Publications by authors named "Kosuke Yusa"

61 Publications

Minimal genome-wide human CRISPR-Cas9 library.

Genome Biol 2021 Jan 21;22(1):40. Epub 2021 Jan 21.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, UK.

CRISPR guide RNA libraries have been iteratively improved to provide increasingly efficient reagents, although their large size is a barrier for many applications. We design an optimised minimal genome-wide human CRISPR-Cas9 library (MinLibCas9) by mining existing large-scale gene loss-of-function datasets, resulting in a greater than 42% reduction in size compared to other CRISPR-Cas9 libraries while preserving assay sensitivity and specificity. MinLibCas9 provides backward compatibility with existing datasets, increases the dynamic range of CRISPR-Cas9 screens and extends their application to complex models and assays.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13059-021-02268-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7818936PMC
January 2021

KAT7 is a genetic vulnerability of acute myeloid leukemias driven by MLL rearrangements.

Leukemia 2021 04 6;35(4):1012-1022. Epub 2020 Aug 6.

Stem Cell Genetics, Wellcome Sanger Institute, Hinxton, Cambridge, UK.

Histone acetyltransferases (HATs) catalyze the transfer of an acetyl group from acetyl-CoA to lysine residues of histones and play a central role in transcriptional regulation in diverse biological processes. Dysregulation of HAT activity can lead to human diseases including developmental disorders and cancer. Through genome-wide CRISPR-Cas9 screens, we identified several HATs of the MYST family as fitness genes for acute myeloid leukemia (AML). Here we investigate the essentiality of lysine acetyltransferase KAT7 in AMLs driven by the MLL-X gene fusions. We found that KAT7 loss leads to a rapid and complete loss of both H3K14ac and H4K12ac marks, in association with reduced proliferation, increased apoptosis, and differentiation of AML cells. Acetyltransferase activity of KAT7 is essential for the proliferation of these cells. Mechanistically, our data propose that acetylated histones provide a platform for the recruitment of MLL-fusion-associated adaptor proteins such as BRD4 and AF4 to gene promoters. Upon KAT7 loss, these factors together with RNA polymerase II rapidly dissociate from several MLL-fusion target genes that are essential for AML cell proliferation, including MEIS1, PBX3, and SENP6. Our findings reveal that KAT7 is a plausible therapeutic target for this poor prognosis AML subtype.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41375-020-1001-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610570PMC
April 2021

Low rates of mutation in clinical grade human pluripotent stem cells under different culture conditions.

Nat Commun 2020 03 23;11(1):1528. Epub 2020 Mar 23.

The Centre for Stem Cell Biology, Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield, S10 2TN, UK.

The occurrence of repetitive genomic changes that provide a selective growth advantage in pluripotent stem cells is of concern for their clinical application. However, the effect of different culture conditions on the underlying mutation rate is unknown. Here we show that the mutation rate in two human embryonic stem cell lines derived and banked for clinical application is low and not substantially affected by culture with Rho Kinase inhibitor, commonly used in their routine maintenance. However, the mutation rate is reduced by >50% in cells cultured under 5% oxygen, when we also found alterations in imprint methylation and reversible DNA hypomethylation. Mutations are evenly distributed across the chromosomes, except for a slight increase on the X-chromosome, and an elevation in intergenic regions suggesting that chromatin structure may affect mutation rate. Overall the results suggest that pluripotent stem cells are not subject to unusually high rates of genetic or epigenetic alterations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-15271-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7089967PMC
March 2020

ARID1A influences HDAC1/BRD4 activity, intrinsic proliferative capacity and breast cancer treatment response.

Nat Genet 2020 02 6;52(2):187-197. Epub 2020 Jan 6.

CRUK Cambridge Institute, University of Cambridge, Cambridge, UK.

Using genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screens to understand endocrine drug resistance, we discovered ARID1A and other SWI/SNF complex components as the factors most critically required for response to two classes of estrogen receptor-alpha (ER) antagonists. In this context, SWI/SNF-specific gene deletion resulted in drug resistance. Unexpectedly, ARID1A was also the top candidate in regard to response to the bromodomain and extraterminal domain inhibitor JQ1, but in the opposite direction, with loss of ARID1A sensitizing breast cancer cells to bromodomain and extraterminal domain inhibition. We show that ARID1A is a repressor that binds chromatin at ER cis-regulatory elements. However, ARID1A elicits repressive activity in an enhancer-specific, but forkhead box A1-dependent and active, ER-independent manner. Deletion of ARID1A resulted in loss of histone deacetylase 1 binding, increased histone 4 lysine acetylation and subsequent BRD4-driven transcription and growth. ARID1A mutations are more frequent in treatment-resistant disease, and our findings provide mechanistic insight into this process while revealing rational treatment strategies for these patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41588-019-0541-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116647PMC
February 2020

Agreement between two large pan-cancer CRISPR-Cas9 gene dependency data sets.

Nat Commun 2019 12 20;10(1):5817. Epub 2019 Dec 20.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

Genome-scale CRISPR-Cas9 viability screens performed in cancer cell lines provide a systematic approach to identify cancer dependencies and new therapeutic targets. As multiple large-scale screens become available, a formal assessment of the reproducibility of these experiments becomes necessary. We analyze data from recently published pan-cancer CRISPR-Cas9 screens performed at the Broad and Sanger Institutes. Despite significant differences in experimental protocols and reagents, we find that the screen results are highly concordant across multiple metrics with both common and specific dependencies jointly identified across the two studies. Furthermore, robust biomarkers of gene dependency found in one data set are recovered in the other. Through further analysis and replication experiments at each institute, we show that batch effects are driven principally by two key experimental parameters: the reagent library and the assay length. These results indicate that the Broad and Sanger CRISPR-Cas9 viability screens yield robust and reproducible findings.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-13805-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925302PMC
December 2019

Functional linkage of gene fusions to cancer cell fitness assessed by pharmacological and CRISPR-Cas9 screening.

Nat Commun 2019 05 16;10(1):2198. Epub 2019 May 16.

Wellcome Sanger Institute, Wellcome Genome Campus, Cambridge, CB10 1SA, UK.

Many gene fusions are reported in tumours and for most their role remains unknown. As fusions are used for diagnostic and prognostic purposes, and are targets for treatment, it is crucial to assess their function in cancer. To systematically investigate the role of fusions in tumour cell fitness, we utilized RNA-sequencing data from 1011 human cancer cell lines to functionally link 8354 fusion events with genomic data, sensitivity to >350 anti-cancer drugs and CRISPR-Cas9 loss-of-fitness effects. Established clinically-relevant fusions were identified. Overall, detection of functional fusions was rare, including those involving cancer driver genes, suggesting that many fusions are dispensable for tumour fitness. Therapeutically actionable fusions involving RAF1, BRD4 and ROS1 were verified in new histologies. In addition, recurrent YAP1-MAML2 fusions were identified as activators of Hippo-pathway signaling in multiple cancer types. Our approach discriminates functional fusions, identifying new drivers of carcinogenesis and fusions that could have clinical implications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-09940-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6522557PMC
May 2019

Genome-wide CRISPR-Cas9 screening in mammalian cells.

Methods 2019 07 26;164-165:29-35. Epub 2019 Apr 26.

Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan.

Forward genetic screens are a powerful and unbiased approach for uncovering the genetic basis behind a specific phenotype. Genome-wide mutagenesis followed by phenotypic screening represents the ultimate manifestation of this method, directly linking biological phenomena to its corresponding genetic cause. Whilst this has been successful in lower organisms, deployment of genome-wide screens in mammalian systems has been hampered by both limitations of scale and inefficient bi-allelic mutagenesis. CRISPR-Cas9 technology has now largely resolved these issues, whereby delivery of genome-scale gRNA libraries in the presence of gRNA-guided Cas9 endonuclease enables the generation of mutant cell libraries; the perfect platform for performing phenotypic screens. Although the tools are now available for virtually any molecular biology laboratory to conduct such screens, many researchers are daunted by the sheer complexity and scale at which such experiments are performed. This Review will address these concerns, presenting a contextual and practical guide to deploying CRISPR-KO screens in mammalian systems. We will discuss key considerations required in all aspects of screening from initiation to conclusion, which will enable researchers to conduct screens of their own, maximising the potential of this powerful technology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymeth.2019.04.015DOI Listing
July 2019

Prioritization of cancer therapeutic targets using CRISPR-Cas9 screens.

Nature 2019 04 10;568(7753):511-516. Epub 2019 Apr 10.

Wellcome Sanger Institute, Cambridge, UK.

Functional genomics approaches can overcome limitations-such as the lack of identification of robust targets and poor clinical efficacy-that hamper cancer drug development. Here we performed genome-scale CRISPR-Cas9 screens in 324 human cancer cell lines from 30 cancer types and developed a data-driven framework to prioritize candidates for cancer therapeutics. We integrated cell fitness effects with genomic biomarkers and target tractability for drug development to systematically prioritize new targets in defined tissues and genotypes. We verified one of our most promising dependencies, the Werner syndrome ATP-dependent helicase, as a synthetic lethal target in tumours from multiple cancer types with microsatellite instability. Our analysis provides a resource of cancer dependencies, generates a framework to prioritize cancer drug targets and suggests specific new targets. The principles described in this study can inform the initial stages of drug development by contributing to a new, diverse and more effective portfolio of cancer drug targets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-019-1103-9DOI Listing
April 2019

CRISPR-Knockout Screen Identifies Dmap1 as a Regulator of Chemically Induced Reprogramming and Differentiation of Cardiac Progenitors.

Stem Cells 2019 07 23;37(7):958-972. Epub 2019 Apr 23.

Stem Cell Genetics, Wellcome Sanger Institute, Hinxton, Cambridge, United Kingdom.

Direct in vivo reprogramming of cardiac fibroblasts into myocytes is an attractive therapeutic intervention in resolving myogenic deterioration. Current transgene-dependent approaches can restore cardiac function, but dependence on retroviral delivery and persistent retention of transgenic sequences are significant therapeutic hurdles. Chemical reprogramming has been established as a legitimate method to generate functional cell types, including those of the cardiac lineage. Here, we have extended this approach to generate progenitor cells that can differentiate into endothelial cells and cardiomyocytes using a single inhibitor protocol. Depletion of terminally differentiated cells and enrichment for proliferative cells result in a second expandable progenitor population that can robustly give rise to myofibroblasts and smooth muscle. Deployment of a genome-wide knockout screen with clustered regularly interspaced short palindromic repeats-guide RNA library to identify novel mediators that regulate the reprogramming revealed the involvement of DNA methyltransferase 1-associated protein 1 (Dmap1). Loss of Dmap1 reduced promoter methylation, increased the expression of Nkx2-5, and enhanced the retention of self-renewal, although further differentiation is inhibited because of the sustained expression of Cdh1. Our results hence establish Dmap1 as a modulator of cardiac reprogramming and myocytic induction. Stem Cells 2019;37:958-972.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/stem.3012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6767549PMC
July 2019

Structural rearrangements generate cell-specific, gene-independent CRISPR-Cas9 loss of fitness effects.

Genome Biol 2019 02 5;20(1):27. Epub 2019 Feb 5.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, UK.

Background: CRISPR-Cas9 genome editing is widely used to study gene function, from basic biology to biomedical research. Structural rearrangements are a ubiquitous feature of cancer cells and their impact on the functional consequences of CRISPR-Cas9 gene-editing has not yet been assessed.

Results: Utilizing CRISPR-Cas9 knockout screens for 250 cancer cell lines, we demonstrate that targeting structurally rearranged regions, in particular tandem or interspersed amplifications, is highly detrimental to cellular fitness in a gene-independent manner. In contrast, amplifications caused by whole chromosomal duplication have little to no impact on fitness. This effect is cell line specific and dependent on the ploidy status. We devise a copy-number ratio metric that substantially improves the detection of gene-independent cell fitness effects in CRISPR-Cas9 screens. Furthermore, we develop a computational tool, called Crispy, to account for these effects on a single sample basis and provide corrected gene fitness effects.

Conclusion: Our analysis demonstrates the importance of structural rearrangements in mediating the effect of CRISPR-Cas9-induced DNA damage, with implications for the use of CRISPR-Cas9 gene-editing in cancer cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13059-019-1637-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6362594PMC
February 2019

JACKS: joint analysis of CRISPR/Cas9 knockout screens.

Genome Res 2019 03 23;29(3):464-471. Epub 2019 Jan 23.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, United Kingdom.

Genome-wide CRISPR/Cas9 knockout screens are revolutionizing mammalian functional genomics. However, their range of applications remains limited by signal variability from different guide RNAs that target the same gene, which confounds gene effect estimation and dictates large experiment sizes. To address this problem, we report JACKS, a Bayesian method that jointly analyzes screens performed with the same guide RNA library. Modeling the variable guide efficacies greatly improves hit identification over processing a single screen at a time and outperforms existing methods. This more efficient analysis gives additional hits and allows designing libraries with a 2.5-fold reduction in required cell numbers without sacrificing performance compared to current analysis standards.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gr.238923.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6396427PMC
March 2019

Genome-wide CRISPR Screens in T Helper Cells Reveal Pervasive Crosstalk between Activation and Differentiation.

Cell 2019 02 10;176(4):882-896.e18. Epub 2019 Jan 10.

Wellcome Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK; EMBL-European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SD, UK; Theory of Condensed Matter, Cavendish Laboratory, 19 JJ Thomson Ave, Cambridge CB3 0HE, UK. Electronic address:

T helper type 2 (Th2) cells are important regulators of mammalian adaptive immunity and have relevance for infection, autoimmunity, and tumor immunology. Using a newly developed, genome-wide retroviral CRISPR knockout (KO) library, combined with RNA-seq, ATAC-seq, and ChIP-seq, we have dissected the regulatory circuitry governing activation and differentiation of these cells. Our experiments distinguish cell activation versus differentiation in a quantitative framework. We demonstrate that these two processes are tightly coupled and are jointly controlled by many transcription factors, metabolic genes, and cytokine/receptor pairs. There are only a small number of genes regulating differentiation without any role in activation. By combining biochemical and genetic data, we provide an atlas for Th2 differentiation, validating known regulators and identifying factors, such as Pparg and Bhlhe40, as part of the core regulatory network governing Th2 helper cell fates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2018.11.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6370901PMC
February 2019

ATM orchestrates the DNA-damage response to counter toxic non-homologous end-joining at broken replication forks.

Nat Commun 2019 01 8;10(1):87. Epub 2019 Jan 8.

The Wellcome Trust and Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK.

Mutations in the ATM tumor suppressor gene confer hypersensitivity to DNA-damaging chemotherapeutic agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase I inhibitor topotecan. Thus, we here establish that inactivating terminal components of the non-homologous end-joining (NHEJ) machinery or of the BRCA1-A complex specifically confer topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase (PARP) inhibitor olaparib reflects delayed engagement of homologous recombination at DNA-replication-fork associated single-ended double-strand breaks (DSBs), allowing some to be subject to toxic NHEJ. Preventing DSB ligation by NHEJ, or enhancing homologous recombination by BRCA1-A complex disruption, suppresses this toxicity, highlighting a crucial role for ATM in preventing toxic LIG4-mediated chromosome fusions. Notably, suppressor mutations in ATM-mutant backgrounds are different to those in BRCA1-mutant scenarios, suggesting new opportunities for patient stratification and additional therapeutic vulnerabilities for clinical exploitation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-07729-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6325118PMC
January 2019

SRPK1 maintains acute myeloid leukemia through effects on isoform usage of epigenetic regulators including BRD4.

Nat Commun 2018 12 19;9(1):5378. Epub 2018 Dec 19.

Haematological Cancer Genetics, Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK.

We recently identified the splicing kinase gene SRPK1 as a genetic vulnerability of acute myeloid leukemia (AML). Here, we show that genetic or pharmacological inhibition of SRPK1 leads to cell cycle arrest, leukemic cell differentiation and prolonged survival of mice transplanted with MLL-rearranged AML. RNA-seq analysis demonstrates that SRPK1 inhibition leads to altered isoform levels of many genes including several with established roles in leukemogenesis such as MYB, BRD4 and MED24. We focus on BRD4 as its main isoforms have distinct molecular properties and find that SRPK1 inhibition produces a significant switch from the short to the long isoform at the mRNA and protein levels. This was associated with BRD4 eviction from genomic loci involved in leukemogenesis including BCL2 and MYC. We go on to show that this switch mediates at least part of the anti-leukemic effects of SRPK1 inhibition. Our findings reveal that SRPK1 represents a plausible new therapeutic target against AML.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-07620-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6300607PMC
December 2018

Pooled extracellular receptor-ligand interaction screening using CRISPR activation.

Genome Biol 2018 11 26;19(1):205. Epub 2018 Nov 26.

Cell Surface Signalling Laboratory, Wellcome Sanger Institute, Cambridge, CB10 1SA, UK.

Extracellular interactions between cell surface receptors are necessary for signaling and adhesion but identifying them remains technically challenging. We describe a cell-based genome-wide approach employing CRISPR activation to identify receptors for a defined ligand. We show receptors for high-affinity antibodies and low-affinity ligands can be unambiguously identified when used in pools or as individual binding probes. We apply this technique to identify ligands for the adhesion G-protein-coupled receptors and show that the Nogo myelin-associated inhibitory proteins are ligands for ADGRB1. This method will enable extracellular receptor-ligand identification on a genome-wide scale.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13059-018-1581-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6258485PMC
November 2018

Unsupervised correction of gene-independent cell responses to CRISPR-Cas9 targeting.

BMC Genomics 2018 Aug 13;19(1):604. Epub 2018 Aug 13.

Wellcome Sanger Institute, Cambridge, UK.

Background: Genome editing by CRISPR-Cas9 technology allows large-scale screening of gene essentiality in cancer. A confounding factor when interpreting CRISPR-Cas9 screens is the high false-positive rate in detecting essential genes within copy number amplified regions of the genome. We have developed the computational tool CRISPRcleanR which is capable of identifying and correcting gene-independent responses to CRISPR-Cas9 targeting. CRISPRcleanR uses an unsupervised approach based on the segmentation of single-guide RNA fold change values across the genome, without making any assumption about the copy number status of the targeted genes.

Results: Applying our method to existing and newly generated genome-wide essentiality profiles from 15 cancer cell lines, we demonstrate that CRISPRcleanR reduces false positives when calling essential genes, correcting biases within and outside of amplified regions, while maintaining true positive rates. Established cancer dependencies and essentiality signals of amplified cancer driver genes are detectable post-correction. CRISPRcleanR reports sgRNA fold changes and normalised read counts, is therefore compatible with downstream analysis tools, and works with multiple sgRNA libraries.

Conclusions: CRISPRcleanR is a versatile open-source tool for the analysis of CRISPR-Cas9 knockout screens to identify essential genes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12864-018-4989-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6088408PMC
August 2018

Genome-wide CRISPR-KO Screen Uncovers mTORC1-Mediated Gsk3 Regulation in Naive Pluripotency Maintenance and Dissolution.

Cell Rep 2018 07;24(2):489-502

Wellcome Sanger Institute, Hinxton, Cambridge CB10 1SA, UK. Electronic address:

The genetic basis of naive pluripotency maintenance and loss is a central question in embryonic stem cell biology. Here, we deploy CRISPR-knockout-based screens in mouse embryonic stem cells to interrogate this question through a genome-wide, non-biased approach using the Rex1GFP reporter as a phenotypic readout. This highly sensitive and efficient method identified genes in diverse biological processes and pathways. We uncovered a key role for negative regulators of mTORC1 in maintenance and exit from naive pluripotency and provided an integrated account of how mTORC1 activity influences naive pluripotency through Gsk3. Our study therefore reinforces Gsk3 as the central node and provides a comprehensive, data-rich resource that will improve our understanding of mechanisms regulating pluripotency and stimulate avenues for further mechanistic studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2018.06.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6057492PMC
July 2018

Genome-scale identification of cellular pathways required for cell surface recognition.

Genome Res 2018 09 18;28(9):1372-1382. Epub 2018 Jun 18.

Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Cambridge CB10 1SA, United Kingdom.

Interactions mediated by cell surface receptors initiate important instructive signaling cues but can be difficult to detect in biochemical assays because they are often highly transient and membrane-embedded receptors are difficult to solubilize in their native conformation. Here, we address these biochemical challenges by using a genome-scale, cell-based genetic screening approach using CRISPR gene knockout technology to identify cellular pathways required for specific cell surface recognition events. By using high-affinity monoclonal antibodies and low-affinity ligands, we determined the necessary screening parameters, including the importance of establishing binding contributions from the glycocalyx, that permitted the unequivocal identification of genes encoding directly interacting membrane-embedded receptors with high statistical confidence. Importantly, we show that this genome-wide screening approach additionally identified receptor-specific pathways that are required for functional display of receptors on the cell surface that included chaperones, enzymes that add post-translational modifications, trafficking proteins, and transcription factors. Finally, we demonstrate the utility of the approach by identifying IGF2R (insulin like growth factor 2 receptor) as a binding partner for the R2 subunit of GABA receptors. We show that this interaction is direct and is critically dependent on mannose-6-phosphate, providing a mechanism for the internalization and regulation of GABA receptor signaling. We conclude that this single approach can reveal both the molecular nature and the genetic pathways required for functional cell surface display of receptors recognized by antibodies, secreted proteins, and membrane-embedded ligands without the need to make any prior assumptions regarding their biochemical properties.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gr.231183.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120632PMC
September 2018

Genome-wide and high-density CRISPR-Cas9 screens identify point mutations in PARP1 causing PARP inhibitor resistance.

Nat Commun 2018 05 10;9(1):1849. Epub 2018 May 10.

The CRUK Gene Function Laboratory, The Institute of Cancer Research, London, SW3 6JB, UK.

Although PARP inhibitors (PARPi) target homologous recombination defective tumours, drug resistance frequently emerges, often via poorly understood mechanisms. Here, using genome-wide and high-density CRISPR-Cas9 "tag-mutate-enrich" mutagenesis screens, we identify close to full-length mutant forms of PARP1 that cause in vitro and in vivo PARPi resistance. Mutations both within and outside of the PARP1 DNA-binding zinc-finger domains cause PARPi resistance and alter PARP1 trapping, as does a PARP1 mutation found in a clinical case of PARPi resistance. This reinforces the importance of trapped PARP1 as a cytotoxic DNA lesion and suggests that PARP1 intramolecular interactions might influence PARPi-mediated cytotoxicity. PARP1 mutations are also tolerated in cells with a pathogenic BRCA1 mutation where they result in distinct sensitivities to chemotherapeutic drugs compared to other mechanisms of PARPi resistance (BRCA1 reversion, 53BP1, REV7 (MAD2L2) mutation), suggesting that the underlying mechanism of PARPi resistance that emerges could influence the success of subsequent therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-03917-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5945626PMC
May 2018

A CRISPR knockout screen identifies SETDB1-target retroelement silencing factors in embryonic stem cells.

Genome Res 2018 06 4;28(6):846-858. Epub 2018 May 4.

Cellular Memory Laboratory, Cluster for Pioneering Research, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.

In mouse embryonic stem cells (mESCs), the expression of provirus and endogenous retroelements is epigenetically repressed. Although many cellular factors involved in retroelement silencing have been identified, the complete molecular mechanism remains elusive. In this study, we performed a genome-wide CRISPR screen to advance our understanding of retroelement silencing in mESCs. The Moloney murine leukemia virus (MLV)-based retroviral vector MSCV-, which is repressed by the SETDB1TRIM28 pathway in mESCs, was used as a reporter provirus, and we identified more than 80 genes involved in this process. In particular, ATF7IP and the BAF complex components are linked with the repression of most of the SETDB1 targets. We characterized two factors, MORC2A and RESF1, of which RESF1 is a novel molecule in retroelement silencing. Although both factors are recruited to repress provirus, their roles in repression are different. MORC2A appears to function dependent on repressive epigenetic modifications, while RESF1 regulates repressive epigenetic modifications associated with SETDB1. Our genome-wide CRISPR screen cataloged genes which function at different levels in silencing of SETDB1-target retroelements and provides a useful resource for further molecular studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gr.227280.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5991520PMC
June 2018

Author Correction: Optimised metrics for CRISPR-KO screens with second-generation gRNA libraries.

Sci Rep 2018 Apr 12;8(1):6136. Epub 2018 Apr 12.

Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-24092-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5895590PMC
April 2018

Constitutively Active SMAD2/3 Are Broad-Scope Potentiators of Transcription-Factor-Mediated Cellular Reprogramming.

Cell Stem Cell 2017 Dec 22;21(6):791-805.e9. Epub 2017 Nov 22.

MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh BioQuarter, 5 Little France Drive, Edinburgh EH16 4UU, Scotland, UK. Electronic address:

Reprogramming of cellular identity using exogenous expression of transcription factors (TFs) is a powerful and exciting tool for tissue engineering, disease modeling, and regenerative medicine. However, generation of desired cell types using this approach is often plagued by inefficiency, slow conversion, and an inability to produce mature functional cells. Here, we show that expression of constitutively active SMAD2/3 significantly improves the efficiency of induced pluripotent stem cell (iPSC) generation by the Yamanaka factors. Mechanistically, SMAD3 interacts with reprogramming factors and co-activators and co-occupies OCT4 target loci during reprogramming. Unexpectedly, active SMAD2/3 also markedly enhances three other TF-mediated direct reprogramming conversions, from B cells to macrophages, myoblasts to adipocytes, and human fibroblasts to neurons, highlighting broad and general roles for SMAD2/3 as cell-reprogramming potentiators. Our results suggest that co-expression of active SMAD2/3 could enhance multiple types of TF-based cell identity conversion and therefore be a powerful tool for cellular engineering.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stem.2017.10.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5732323PMC
December 2017

Molecular synergy underlies the co-occurrence patterns and phenotype of -mutant acute myeloid leukemia.

Blood 2017 10 23;130(17):1911-1922. Epub 2017 Aug 23.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, United Kingdom.

mutations define the commonest subgroup of acute myeloid leukemia (AML) and frequently co-occur with internal tandem duplications (ITD) or, less commonly, or mutations. Co-occurrence of mutant with carries a significantly worse prognosis than combinations. To understand the molecular basis of these observations, we compare the effects of the 2 combinations on hematopoiesis and leukemogenesis in knock-in mice. Early effects of these mutations on hematopoiesis show that compound or share a number of features: gene overexpression, enhanced self-renewal, expansion of hematopoietic progenitors, and myeloid differentiation bias. However, mutants displayed significantly higher peripheral leukocyte counts, early depletion of common lymphoid progenitors, and a monocytic bias in comparison with the granulocytic bias in mutants. Underlying this was a striking molecular synergy manifested as a dramatically altered gene expression profile in , but not , progenitors compared with wild-type. Both double-mutant models developed high-penetrance AML, although latency was significantly longer with During AML evolution, both models acquired additional copies of the mutant or alleles, but only mice showed acquisition of other human AML mutations, including R132Q. We also find, using primary Cas9-expressing AMLs, that genes and selected interactors or downstream targets are required for survival of both types of double-mutant AML. Our results show that molecular complementarity underlies the higher frequency and significantly worse prognosis associated with c/ vs mutant AML and functionally confirm the role of genes in NPM1c-driven AML.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2017-01-760595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5672315PMC
October 2017

Optimised metrics for CRISPR-KO screens with second-generation gRNA libraries.

Sci Rep 2017 08 7;7(1):7384. Epub 2017 Aug 7.

Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK.

Genome-wide CRISPR-based knockout (CRISPR-KO) screening is an emerging technique which enables systematic genetic analysis of a cellular or molecular phenotype in question. Continuous improvements, such as modifications to the guide RNA (gRNA) scaffold and the development of gRNA on-target prediction algorithms, have since been made to increase their screening performance. We compared the performance of three available second-generation human genome-wide CRISPR-KO libraries that included at least one of the improvements, and examined the effect of gRNA scaffold, number of gRNAs per gene and number of replicates on screen performance. We identified duplicated screens using a library with 6 gRNAs per gene as providing the best trade-off. Despite the improvements, we found that each improved library still has library-specific false negatives and, for the first time, estimated the false negative rates of CRISPR-KO screens, which are between 10% and 20%. Our newly-defined optimal screening parameters would be helpful in designing screens and constructing bespoke gRNA libraries.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-07827-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5547152PMC
August 2017

Applications of CRISPR genome editing technology in drug target identification and validation.

Expert Opin Drug Discov 2017 Jun 23;12(6):541-552. Epub 2017 Apr 23.

b Target Sciences, GlaxoSmithKline R&D , Stevenage , UK.

Introduction: The analysis of pharmaceutical industry data indicates that the major reason for drug candidates failing in late stage clinical development is lack of efficacy, with a high proportion of these due to erroneous hypotheses about target to disease linkage. More than ever, there is a requirement to better understand potential new drug targets and their role in disease biology in order to reduce attrition in drug development. Genome editing technology enables precise modification of individual protein coding genes, as well as noncoding regulatory sequences, enabling the elucidation of functional effects in human disease relevant cellular systems. Areas covered: This article outlines applications of CRISPR genome editing technology in target identification and target validation studies. Expert opinion: Applications of CRISPR technology in target validation studies are in evidence and gaining momentum. Whilst technical challenges remain, we are on the cusp of CRISPR being applied in complex cell systems such as iPS derived differentiated cells and stem cell derived organoids. In the meantime, our experience to date suggests that precise genome editing of putative targets in primary cell systems is possible, offering more human disease relevant systems than conventional cell lines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/17460441.2017.1317244DOI Listing
June 2017

A CRISPR Dropout Screen Identifies Genetic Vulnerabilities and Therapeutic Targets in Acute Myeloid Leukemia.

Cell Rep 2016 10;17(4):1193-1205

Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK. Electronic address:

Acute myeloid leukemia (AML) is an aggressive cancer with a poor prognosis, for which mainstream treatments have not changed for decades. To identify additional therapeutic targets in AML, we optimize a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening platform and use it to identify genetic vulnerabilities in AML cells. We identify 492 AML-specific cell-essential genes, including several established therapeutic targets such as DOT1L, BCL2, and MEN1, and many other genes including clinically actionable candidates. We validate selected genes using genetic and pharmacological inhibition, and chose KAT2A as a candidate for downstream study. KAT2A inhibition demonstrated anti-AML activity by inducing myeloid differentiation and apoptosis, and suppressed the growth of primary human AMLs of diverse genotypes while sparing normal hemopoietic stem-progenitor cells. Our results propose that KAT2A inhibition should be investigated as a therapeutic strategy in AML and provide a large number of genetic vulnerabilities of this leukemia that can be pursued in downstream studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2016.09.079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5081405PMC
October 2016

Mutational History of a Human Cell Lineage from Somatic to Induced Pluripotent Stem Cells.

PLoS Genet 2016 Apr 7;12(4):e1005932. Epub 2016 Apr 7.

Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom.

The accuracy of replicating the genetic code is fundamental. DNA repair mechanisms protect the fidelity of the genome ensuring a low error rate between generations. This sustains the similarity of individuals whilst providing a repertoire of variants for evolution. The mutation rate in the human genome has recently been measured to be 50-70 de novo single nucleotide variants (SNVs) between generations. During development mutations accumulate in somatic cells so that an organism is a mosaic. However, variation within a tissue and between tissues has not been analysed. By reprogramming somatic cells into induced pluripotent stem cells (iPSCs), their genomes and the associated mutational history are captured. By sequencing the genomes of polyclonal and monoclonal somatic cells and derived iPSCs we have determined the mutation rates and show how the patterns change from a somatic lineage in vivo through to iPSCs. Somatic cells have a mutation rate of 14 SNVs per cell per generation while iPSCs exhibited a ten-fold lower rate. Analyses of mutational signatures suggested that deamination of methylated cytosine may be the major mutagenic source in vivo, whilst oxidative DNA damage becomes dominant in vitro. Our results provide insights for better understanding of mutational processes and lineage relationships between human somatic cells. Furthermore it provides a foundation for interpretation of elevated mutation rates and patterns in cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1005932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4824386PMC
April 2016

piggyBac Transposon.

Authors:
Kosuke Yusa

Microbiol Spectr 2015 Apr;3(2):MDNA3-0028-2014

Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.

The piggyBac transposon was originally isolated from the cabbage looper moth, Trichoplusia ni, in the 1980s. Despite its early discovery and dissimilarity to the other DNA transposon families, the piggyBac transposon was not recognized as a member of a large transposon superfamily for a long time. Initially, the piggyBac transposon was thought to be a rare transposon. This view, however, has now been completely revised as a number of fully sequenced genomes have revealed the presence of piggyBac-like repetitive elements. The isolation of active copies of the piggyBac-like elements from several distinct species further supported this revision. This includes the first isolation of an active mammalian DNA transposon identified in the bat genome. To date, the piggyBac transposon has been deeply characterized and it represents a number of unique characteristics. In general, all members of the piggyBac superfamily use TTAA as their integration target sites. In addition, the piggyBac transposon shows precise excision, i.e., restoring the sequence to its preintegration state, and can transpose in a variety of organisms such as yeasts, malaria parasites, insects, mammals, and even in plants. Biochemical analysis of the chemical steps of transposition revealed that piggyBac does not require DNA synthesis during the actual transposition event. The broad host range has attracted researchers from many different fields, and the piggyBac transposon is currently the most widely used transposon system for genetic manipulations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/microbiolspec.MDNA3-0028-2014DOI Listing
April 2015

Simulation and estimation of gene number in a biological pathway using almost complete saturation mutagenesis screening of haploid mouse cells.

BMC Genomics 2014 Nov 24;15:1016. Epub 2014 Nov 24.

Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan.

Background: Genome-wide saturation mutagenesis and subsequent phenotype-driven screening has been central to a comprehensive understanding of complex biological processes in classical model organisms such as flies, nematodes, and plants. The degree of "saturation" (i.e., the fraction of possible target genes identified) has been shown to be a critical parameter in determining all relevant genes involved in a biological function, without prior knowledge of their products. In mammalian model systems, however, the relatively large scale and labor intensity of experiments have hampered the achievement of actual saturation mutagenesis, especially for recessive traits that require biallelic mutations to manifest detectable phenotypes.

Results: By exploiting the recently established haploid mouse embryonic stem cells (ESCs), we present an implementation of almost complete saturation mutagenesis in a mammalian system. The haploid ESCs were mutagenized with the chemical mutagen N-ethyl-N-nitrosourea (ENU) and processed for the screening of mutants defective in various steps of the glycosylphosphatidylinositol-anchor biosynthetic pathway. The resulting 114 independent mutant clones were characterized by a functional complementation assay, and were shown to be defective in any of 20 genes among all 22 known genes essential for this well-characterized pathway. Ten mutants were further validated by whole-exome sequencing. The predominant generation of single-nucleotide substitutions by ENU resulted in a gene mutation rate proportional to the length of the coding sequence, which facilitated the experimental design of saturation mutagenesis screening with the aid of computational simulation.

Conclusions: Our study enables mammalian saturation mutagenesis to become a realistic proposition. Computational simulation, combined with a pilot mutagenesis experiment, could serve as a tool for the estimation of the number of genes essential for biological processes such as drug target pathways when a positive selection of mutants is available.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2164-15-1016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4301880PMC
November 2014