AMLs. Importantly, sgRNAs targeting the Xpo7 UTRs were unchanged/neutral regardless of experimental conditions (e.g. Trp53 status, MDM2 inhibitor treatment), serving as valid control for the experiments. Since Xpo7 presumably mediates the nuclear import and export of proteins, we hypothesized that Xpo7 promotes retention of Trp53 protein in the nucleus in Trp53-WT AML cells. As expected, Trp53 protein levels in the nucleus were significantly decreased, while those in the cytoplasm were increased upon Xpo7 depletion, revealed by Western blot. These data suggest that Xpo7 retains WT-Trp53 in the nucleus and functions as a Trp53-dependent tumor suppressor in Trp53-WT AML. Finally, to explore functional significance of XPO7 in human AML, we assessed correlation between XPO7 expression levels and AML subtypes and/or genetic background using publicly-available datasets. XPO7 mRNA levels were significantly upregulated in TP53-mutated AMLs in the TCGA datasets (Ley et al. NEJM 2013). XPO7 mRNAs levels were remarkably high in acute erythroid leukemia (AEL) cases, where TP53 mutations are frequently observed (Tyner et al. Nature 2018 and Iacobucci et al. Nat Genet. 2019). In fact, shRNA-mediated XPO7 knockdown significantly suppressed proliferation of HEL, a human AEL cell line harboring TP53 mutation. In summary, we identified a synthetic lethal relationship between TP53 and XPO7. Our study may facilitate the development of novel therapeutic strategies for TP53-mutated AML, such as AEL. Disclosures Akashi: Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding; Sumitomo Dainippon, Kyowa Kirin: Consultancy.

Mouse models represent invaluable tools for the systematic evaluation of cancer drivers, yet models that address the impact of putative genetic drivers of chronic lymphocytic leukemia (CLL) on B cell development and function are largely lacking. To study recurrent loss-of-function (LOF) mutations observed in human CLL, we established a transplant model that can rapidly evaluate genetic lesions. First, we crossed mice carrying B-cell restricted Cre expression (Cd19-cre) with mice carrying conditional Cas9-GFP, to generate a strain expressing B cell-restricted Cas9 (Cd19-Cas9). Next, we optimized methods for in vitro engineering of early stem and progenitor cells (Lin- Sca-1+ c-kit+ [LSK]) from Cd19-Cas9 mice using lentivirus expressing sgRNAs (mCherry+)targeting Atm, Trp53, Chd2, Birc3, Mga, or Samhd1. We chose LSKs because of their high transducibility and long-term repopulating potential. Last, we transplanted the single sgRNA-expressing LSKs into sub-lethally irradiated CD45.1 recipient mice, and then confirmed presence of ~45-85% gene-edited sequences (>70% carrying frameshift mutations) in edited B cells (GFP+mCherry+) at 2 months post-transplant, by PCR-based targeted deep sequencing and CRISPResso software analysis. We also verified presence of gene alterations (and putative off-target lesions) at the single cell DNA level (targeted sequencing by Tapestri, Mission Bio). We first asked whether presence of the 6 LOFs could impact B cell developmental trajectories in marrow, spleen and peritoneum at 4 months post-transplant, a time point by which B cells are considered to achieve optimal host reconstitution (n=5/group, including a non-targeting control group). No marked changes were observed in mice with Atmindel, Trp53indel, Chd2indel, Birc3indel or Samhd1indel, as analyzed by flow cytometry. Of interest, however, Mgaindel mice were detected to have increased germinal center (B220+CD95+CD38-) and marginal zone (B220+CD21highCD23-) splenic B cells, and also showed increased B1a (CD5+ B220low CD23- CD43+) and decreased B1b (CD5- B220low CD23- CD43+) cells in the peritoneum (p The overall abundance of edited B cells in spleen and blood of each group was higher (overall median: 17.0%; 90%CI 6.7-58.8%) than the non-targeting control (8.4%; 90%CI 1.6-14.2%) at 4 months post-transplant (n=8/group, p To address this question, we analyzed the transcriptional profiles of edited B cell splenocytes (n=3/group), and compared them to their non-edited counterparts (GFP+mCherry- splenocytes from the same animal), identifying a total of ~3900 differentially expressed genes among the 6 groups (p0.37 for each of the 10 pairs of 5 groups), with the exception of Mgaindel, consistent with its unique phenotype, observed in developmental studies. Gene ontology analyses using Enrichr confirmed commonalities in pathway dysregulations across the 5 similar groups of mice (p In conclusion, we demonstrate that common LOFs typical of patients with CLL lead to increased cellular fitness in B-cell restricted mouse models, while dysregulating pro-survival pathways relevant to B cell development, CLL pathogenesis and more broadly to tumorigenesis. We are currently exploring phenotypic similarities and differences through tailored functional assays, while addressing the relative contribution of each alteration to CLL development in multiplexed edited mouse lines. Disclosures Wang: Mission Bio Inc.: Employment. Jacob:Mission Bio Inc.: Employment. Flynn:Mission Bio Inc.: Employment. Ruff:Mission Bio Inc.: Employment. Jones:Mission Bio Inc.: Employment. Neuberg:Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Wu:Neon Therapeutics: Other: Member, Advisory Board; Pharmacyclics: Research Funding.

Severe congenital neutropenia (SCN) is a life-threatening disorder of insufficient granulocytes. Lifelong granulocyte colony-stimulating factor (G-CSF) injections are the mainstay of treatment, yet there remains a high risk of myelodysplastic syndrome and acute myeloid leukemia. The most common etiology of SCN is germline ELANE mutation. These dominantly acting mutations preserve expression but alter the structure of the neutrophil elastase protein product resulting in altered protein folding and/or trafficking with excess cell death at the promyelocyte/myelocyte stage of maturation. Recent advances in gene editing technologies have enabled targeted genetic modification of hematopoietic stem cells (HSCs); nonetheless genetic repair of specific disease-associated mutations remains challenging. We hypothesized that introduction of premature termination codons (PTCs) by nuclease-mediated frameshift mutations within early exons of ELANE could constitute a universal, highly efficient, simple therapeutic approach for ELANE-associated SCN. We predicted that the PTCs would trigger nonsense mediated decay (NMD) of the mutant transcript resulting in its loss of expression and thus bypassing neutrophil precursor cell death and consequent neutropenia. The mild phenotype observed in the Papillon-Lefevre syndrome, characterized by combined serine protease deficiency, suggests that isolated neutrophil elastase deficiency would not result in clinically significant immunodeficiency. We delivered 3xNLS-SpCas9 and ELANE targeting sgRNA as ribonucleoprotein (RNP) complexes to primary human CD34+ hematopoietic stem and progenitor cells (HSPCs) and conducted in vitro neutrophil maturation culture. Introducing indels at exon 2 of ELANE efficiently triggered NMD. Edited cells were fully competent for neutrophil maturation similar to neutral locus targeted control cells. Using three human donors, we found that ELANE exon 2 edited HSPCs produced similar human bone marrow (BM) chimerism as unedited cells in NBSGW recipient mice 16 weeks following infusion. We found similar lymphoid, erythroid, and myeloid engraftment including similar fraction of human neutrophils (13.4% of total human BM cells in unedited and 13.7% in ELANE exon 2 edited, despite 97.3% on-target indel frequency and 84.3% reduction in ELANE expression in the latter). Using CD34+ HSPCs from four ELANE mutant SCN patient donors, we demonstrated that exon 2 targeting RNPs achieve highly efficient editing exceeding 95% indel frequency, trigger ELANE transcript decay, and rescue promyelocyte stage maturation arrest. In contrast to these ameliorating early exon frameshifts, naturally occurring SCN-associated frameshifts affect late exons of ELANE, suggesting that these mutations might escape NMD. Indeed we found that targeting ELANE exon 5 in HSPCs resulted in robust indels (93.5%), preserving ELANE expression but resulting in cell death at the promyelocyte/myelocyte stages of development, recapitulating an SCN phenotype. To our surprise, we found that only -1 frameshifts and not -2 frameshifts induced by gene editing with NHEJ repair led to the SCN-like phenotype, although we noted that all 23 reported naturally occurring SCN-associated ELANE frameshift mutations result from -1 but not -2 bp frameshifts. Using xenograft of NBSGW recipients, we found that an RNP complex leading to efficient -1 frame indels in ELANE exon 5 produced profound neutrophil maturation block, with reduction from 13.4% neutrophils in controls to 0.5% neutrophils in ELANE exon 5 targeted recipients, with otherwise indistinguishable human monocyte, lymphoid, and erythroid reconstitution as compared to controls. This dramatic phenotype contrasts with mice engineered to express SCN-associated Elane mutations that do not exhibit neutropenia, indicating species differences in granulopoiesis. Together these results support the development of ELANE early exon targeting as a highly efficient universal therapy for ELANE mutant SCN, feasible with existing gene editing technology. Moreover, by late exon ELANE gene editing we have developed a robust new model of SCN using primary human HSPCs that recapitulates neutropenia in vivo following xenotransplant, refines the molecular genetics of mutant ELANE induced neutrophil maturation arrest, and offers opportunities to explore novel therapeutic approaches. Disclosures Newburger: TransCytos LLC: Consultancy; X4 Pharmaceuticals: Consultancy, Honoraria.

Progress has been made in deciphering molecular mechanisms underlying AML pathogenesis due in part to near-complete understanding of AML genomes. However, AML is yet a devastating disease with a long-term survival rate of less than 30%, underscoring an urgent need for the development of additional therapeutic modalities. To identify novel targets for AML therapy, we performed genome-wide CRISPR-Cas9 dropout screens employing two mouse AML cell lines (CALM/AF10 and MLL/AF9), followed by a second screen in vivo. These two cell lines, which we established, harbor wild-type (WT) Trp53with normal karyotype, enabling us to interpret screening results more easily due to a "clean" genetic background. We then validated our findings using human AML cell lines and patient-derived xenograft (PDX) models (Yamauchi et al. Cancer Cell 2018). In the current study, we assessed the screening results furtherusing MAGeCK MLE program (Li et al. Genome Biology 2015)and the DepMap (https://depmap.org/), a publicly available genome-wide CRISPR-Cas9 screen datasets of cancer cell lines including 15 human AML cell lines. We show that PAICS (Phosphoribosylaminoimidazole carboxylase), which encodes an enzyme involved in de novo purine biosynthesis, is a molecule essential for AML cell survival. MRT252040, a newly-developed PAICS inhibitor (PAICSi), efficiently killed AML cell lines with different genetic backgrounds and significantly prolonged survival of AML PDX models. Furthermore, we investigated the mechanism action of PAICSi employing additional functional screens: CRISPR-Cas9 mutagenesis scan of all Paicscoding exons and a genome-wide CRISPR/Cas9 dropout screen in the presence of PAICSi. Read counts for each Paics-targeted single-guide RNA (sgRNA) significantly decreased in vitro (AML cell lines) and in vivo (mouse AML model). We then assessed the functional significance of PAICS inhibition in AML cell survival via shRNA-mediated PAICSknockdown. AML cells expressing PAICS shRNA exhibited a proliferative disadvantage compared to non-transduced cells or those expressing scrambled shRNA, indicating a toxic effect of PAICS depletion in AML cells. We next asked whether inhibition of PAICS enzymatic activity affects AML cell proliferation and/or apoptosis using PAICSi. We assessed AML growth rate, cell cycle status and apoptosis and found that inhibition of PAICS enzymatic activity delays AML cell proliferation via inducing cell cycle arrest and apoptosis. As expected, CRISPR-Cas9 mutagenesis scan showed that sgRNAs targeting the exonic regions relevant to PAICS enzymatic activity were significantly decreased after the 16-day incubation. We next performed genome-wide CRISPR-Cas9 screens in the presence of PAICSi, followed by second screens using a small-scale sgRNA library containing 8-10 sgRNAs per candidate gene.We identified genes potentially involved in PAICSi resistance as well as those whose loss are synthetic lethal to PAICS inhibition. X-box-binding protein 1 (Xbp1) was among the top hits in the genes relevant to PAICSi resistance genes, and sgRNAs targeting Xbp1significantly enriched in the presence of PAICSi. In contrast, sgRNAs targeting Slc43a3or Hprt, both of which are implicated in the purine salvage pathway, were significantly dropped-out, indicating that PAICSi-mediated anti-leukemia effects can be enhanced upon concurrentinhibition of the purine salvage pathway. Finally, we explored potential anti-leukemia effects of PAICSi in vivo using AML PDX models established from two human AML lines. PAICSi exhibited anti-leukemic activity, as evidenced by the lower leukemia burden in peripheral blood and bone marrow of PAICSi-treated mice. They survived significantly longer than vehicle-treated mice, indicative of therapeutic efficacy of PAICSimonotherapy against AML in vivo. In summary, we identified PAICS as an essential gene for AML cell survival. We propose that pharmacological targeting of the de-novo purine synthesis pathway via PAICSi is a potential therapeutic strategy for AML therapy. Disclosures Akashi: Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding; Sumitomo Dainippon, Kyowa Kirin: Consultancy.

Summary Self-renewal and pluripotency of the embryonic stem cell (ESC) state are established and maintained by multiple regulatory networks that comprise transcription factors and epigenetic regulators. While much has been learned regarding transcription factors, the function of epigenetic regulators in these networks is less well defined. We conducted a CRISPR-Cas9-mediated loss-of-function genetic screen that identified two epigenetic regulators, TAF5L and TAF6L, components or co-activators of the GNAT-HAT complexes for the mouse ESC (mESC) state. Detailed molecular studies demonstrate that TAF5L/TAF6L transcriptionally activate c-Myc and Oct4 and their corresponding MYC and CORE regulatory networks. Besides, TAF5L/TAF6L predominantly regulate their target genes through H3K9ac deposition and c-MYC recruitment that eventually activate the MYC regulatory network for self-renewal of mESCs. Thus, our findings uncover a role of TAF5L/TAF6L in directing the MYC regulatory network that orchestrates gene expression programs to control self-renewal for the maintenance of mESC state.

Developmental silencing of fetal globins serves as both a paradigm of spatiotemporal gene regulation and an opportunity for β-hemoglobinopathy therapeutic intervention. The NuRD chromatin complex participates in γ-globin repression. Here we use pooled CRISPR screening to comprehensively disrupt NuRD protein coding sequences in human adult erythroid precursors. We find essential for fetal hemoglobin (HbF) control a nonredundant subcomplex of NuRD protein family paralogs, whose composition we corroborate by affinity chromatography and proximity labeling mass spectrometry proteomics. Mapping top functional guide RNAs identifies key protein interfaces where in-frame alleles result in loss-of-function due to destabilization or altered function of subunits. We ascertain mutations of CHD4 that dissociate its requirement for cell fitness from HbF repression in both primary human erythroid precursors and transgenic mice. Finally we demonstrate that sequestering CHD4 from NuRD phenocopies these mutations. This work indicates a generalizable approach to discover protein complex features amenable to rational biochemical targeting., Editorial summary: Comprehensive CRISPR mutagenesis targeting all members of the NuRD complex identifies a specific sub-complex required for fetal globin silencing and informs a rational targeting strategy for elevating globin levels while avoiding cytotoxicity.

Inappropriate activation of the transcription factors STAT3 and STAT5 has been shown to drive cancer pathogenesis through dysregulation of genes involved in cell survival, growth, and differentiation. Although STAT3 and STAT5 are structurally related, they can have opposite effects on key genes, including BCL6. BCL6, a transcriptional repressor, has been shown to be oncogenic in diffuse large B cell lymphoma. BCL6 also plays an important role in breast cancer pathogenesis, a disease in which STAT3 and STAT5 can be activated individually or concomitantly. To determine the mechanism by which these oncogenic transcription factors regulate BCL6 transcription, we analyzed their effects at the levels of chromatin and gene expression. We found that STAT3 increases expression of BCL6 and enhances recruitment of RNA polymerase II phosphorylated at a site associated with transcriptional initiation. STAT5, in contrast, represses BCL6 expression below basal levels and decreases the association of RNA polymerase II at the gene. Furthermore, the repression mediated by STAT5 is dominant over STAT3-mediated induction. STAT5 exerts this effect by displacing STAT3 from one of the two regulatory regions to which it binds. These findings may underlie the divergent biology of breast cancers containing activated STAT3 alone or in conjunction with activated STAT5.

Invariant natural killer T cells (iNKT cells) are innate-like lymphocytes that protect against infection, autoimmune disease and cancer. However, little is known about the epigenetic regulation of iNKT cell development. Here we found that the H3K27me3 histone demethylase UTX was an essential cell-intrinsic factor that controlled an iNKT-cell lineage-specific gene-expression program and epigenetic landscape in a demethylase-activity-dependent manner. UTX-deficient iNKT cells exhibited impaired expression of iNKT cell signature genes due to a decrease in activation-associated H3K4me3 marks and an increase in repressive H3K27me3 marks within the promoters occupied by UTX. We found that JunB regulated iNKT cell development and that the expression of genes that were targets of both JunB and the iNKT cell master transcription factor PLZF was UTX dependent. We identified iNKT cell super-enhancers and demonstrated that UTX-mediated regulation of super-enhancer accessibility was a key mechanism for commitment to the iNKT cell lineage. Our findings reveal how UTX regulates the development of iNKT cells through multiple epigenetic mechanisms.

Recent progress in genome editing technologies, in particular the CRISPR-Cas9 system, has provided new opportunities to investigate the biological functions of genomic sequences by targeted mutagenesis. Double strand breaks (DSBs) resulting from site-specific Cas9 cleavage can be resolved by endogenous DNA repair pathways such as non-homologous end joining (NHEJ) or homology-directed repair (HDR). Deep sequencing of amplified genomic regions allows for quantitative and sensitive detection of targeted mutations. However, no standard analytic tool to date has been developed to systematically enumerate and visualize these events, and to solve challenging issues such as amplification or sequencing errors, experimental variation in sequence quality, ambiguous alignment of variable length indels, and difficulty in deconvoluting mixed HDR/NHEJ outcomes. To address these issues we developed CRISPResso, a robust computational pipeline that enables accurate quantification and visualization of CRISPR-Cas9 outcomes as well as comprehensive evaluation of effects on coding sequences, noncoding elements and selected off-target sites.

Background. Numerous human genes encode potentially active DNA transposases or recombinases, but our understanding of their functions remains limited due to shortage of methods to profile their activities on endogenous genomic substrates. Results. To enable functional analysis of human transposase-derived genes, we combined forward chemical genetic hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) screening with massively parallel paired-end DNA sequencing and structural variant genome assembly and analysis. Here, we report the HPRT1 mutational spectrum induced by the human transposase PGBD5, including PGBD5-specific signal sequences (PSS) that serve as potential genomic rearrangement substrates. Conclusions. The discovered PSS motifs and high-throughput forward chemical genomic screening approach should prove useful for the elucidation of endogenous genome remodeling activities of PGBD5 and other domesticated human DNA transposases and recombinases.

High-throughput single-cell technologies have great potential to discover new cell types; however, it remains challenging to detect rare cell types that are distinct from a large population. We present a novel computational method, called GiniClust, to overcome this challenge. Validation against a benchmark dataset indicates that GiniClust achieves high sensitivity and specificity. Application of GiniClust to public single-cell RNA-seq datasets uncovers previously unrecognized rare cell types, including Zscan4-expressing cells within mouse embryonic stem cells and hemoglobin-expressing cells in the mouse cortex and hippocampus. GiniClust also correctly detects a small number of normal cells that are mixed in a cancer cell population. Electronic supplementary material The online version of this article (doi:10.1186/s13059-016-1010-4) contains supplementary material, which is available to authorized users.

The evolutionarily conserved ATP-dependent nucleosome remodelling factor ISWI can space nucleosomes affecting a variety of nuclear processes. In Drosophila, loss of ISWI leads to global transcriptional defects and to dramatic alterations in higher-order chromatin structure, especially on the male X chromosome. In order to understand if chromatin condensation and gene expression defects, observed in ISWI mutants, are directly correlated with ISWI nucleosome spacing activity, we conducted a genome-wide survey of ISWI binding and nucleosome positioning in wild-type and ISWI mutant chromatin. Our analysis revealed that ISWI binds both genic and intergenic regions. Remarkably, we found that ISWI binds genes near their promoters causing specific alterations in nucleosome positioning at the level of the Transcription Start Site, providing an important insights in understanding ISWI role in higher eukaryote transcriptional regulation. Interestingly, differences in nucleosome spacing, between wild-type and ISWI mutant chromatin, tend to accumulate on the X chromosome for all ISWI-bound genes analysed. Our study shows how in higher eukaryotes the activity of the evolutionarily conserved nucleosome remodelling factor ISWI regulates gene expression and chromosome organization genome-wide.

Although therapeutic genome editing of autologous hematopoietic stem cells (HSCs) in principle could cure β-hemoglobinopathies, CRISPR-Cas9 mediated gene modification has demonstrated variable efficiency, specificity, and persistence in HSCs. Here we demonstrate selection-free on-target editing of the BCL11A erythroid enhancer by Cas9:sgRNA ribonucleoprotein in patient-derived HSCs as a nearly complete reaction lacking detectable genotoxicity or deleterious impact on stem cell function. First we screened a set of 20 guide RNAs targeting the functional core of the +58 BCL11A enhancer for maximal HbF induction by RNP delivery. We used SpCas9 protein with additional NLS sequences, synthetic modified sgRNA, and optimized electroporation buffer to produce >95% on-target indels disrupting a critical GATA1 binding site within the +58 BCL11A enhancer in CD34+ HSPCs. Clonal analysis showed that even 1 bp indels around the cleavage site were sufficient for HbF reactivation. Specificity was evaluated by CIRCLE-seq, a method to define genome-wide target sequences susceptible to RNP cleavage in vitro. Amplicon deep sequencing of 24 possible off-target sites from edited CD34+ cells did not reveal any off-target editing with limit of detection 0.1% allele frequency. Despite transient induction of a p53 transcriptional response peaking at 4-8 hours after RNP electroporation, we found no evidence of selection for TP53 or 94 other hematologic malignancy associated mutations by targeted deep sequencing. Edited CD34+ HSPCs from healthy donors contributed to multilineage engraftment of primary and secondary immunodeficient mouse recipients at similar levels as unedited control cells. Likewise we found that edited CD34+ HSPCs from a plerixafor-mobilized SCD donor contributed to marrow engraftment and multilineage hematopoiesis in immunodeficient NBSGW mice after 16 weeks at similar levels as unedited cells, with ~95% indel frequency for engrafting healthy and SCD donor cells. Edited engrafting SCD cells were similarly competent for secondary transplantation as unedited controls. Erythroid progeny of edited engrafting sickle cell disease HSCs expressed therapeutic levels of fetal hemoglobin (HbF) and resisted sickling. Erythroid progeny of edited CD34+ HSPCs from 7 transfusion-dependent ß-thalassemia donors showed restored globin chain balance and amelioration of microcytosis and poikilocytosis. We found that compared to the bulk CD34+ HSPC pool, HSCs preferentially underwent nonhomologous as compared to microhomology mediated end-joining repair based on three assays: sorting of CD34+ CD38- CD90+ CD45RA- cells; isolation of cells in G0, G1, S, and G2/M cell cycle phases; and evaluation of long-term engrafting cells in immunodeficient mice. Together these data show that NHEJ-based BCL11A enhancer editing approaching complete allelic disruption is a practicable therapeutic strategy to produce durable HbF induction in SCD and ß-thalassemia. Disclosures Esrick: Bluebird Bio: Honoraria. Williams:Bluebird Bio: Research Funding.

Sickle cell disease and β-thalassemia are major hemoglobin disorders for which induction of fetal hemoglobin (HbF) can mitigate disease severity. However, the molecular mechanisms underlying the developmental repression of HbF remain incompletely understood. The nucleosome remodeling and deacetylase (NuRD) complex is a major negative regulator of HbF level. In this study, we sought to identify possible rational therapeutic strategies targeting critical NuRD determinants. We employed comprehensive dense mutagenesis using pooled CRISPR screening in HUDEP-2 human erythroid precursors to disrupt protein coding sequences of all 13 genes of the NuRD complex, including CHD, MTA, GATAD2, HDAC, MBD, and RBBP family members. The custom sgRNA library included 5,038 sgRNAs. We found that only 5 genes, CHD4, MTA2, GATAD2A, HDAC2, and MBD2, were required for HbF repression, suggesting that a non-redundant NuRD sub-complex contributes to HbF silencing. We validated the existence of this NuRD sub-complex by mass spectrometry analysis after immunoprecipitation of CHD4 and MTA2 as well as MTA2-BioID2 mediated proximity labeling. Remarkably, 5 of the 6 NuRD subunit proteins commonly detected by these three methods were identified as functional by CRISPR screening (MTA2, RBBP4, CHD4, GATAD2A, HDAC2). Disruption of CHD4 resulted in the highest HbF induction of any of the NuRD subunits. However, unlike the other NuRD genes, CHD4 disruption also led to cellular toxicity. We observed a small group of sgRNAs within the CHDCT2 domain of CHD4 associated with high HbF induction yet relatively modest negative fitness. We validated by electroporation of Cas9:sgRNA to CD34+ HSPC primary erythroid precursors that in-frame mutations of CHD4 CHDCT2 escape cellular toxicity while inducing HbF. Similarly, we targeted homologous amino acid residues within mouse Chd4 CHDCT2 domain by Cas9 mutagenesis in mouse oocytes. While loss of Chd4 is lethal at the blastocyst stage, homozygous in-frame deletions within the Chd4 CHDCT2 domain are tolerated in mouse embryos and result in increased γ-globin expression in mid-gestation embryos bearing transgenic human β-globin gene clusters. To investigate the mechanism whereby in-frame deletions at CHD4 CHDCT2 impact NuRD, we performed glycerol gradient density sedimentation, which revealed that these in-frame mutations impair the recruitment of CHD4 to the NuRD complex. A recent study demonstrated that the previously poorly characterized CHD4 CHDCT2 domain directly binds to GATAD2 factors (Torrado et al, FEBS J, 2017). We observed a cluster of sgRNAs associated with heightened HbF enrichment scores at the C-terminal region of GATAD2A encompassing a C2C2-type GATA zinc finger. We hypothesized that ectopic expression of this GATAD2A zinc finger might competitively bind to CHD4 and displace CHD4 from NuRD. Overexpression of the GATAD2A zinc finger in both HUDEP-2 and CD34+ HSPC derived primary erythroid precursors led to robust induction of HbF without negatively impacting cellular fitness. Immunoprecipitation of the GATAD2A zinc finger enriched CHD4 but not other endogenous NuRD components, such as GATAD2A or MBD2. Moreover, glycerol gradient density sedimentation showed that the GATAD2A zinc finger co-sedimented with sub-NuRD fractions of CHD4. Together these data suggest that expression of the GATAD2A zinc finger sequesters CHD4 from NuRD, yet spares cytotoxicity. In summary, we show that biochemical disruption of the CHD4-GATAD2A interaction could serve as a rational therapeutic strategy to potently induce HbF for the β-hemoglobin disorders while preventing cellular toxicity associated with complete CHD4 inhibition. Disclosures No relevant conflicts of interest to declare.

Discovery of molecular mechanisms responsible for trait associations as discovered by genome-wide association studies (GWAS) is hampered by difficulty in identifying causal genetic variants due to linkage disequilibrium. Typical assays of genetic function are low throughput or evaluate sequences in heterologous ectopic settings. Genome editing enables perturbation of trait-associated genetic sequences within relevant genomic, chromatin and cellular context. Here we perform comprehensive analysis of genetic variants associated with red blood cell traits by pooled CRISPR screening. We performed a genome-wide Cas9 gene knockout screen in immortalized erythroid precursors (HUDEP-2 cells) during erythroid maturation to define functional erythroid genes required for cell growth or differentiation. We evaluated 952 loci associated with nine red blood cell traits (Astle et al, Cell 2016) comprising 24,843 SNPs. We linked 7,187 (28.9%) of these SNPs to genes by at least one of four routes: sharing topological associated domain, physical proximity ( Disclosures No relevant conflicts of interest to declare.

In Fig. 4e of this Article, the labels for ‘Control’ and ‘HFD’ were reversed (‘Control’ should have been labelled blue rather than purple, and ‘HFD’ should have been labelled purple rather than blue). Similarly, in Fig. 4f of this Article, the labels for ‘V’ and ‘GW’ were reversed (‘V’ should have been labelled blue rather than purple, and ‘GW’ should have been labelled purple instead of blue). The original figure has been corrected online.

The basic unit of eukaryotic chromatin is the nucleosome, consisting of about 150 bp of DNA wrapped around a protein core made of histone proteins. Nucleosomes position is modulated in vivo to regulate fundamental nuclear processes. To measure nucleosome positions on a genomic scale both theoretical and experimental approaches have been recently reported. We have developed a new method, Multi-Layer Model (MLM), for the analysis of nucleosome position data obtained with microarray-based approach. The MLM is a feature extraction method in which the input data is processed by a classifier to distinguish between several kinds of patterns. We applied our method to simulated-synthetic and experimental nucleosome position data and found that besides a high nucleosome recognition and a strong agreement with standard statistical methods, the MLM can identify distinct classes of nucleosomes, making it an important tool for the genome wide analysis of nucleosome position and function. In conclusion, the MLM allows a better representation of nucleosome position data and a significant reduction in computational time.

Bivalent promoters in embryonic stem cells (ESCs) carry methylation marks on two lysine residues, K4 and K27, in histone3 (H3). K4me2/3 is generally considered to promote transcription, and Polycomb Repressive Complex 2 (PRC2) places K27me3, which is erased at lineage-restricted genes when ESCs differentiate in culture. Molecular defects in various PRC2 null adult tissues lack a unifying explanation. We found that epigenomes in adult mouse intestine and other self-renewing tissues show fewer and distinct bivalent promoters compared to ESCs. Groups of tissue-specific genes that carry bivalent marks are repressed, despite the presence of promoter H3K4me2/3. These are the predominant genes de-repressed in PRC2-deficient adult cells, where aberrant expression is proportional to the H3K4me2/3 levels observed at their promoters in wild-type cells. Thus, in adult animals, PRC2 specifically represses genes with acquired, tissue-restricted promoter bivalency. These findings provide new insights into specificity in chromatin-based gene regulation.

Recent progress in genome editing technologies, in particular the CRISPR-Cas9 system, has provided new opportunities to investigate the biological functions of genomic sequences by targeted mutagenesis. Double strand breaks (DSBs) resulting from site-specific Cas9 cleavage can be resolved by endogenous DNA repair pathways such as non-homologous end joining (NHEJ) or homology-directed repair (HDR). Deep sequencing of amplified genomic regions allows for quantitative and sensitive detection of targeted mutations. However, no standard analytic tool to date has been developed to systematically enumerate and visualize these events, and to solve challenging issues such as amplification or sequencing errors, experimental variation in sequence quality, ambiguous alignment of variable length indels, and difficulty in deconvoluting mixed HDR/NHEJ outcomes. To address these issues we developed CRISPResso, a robust computational pipeline that enables accurate quantification and visualization of CRISPR-Cas9 outcomes as well as comprehensive evaluation of effects on coding sequences, noncoding elements and selected off-target sites.

Background The cohesin complex consists of multiple core subunits that play critical roles in mitosis and transcriptional regulation. The cohesin-associated protein Wapal plays a central role in off-loading cohesin to facilitate sister chromatid separation, but its role in regulating mammalian gene expression is not understood. We used embryonic stem cells as a model, given that the well-defined transcriptional regulatory circuits were established through master transcription factors and epigenetic pathways that regulate their ability to maintain a pluripotent state. Results RNAi-mediated depletion of Wapal causes a loss of pluripotency, phenocopying loss of core cohesin subunits. Using chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq), we determine that Wapal occupies genomic sites distal to genes in combination with CTCF and core cohesin subunits such as Rad21. Interestingly, genomic sites occupied by Wapal appear enriched for cohesin, implying that Wapal does not off-load cohesin at regions it occupies. Wapal depletion induces derepression of Polycomb group (PcG) target genes without altering total levels of Polycomb-mediated histone modifications, implying that PcG enzymatic activity is preserved. By integrating ChIP-seq and gene expression changes data, we identify that Wapal binding is enriched at the promoters of PcG-silenced genes and is required for proper Polycomb repressive complex 2 (PRC2) recruitment. Lastly, we demonstrate that Wapal is required for the interaction of a distal cis-regulatory element (CRE) with the c-Fos promoter. Conclusions Collectively, this work indicates that Wapal plays a critical role in silencing of PcG target genes through the interaction of distal CREs with promoters. Electronic supplementary material The online version of this article (doi:10.1186/s13072-016-0063-7) contains supplementary material, which is available to authorized users.

Genome-wide mapping of three dimensional chromatin organization is an important yet technically challenging task. To aid experimental effort and to understand the determinants of long-range chromatin interactions, we have developed a computational model integrating Hi-C and histone mark ChIP-seq data to predict two important features of chromatin organization: chromatin interaction hubs and topologically associated domain (TAD) boundaries. Our model accurately and robustly predicts these features across datasets and cell types. Cell-type specific histone mark information is required for prediction of chromatin interaction hubs but not for TAD boundaries. Our predictions provide a useful guide for the exploration of chromatin organization. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0740-z) contains supplementary material, which is available to authorized users.

Little is known about how pro-obesity diets regulate tissue stem and progenitor cell function. Here we find that high fat diet (HFD)-induced obesity augments the numbers and function of Lgr5[superscript +] intestinal stem-cells (ISCs) of the mammalian intestine. Mechanistically, HFD induces a robust peroxisome proliferator-activated receptor delta (PPAR-d) signature in intestinal stem and (nonISC) progenitor cells, and pharmacologic activation of PPAR-d recapitulates the effects of a HFD on these cells. Like a HFD, ex vivo treatment of intestinal organoid cultures with fatty acid constituents of the HFD enhances the self-renewal potential of these organoid bodies in a PPAR-d dependent manner. Interestingly, HFD- and agonist-activated PPAR-d signaling endow organoidinitiating capacity to progenitors, and enforced PPAR-d signaling permits these progenitors to form in vivo tumors upon loss of the tumor suppressor Apc. These findings highlight how dietmodulated PPAR-d activation alters not only the function of intestinal stem and progenitor cells, but also their capacity to initiate tumors., Howard Hughes Medical Institute, Ellison Medical Foundation (Aging Grant), United States. Department of Defense (PRCRP Career Development Award CA120198), National Institutes of Health (U.S.) (Grants R01 CA103866, AI47389, K08 CA198002, R00 AG045144, R00 AG041765, and DK043351), National Institutes of Health (U.S.) (Cancer Center Support Core Grant P30-CA14051), Kathy and Curt Marble Cancer Research Fund, American Federation for Aging Research, V Foundation for Cancer Research (Scholar Grant), Massachusetts Institute of Technology. Ludwig Center for Molecular Oncology (Post-doctoral Fellowship), Massachusetts General Hospital (Fellowship T32DK007191), Damon Runyon Cancer Research Foundation (Robert Black Fellowship)

Polycomb Repressive Complex 2 (PRC2) plays crucial roles in transcriptional regulation and stem cell development. However, the context-specific functions associated with alternative subunits remain largely unexplored. Here we show that the related enzymatic subunits EZH1 and EZH2 undergo an expression switch during blood cell development. An erythroid-specific enhancer mediates transcriptional activation of EZH1, and a switch from GATA2 to GATA1 controls the developmental EZH1/2 switch by differential association with EZH1 enhancers. We further examine the in vivo stoichiometry of the PRC2 complexes by quantitative proteomics and reveal the existence of an EZH1-SUZ12 sub-complex lacking EED. EZH1 together with SUZ12 form a non-canonical PRC2 complex, occupy active chromatin, and positively regulate gene expression. Loss of EZH2 expression leads to repositioning of EZH1 to EZH2 targets. Thus, the lineage- and developmental stage-specific regulation of PRC2 subunit composition leads to a switch from canonical silencing to non-canonical functions during blood stem cell specification.