Abstract 403: CRISPR/Cas9 genome-wide sgRNA libraries for loss-of-function and gain-of-function genetic screens

AbstractIdentifying essential genes in genome-wide loss of function screens is a critical step in functional genomics and cancer target finding. We previously described the Bayesian Analysis of Gene Essentiality (BAGEL) algorithm for accurate classification of gene essentiality from short hairpin RNA and CRISPR/Cas9 genome wide genetic screens. Here, we introduce an updated version, BAGEL2, which employs an improved model that offers greater dynamic range of Bayes Factors, enabling detection of tumor suppressor genes, and a multi-target correction that reduces false positives from off-target CRISPR guide RNA. We also suggest a metric for screen quality at the replicate level and demonstrate how different algorithms handle lower-quality data in substantially different ways. BAGEL2 is written in Python 3 and source code, along with all supporting files, are available on github (https://github.com/hart-lab/bagel).

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Genome-Wide Analysis of MDS/MPD Disclosed Frequent Homozygous C-Cbl mutations Tightly Associated with 11q-UPD

Blood ◽

10.1182/blood.v112.11.855.855 ◽

2008 ◽

Vol 112 (11) ◽

pp. 855-855 ◽

Cited By ~ 1

Author(s):

Sanada Masashi ◽

Shih Lee Yung ◽

Takahiro Suzuki ◽

Motohiro Kato ◽

Mamiko Yanagimoto Sakata ◽

...

Keyword(s):

Bone Marrow Cells ◽

Snp Array ◽

Uniparental Disomy ◽

Point Mutations ◽

Loss Of Function ◽

Hematopoietic Progenitors ◽

Gain Of Function ◽

Gene Target ◽

Genome Wide ◽

Homozygous Mutations

Abstract Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic progenitors characterized by ineffective hematopoiesis and high propensity to leukemias. Although a number of gene targets have been identified, in many MDS cases, particular genetic targets are unknown. In this study, we performed genome-wide profiling of copy number (CN) abnormalities and allelic imbalances in MDS genomes in order to clarify the distribution of LOH (loss of heterozygosity) and to identify their gene targets. We analyzed a total of 171MDS and MDS/MPD specimens, including 7 RA/RARS, 23 RCMD/RCMD-RS, 6 5q-syndrome, 30 RAEB-1, 40 RAEB-2, 4 therapy related-MDS/AML, 5 MDSu, 17 CMML-1, 16 CMML-2, 24 overt AML, using high-density SNP arrays. The data were analyzed by CNAG/AsCNAR software, which enabled allele-specific CN analysis and sensitive LOH detection. MDS showed characteristic CN profiles in SNP array analysis. Of particular interest is the finding of high frequency of CN-neutral LOH (Uniparental disomy,UPD) observed in 51 of 171 (30%) MDS cases. They preferentially involved 1p, 1q, 4q, 7q, 11q, 17p and other chromosomal segments, which were associated with homozygous mutations of both loss-of-function mutations and gain-of function mutations of tumor suppressor genes and cellular oncogenes, including TP53 (17p UPD), AML1/RUNX1 (21q UPD), Nras and cMPL (1p UPD), JAK-2 (9p UPD), and FLT3 (13q UPD). Next we tried to identify a new gene target in 11q UPD, which was most common UPD region in this study and many of these cases were CMML with a normal karyotype. The minimum 11q UPD segment is about 2Mb which existed in 11q23. We sequenced coding exons of c-cbl and detected homozygous mutations in 8 of 9 MDS cases with 11q UPD (CMML=5, RAEB=3, overt leukemia=1), but very rare in cases without 11q UPD (1/162), demonstrating that the mutation is tightly linked to 11q UPD. These mutations were 8 point mutations and 1 micro-deletion, they were accumulated in the linker or RING domain. These c-cbl mutants transformed NIH3T3 in a dominant fashion, in which they were phosphorylated and activate PI3K-Akt pathway. To investigate the functions of these mutants in hematopoietic cells, we introduced these mutants into c-kit(+)Sca1(+)Lin(−) murine bone marrow cells, it prolonged replating capacity of these hematopoietic progenitors, suggesting involvement of aberrant c-cbl functions in the myeloproliferative phenotypes frequently found in 11q-UPD positive cases. In conclusion, UPD is an important mechanism of development of MDS, in which both gain-of-function and loss-of-function mutations are duplicated with exclusion of wild-type allele. Analysis of 11q UPD disclosed novel gain-of-function mutations. Identification of the targets of UPDs in 1q, 4q and 7q should also be important to gain a novel insight into the pathogenesis of MDS.

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A simplified transposon mutagenesis method to perform phenotypic forward genetic screens in cultured cells

10.1101/598474 ◽

2019 ◽

Author(s):

Charlotte R. Feddersen ◽

Lexy S. Wadsworth ◽

Eliot Y. Zhu ◽

Hayley R. Vaughn ◽

Andrew P. Voigt ◽

...

Keyword(s):

Cultured Cells ◽

Sequence Data ◽

Sleeping Beauty ◽

Transposon Mutagenesis ◽

Loss Of Function ◽

Genetic Screens ◽

Sleeping Beauty Transposon ◽

Genome Wide ◽

Forward Genetic Screens ◽

The Impact

AbstractThe introduction of genome-wide shRNA and CRISPR libraries has facilitated cell-based screens to identify loss-of-function mutations associated with a phenotype of interest. Approaches to perform analogous gain-of-function screens are less common, although some reports have utilized arrayed viral expression libraries or the CRISPR activation system. However, a variety of technical and logistical challenges make these approaches difficult for many labs to execute. In addition, genome-wide shRNA or CRISPR libraries typically contain of hundreds of thousands of individual engineered elements, and the associated complexity creates issues with replication and reproducibility for these methods. Here we describe a simple, reproducible approach using the Sleeping Beauty transposon system to perform phenotypic cell-based genetic screens. This approach employs only three plasmids to perform unbiased, whole-genome transposon mutagenesis. We also describe a ligation-mediated PCR method that can be used in conjunction with the included software tools to map raw sequence data, identify candidate genes associated with phenotypes of interest, and predict the impact of recurrent transposon insertions on candidate gene function. Finally, we demonstrate the high reproducibility of our approach by having three individuals perform independent replicates of a mutagenesis screen to identify drivers of vemurafenib resistance in cultured melanoma cells. Collectively, our work establishes a facile, adaptable method that can be performed by labs of any size to perform robust, genome-wide screens to identify genes that influence phenotypes of interest.

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Systematic genetic mapping of necroptosis identifies SLC39A7 as modulator of death receptor trafficking

10.1101/290718 ◽

2018 ◽

Author(s):

Astrid Fauster ◽

Manuele Rebsamen ◽

Katharina L. Willmann ◽

Adrian César-Razquin ◽

Enrico Girardi ◽

...

Keyword(s):

Cell Death ◽

Death Receptor ◽

Receptor Trafficking ◽

Dependent Manner ◽

Loss Of Function ◽

Genetic Screens ◽

Gain Of Function ◽

Interacting Protein ◽

Knock Out ◽

Unbiased Gene

ABSTRACTRegulation of cell and tissue homeostasis by programmed cell death is a fundamental process with wide physiological and pathological implications. The advent of scalable somatic cell genetic technologies creates the opportunity to functionally map these essential pathways, thereby identifying potential disease-relevant components. We investigated the genetic basis underlying necroptotic cell death by performing a complementary set of loss- and gain-of-function genetic screens. To this end, we established FADD-deficient haploid human KBM7 cells, which specifically and efficiently undergo necroptosis after a single treatment with either TNFα or the SMAC mimetic compound birinapant. A series of unbiased gene-trap screens identified key signaling mediators, such as TNFR1, RIPK1, RIPK3, and MLKL. Among the novel components, we focused on the zinc transporter SLC39A7, whose knock-out led to necroptosis resistance by affecting TNF receptor trafficking and ER homeostasis. Orthogonal, solute carrier (SLC)-focused CRISPR/Cas9-based genetic screens revealed the exquisite specificity of SLC39A7, among ~ 400 SLC genes, for TNFR1- and FAS-but not TRAIL-R1-mediated responses. The newly established cellular model also allowed genome-wide gain-of-function screening for genes conferring resistance to necroptosis via the CRISPR/Cas9 synergistic activation mediator approach. Among these, we found cIAP1 and cIAP2, and characterized the role of TNIP1 (TNFAIP3-interacting protein 1), which prevented pathway activation in a ubiquitin-binding dependent manner. Altogether, the gain- and loss-of-function screens described here provide a global genetic chart of the molecular factors involved in necroptosis and death receptor signaling, prompting investigation of their individual contribution and potential role in pathological conditions.

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Improved analysis of CRISPR fitness screens and reduced off-target effects with the BAGEL2 gene essentiality classifier

Genome Medicine ◽

10.1186/s13073-020-00809-3 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Eiru Kim ◽

Traver Hart

Keyword(s):

Dynamic Range ◽

Quality Data ◽

Loss Of Function ◽

Genetic Screens ◽

Gene Essentiality ◽

Guide Rna ◽

Genome Wide ◽

And Performance ◽

Improved Model ◽

Sensitivity Specificity

Abstract Background Identifying essential genes in genome-wide loss-of-function screens is a critical step in functional genomics and cancer target finding. We previously described the Bayesian Analysis of Gene Essentiality (BAGEL) algorithm for accurate classification of gene essentiality from short hairpin RNA and CRISPR/Cas9 genome-wide genetic screens. Results We introduce an updated version, BAGEL2, which employs an improved model that offers a greater dynamic range of Bayes Factors, enabling detection of tumor suppressor genes; a multi-target correction that reduces false positives from off-target CRISPR guide RNA; and the implementation of a cross-validation strategy that improves performance ~ 10× over the prior bootstrap resampling approach. We also describe a metric for screen quality at the replicate level and demonstrate how different algorithms handle lower quality data in substantially different ways. Conclusions BAGEL2 substantially improves the sensitivity, specificity, and performance over BAGEL and establishes the new state of the art in the analysis of CRISPR knockout fitness screens. BAGEL2 is written in Python 3 and source code, along with all supporting files, are available on github (https://github.com/hart-lab/bagel).

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Pooled analysis of radiation hybrids identifies loci for growth and drug action in mammalian cells

10.1101/2019.12.16.877365 ◽

2019 ◽

Author(s):

Arshad H. Khan ◽

Andy Lin ◽

Richard T. Wang ◽

Joshua S. Bloom ◽

Kenneth Lange ◽

...

Keyword(s):

Mammalian Cells ◽

Single Gene ◽

Pooled Analysis ◽

Loss Of Function ◽

Genetic Screens ◽

Gene Copies ◽

Radiation Hybrids ◽

Genome Wide ◽

Low Pass ◽

Mammalian Genetic

AbstractGenetic screens in mammalian cells commonly focus on loss-of-function approaches. To evaluate the phenotypic consequences of extra gene copies, we used bulk segregant analysis (BSA) of radiation hybrid (RH) cells. We constructed six pools of RH cells, each consisting of ~2500 independent clones, and placed the pools under selection in media with or without paclitaxel. Low pass sequencing identified 859 growth loci, 38 paclitaxel loci, 62 interaction loci and 3 loci for mitochondrial abundance at genome-wide significance. Resolution was measured as ~30 kb, close to single-gene. Divergent properties were displayed by the RH-BSA growth genes compared to those from loss-of-function screens, refuting the balance hypothesis. In addition, enhanced retention of human centromeres in the RH pools suggests a new approach to functional dissection of these chromosomal elements. Pooled analysis of RH cells showed high power and resolution and should be a useful addition to the mammalian genetic toolkit.

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Faculty Opinions recommendation of Loss-of-Function and Gain-of-Function Mutations in KCNQ5 Cause Intellectual Disability or Epileptic Encephalopathy.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.727772380.793535929 ◽

2017 ◽

Author(s):

Mitsuhiro Kato

Keyword(s):

Intellectual Disability ◽

Epileptic Encephalopathy ◽

Loss Of Function ◽

Gain Of Function

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Gain-of-function GABRB3 variants identified in vigabatrin-hypersensitive epileptic encephalopathies

Brain Communications ◽

10.1093/braincomms/fcaa162 ◽

2020 ◽

Vol 2 (2) ◽

Author(s):

Nathan L Absalom ◽

Vivian W Y Liao ◽

Kavitha Kothur ◽

Dinesh C Indurthi ◽

Bruce Bennetts ◽

...

Keyword(s):

De Novo ◽

Drug Effects ◽

Receptor Expression ◽

Aminobutyric Acid ◽

Loss Of Function ◽

Molecular Phenotype ◽

Gain Of Function ◽

Gene Encoding ◽

Epileptic Encephalopathies ◽

Receptor Phenotype

Abstract Variants in the GABRB3 gene encoding the β3-subunit of the γ-aminobutyric acid type A ( receptor are associated with various developmental and epileptic encephalopathies. Typically, these variants cause a loss-of-function molecular phenotype whereby γ-aminobutyric acid has reduced inhibitory effectiveness leading to seizures. Drugs that potentiate inhibitory GABAergic activity, such as nitrazepam, phenobarbital or vigabatrin, are expected to compensate for this and thereby reduce seizure frequency. However, vigabatrin, a drug that inhibits γ-aminobutyric acid transaminase to increase tonic γ-aminobutyric acid currents, has mixed success in treating seizures in patients with GABRB3 variants: some patients experience seizure cessation, but there is hypersensitivity in some patients associated with hypotonia, sedation and respiratory suppression. A GABRB3 variant that responds well to vigabatrin involves a truncation variant (p.Arg194*) resulting in a clear loss-of-function. We hypothesized that patients with a hypersensitive response to vigabatrin may exhibit a different γ-aminobutyric acid A receptor phenotype. To test this hypothesis, we evaluated the phenotype of de novo variants in GABRB3 (p.Glu77Lys and p.Thr287Ile) associated with patients who are clinically hypersensitive to vigabatrin. We introduced the GABRB3 p.Glu77Lys and p.Thr287Ile variants into a concatenated synaptic and extrasynaptic γ-aminobutyric acid A receptor construct, to resemble the γ-aminobutyric acid A receptor expression by a patient heterozygous for the GABRB3 variant. The mRNA of these constructs was injected into Xenopus oocytes and activation properties of each receptor measured by two-electrode voltage clamp electrophysiology. Results showed an atypical gain-of-function molecular phenotype in the GABRB3 p.Glu77Lys and p.Thr287Ile variants characterized by increased potency of γ-aminobutyric acid A without change to the estimated maximum open channel probability, deactivation kinetics or absolute currents. Modelling of the activation properties of the receptors indicated that either variant caused increased chloride flux in response to low concentrations of γ-aminobutyric acid that mediate tonic currents. We therefore propose that the hypersensitivity reaction to vigabatrin is a result of GABRB3 variants that exacerbate GABAergic tonic currents and caution is required when prescribing vigabatrin. In contrast, drug strategies increasing tonic currents in loss-of-function variants are likely to be a safe and effective therapy. This study demonstrates that functional genomics can explain beneficial and adverse anti-epileptic drug effects, and propose that vigabatrin should be considered in patients with clear loss-of-function GABRB3 variants.

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Emerging Role of ODC1 in Neurodevelopmental Disorders and Brain Development

Genes ◽

10.3390/genes12040470 ◽

2021 ◽

Vol 12 (4) ◽

pp. 470

Author(s):

Jeremy W. Prokop ◽

Caleb P. Bupp ◽

Austin Frisch ◽

Stephanie M. Bilinovich ◽

Daniel B. Campbell ◽

...

Keyword(s):

Brain Development ◽

Neural Progenitor Cells ◽

Neurodevelopmental Disorder ◽

Fetal Brain ◽

Missense Variant ◽

Neural Progenitor ◽

Loss Of Function ◽

Gain Of Function ◽

Systematic Analysis ◽

Function Expression

Ornithine decarboxylase 1 (ODC1 gene) has been linked through gain-of-function variants to a rare disease featuring developmental delay, alopecia, macrocephaly, and structural brain anomalies. ODC1 has been linked to additional diseases like cancer, with growing evidence for neurological contributions to schizophrenia, mood disorders, anxiety, epilepsy, learning, and suicidal behavior. The evidence of ODC1 connection to neural disorders highlights the need for a systematic analysis of ODC1 genotype-to-phenotype associations. An analysis of variants from ClinVar, Geno2MP, TOPMed, gnomAD, and COSMIC revealed an intellectual disability and seizure connected loss-of-function variant, ODC G84R (rs138359527, NC_000002.12:g.10444500C > T). The missense variant is found in ~1% of South Asian individuals and results in 2.5-fold decrease in enzyme function. Expression quantitative trait loci (eQTLs) reveal multiple functionally annotated, non-coding variants regulating ODC1 that associate with psychiatric/neurological phenotypes. Further dissection of RNA-Seq during fetal brain development and within cerebral organoids showed an association of ODC1 expression with cell proliferation of neural progenitor cells, suggesting gain-of-function variants with neural over-proliferation and loss-of-function variants with neural depletion. The linkage from the expression data of ODC1 in early neural progenitor proliferation to phenotypes of neurodevelopmental delay and to the connection of polyamine metabolites in brain function establish ODC1 as a bona fide neurodevelopmental disorder gene.

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CRISPRi enables isoform-specific loss-of-function screens and identification of gastric cancer-specific isoform dependencies

Genome Biology ◽

10.1186/s13059-021-02266-6 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Rebecca Davies ◽

Ling Liu ◽

Sheng Taotao ◽

Natasha Tuano ◽

Richa Chaturvedi ◽

...

Keyword(s):

Gastric Cancer ◽

Functional Dependencies ◽

Loss Of Function ◽

Genetic Screens ◽

Transcript Isoforms ◽

Specific Loss ◽

Current Loss ◽

Specific Transcript ◽

Multiple Promoters ◽

Poor Patient

Abstract Introduction Genes contain multiple promoters that can drive the expression of various transcript isoforms. Although transcript isoforms from the same gene could have diverse and non-overlapping functions, current loss-of-function methodologies are not able to differentiate between isoform-specific phenotypes. Results Here, we show that CRISPR interference (CRISPRi) can be adopted for targeting specific promoters within a gene, enabling isoform-specific loss-of-function genetic screens. We use this strategy to test functional dependencies of 820 transcript isoforms that are gained in gastric cancer (GC). We identify a subset of GC-gained transcript isoform dependencies, and of these, we validate CIT kinase as a novel GC dependency. We further show that some genes express isoforms with opposite functions. Specifically, we find that the tumour suppressor ZFHX3 expresses an isoform that has a paradoxical oncogenic role that correlates with poor patient outcome. Conclusions Our work finds isoform-specific phenotypes that would not be identified using current loss-of-function approaches that are not designed to target specific transcript isoforms.

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