scholarly journals Improved vectors and genome-wide libraries for CRISPR screening

2014 ◽  
Author(s):  
Neville E Sanjana ◽  
Ophir Shalem ◽  
Feng Zhang
Keyword(s):  
Rna Interference ◽  
Viral Titer ◽  
Screening System ◽  
Loss Of Function ◽  
Knock Out ◽  
Genome Wide ◽  
A Cell ◽  
Mouse Cells ◽  
Genome Scale ◽  
Human And Mouse

Genome-wide, targeted loss-of-function pooled screens using the CRISPR (clustered regularly interspaced short palindrome repeats)?associated nuclease Cas9 in human and mouse cells provide an alternative screening system to RNA interference (RNAi). Initial lentiviral delivery systems for CRISPR screening had low viral titer or required a cell line already expressing Cas9, limiting the range of biological systems amenable to screening. In this work, we present 1- and 2-vector lentiCRISPR systems capable of producing higher viral titers and, in these vectors, new human and mouse libraries for genome-scale CRISPR knock-out (GeCKO) screening.

scholarly journals HiCRes: a computational method to estimate and predict the resolution of HiC libraries

2020 ◽  
Author(s):  
Claire Marchal ◽  
Nivedita Singh ◽  
Ximena Corso-Díaz ◽  
Anand Swaroop
Keyword(s):  
Expression Patterns ◽  
Three Dimensional ◽  
Computational Method ◽  
Mathematical Concepts ◽  
Regulate Gene Expression ◽  
Chromatin Interactions ◽  
Genome Wide ◽  
A Cell ◽  
Cell Type Specific ◽  
Human And Mouse

AbstractThree-dimensional (3D) conformation of the chromatin is crucial to stringently regulate gene expression patterns and DNA replication in a cell-type specific manner. HiC is a key technique for measuring 3D chromatin interactions genome wide. Estimating and predicting the resolution of a library is an essential step in any HiC experimental design. Here, we present the mathematical concepts to estimate the resolution of a library and predict whether deeper sequencing would enhance the resolution. We have developed HiCRes, a docker pipeline, by applying these concepts to human and mouse HiC libraries.

scholarly journals Efficient genome-wide first-generation phenotypic screening system in mice using thepiggyBactransposon

2019 ◽  
Vol 116 (37) ◽  
pp. 18507-18516 ◽  
Author(s):  
Hao Chang ◽  
Yukun Pan ◽  
Sean Landrette ◽  
Sheng Ding ◽  
Dong Yang ◽  
...  
Keyword(s):  
First Generation ◽  
Cranial Nerves ◽  
Model Organisms ◽  
Biological Traits ◽  
Screening System ◽  
Loss Of Function ◽  
Developmental Defects ◽  
Genome Wide ◽  
A Genome ◽  
Milk Ingestion

Genome-wide phenotypic screens provide an unbiased way to identify genes involved in particular biological traits, and have been widely used in lower model organisms. However, cost and time have limited the utility of such screens to address biological and disease questions in mammals. Here we report a highly efficientpiggyBac(PB) transposon-based first-generation (F1) dominant screening system in mice that enables an individual investigator to conduct a genome-wide phenotypic screen within a year with fewer than 300 cages. ThePBscreening system uses visually trackable transposons to induce both gain- and loss-of-function mutations and generates genome-wide distributed new insertions in more than 55% of F1 progeny. Using this system, we successfully conducted a pilot F1 screen and identified 5 growth retardation mutations. One of these mutants, a Six1/4PB/+mutant, revealed a role in milk intake behavior. The mutant animals exhibit abnormalities in nipple recognition and milk ingestion, as well as developmental defects in cranial nerves V, IX, and X. ThisPBF1 screening system offers individual laboratories unprecedented opportunities to conduct affordable genome-wide phenotypic screens for deciphering the genetic basis of mammalian biology and disease pathogenesis.

scholarly journals Genome-wide CRISPR/Cas9 screening in human iPS derived cardiomyocytes uncovers novel mediators of doxorubicin cardiotoxicity

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Valerie Sapp ◽  
Aitor Aguirre ◽  
Gayatri Mainkar ◽  
Jeffrey Ding ◽  
Eric Adler ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Types ◽  
Functional Screening ◽  
Loss Of Function ◽  
Ips Cell ◽  
Genome Wide ◽  
Doxorubicin Cardiotoxicity ◽  
Induced Pluripotent ◽  
Genome Scale ◽  
Human Specific

AbstractHuman induced pluripotent stem (iPS) cell technologies coupled with genetic engineering now facilitate the study of the molecular underpinnings of disease in relevant human cell types. Application of CRISPR/Cas9-based approaches for genome-scale functional screening in iPS-derived cells, however, has been limited by technical constraints, including inefficient transduction in pooled format, loss of library representation, and poor cellular differentiation. Herein, we present optimized approaches for whole-genome CRISPR/Cas9 based screening in human iPS derived cardiomyocytes with near genome-wide representation at both the iPS and differentiated cell stages. As proof-of-concept, we perform a screen to investigate mechanisms underlying doxorubicin mediated cell death in iPS derived cardiomyocytes. We identified two poorly characterized, human-specific transporters (SLCO1A2, SLCO1B3) whose loss of function protects against doxorubicin-cardiotoxicity, but does not affect cell death in cancer cells. This study provides a technical framework for genome-wide functional screening in iPS derived cells and identifies new targets to mitigate doxorubicin-cardiotoxicity in humans.

scholarly journals Genome-Scale Crispr-Cas9 Knockout Studies Reveal Mutifactorial and Functionally Overlapping Mechanisms of Myeloma Cell Resistance to Proteasome Inhibition

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 273-273 ◽  
Author(s):  
Michal Sheffer ◽  
Yiguo Hu ◽  
Ophir Shalem ◽  
Neville Sanjana ◽  
Eugen Dhimolea ◽  
...  
Keyword(s):  
Resistance Genes ◽  
Proteasome Inhibitor ◽  
Research Funding ◽  
Molecular Mechanisms ◽  
Patent Application ◽  
Loss Of Function ◽  
Genome Wide ◽  
Cas9 Nuclease ◽  
A Genome ◽  
Genome Scale

Abstract Acquired or de novo resistance to established and investigational therapies represents a major clinical challenge for multiple myeloma (MM) and other neoplasias. Despite extensive efforts, clinically-validated molecular markers that predict for proteasome inhibitor (PSI) resistance in most MM patients remain elusive. This challenge is partly due to limited availability so far of molecular data on MM patients before the start of PSI treatment vs. immediately after resistance to it develops; this challenge may also reflect the heterogeneity of the complex molecular mechanisms regulating MM cell response to PSIs. We hypothesized that resistance to PSIs can be mediated by disruption of several functionally overlapping genes, and that the prevalence of any of these lesions may be too low to detect in datasets available thus far. To examine this latter hypothesis, we performed a genome-wide screen for genes whose loss confers to MM cells resistance against bortezomib, through the use of the CRISPR (clustered regularly interspaced short palindromic repeats)–associated nuclease Cas9 system. Specifically RPMI-8226 MM cells were transduced with lentiviral construct for Cas9 nuclease, followed by lentiviral delivery of a genome-scale pooled library of 123,411 single-guide RNAs (sgRNAs), which selectively align to target sequences at the 5′ constitutive exons of 18,080 genes and direct the Cas9 nuclease to cause double-stranded cleavage and loss of function of the respective gene. From the pool of MM cells transduced with the sgRNA library and treated with bortezomib, treatment-resistant cells were processed for deep sequencing, to identify enriched sgRNAs and their corresponding genes. We identified that loss-of-function of 33 candidate genes is associated with bortezomib resistance. We observed a high level of consistency between independent sgRNAs targeting the same gene, as well as a high rate of hit confirmation across different biological replicates. Notably, this set of candidate bortezomib-resistance genes was distinct from the "hits" we identified through a parallel CRISPR screen on the same cell line for resistance to a different targeted therapy (namely the bromodomain inhibitor JQ1), supporting the ability of this approach to identify treatment-specific resistance genes. These candidate bortezomib-resistance genes have documented or presumed roles in the regulation of extrinsic and intrinsic apoptotic cascades, autophagy, Toll-like receptor and NF-kappaB signaling, aggresome function, heat shock protein expression, chromatin remodeling, nutrient sensing, and tumor suppressor gene networks. Importantly, information from several publically available molecular profiling datasets converge to support the putative clinical relevance of these genes. For instance, gene expression data from tumor cells of bortezomib-naive patients with advanced MM revealed several transcriptional signatures of these candidate genes (defined by low transcript levels for any of the genes in the signature) which correlated with shorter time to disease progression after treatment with bortezomib (p<0.01, log-rank test), but not dexamethasone (p>0.426). Congruent with these findings, the highly bortezomib-responsive clinical setting of newly-diagnosed MM is associated with low cumulative frequency of mutations of these bortezomib-resistance genes (e.g. cumulative mutation rate of 3.9%, 95% confidence interval [CI] 1.25-6.55%). Notably, in other malignancies that are typically PSI-resistant, a higher cumulative frequency of such lesions is observed (average of ~28%, range 0-76%, 95% CI 22.46-32.70%; 57 datasets from 20+ neoplasias examined). In summary, this first application of the CRISPR/Cas9-based technology in MM illustrates its power to interrogate gene function on a genome-wide scale. This approach identifies bortezomib-resistance genes that are associated with pathways linked with the regulation of proteasome inhibitor response. Results from molecularly-annotated clinical samples converge to support a possible role for these genes in bortezomib resistance. This experience supports the value of CRISPR/Cas9-based studies to dissect the molecular mechanisms of treatment resistance in MM and other hematologic neoplasias (* equal contribution of M.S. and Y.H.). Disclosures Shalem: Broad Institute: Patent application for CRISPR technology Patents & Royalties. Sanjana:Broad Institute: Patent application for CRISPR technology Patents & Royalties. Zhang:Broad Institute: Patent application for CRISPR technology Patents & Royalties. Mitsiades:Johnson & Johnson: Research Funding; Amgen: Research Funding; Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals: Consultancy, Honoraria.

scholarly journals Genome-wide RNA interference screening reveals a COPI-MAP2K3 pathway required for YAP regulation

2020 ◽  
Vol 117 (33) ◽  
pp. 19994-20003 ◽  
Author(s):  
Yong Joon Kim ◽  
Eunji Jung ◽  
Eunbie Shin ◽  
Sin-Hyoung Hong ◽  
Hui Su Jeong ◽  
...  
Keyword(s):  
Rna Interference ◽  
Regulatory Mechanism ◽  
Inflammatory Diseases ◽  
Transcriptional Regulator ◽  
Endoplasmic Reticulum Stress Response ◽  
Loss Of Function ◽  
Genome Wide ◽  
A Genome ◽  
Stress Response Pathway ◽  
Kinase Cascade

The transcriptional regulator YAP, which plays important roles in the development, regeneration, and tumorigenesis, is activated when released from inhibition by the Hippo kinase cascade. The regulatory mechanism of YAP in Hippo-low contexts is poorly understood. Here, we performed a genome-wide RNA interference screen to identify genes whose loss of function in a Hippo-null background affects YAP activity. We discovered that the coatomer protein complex I (COPI) is required for YAP nuclear enrichment and that COPI dependency of YAP confers an intrinsic vulnerability to COPI disruption in YAP-driven cancer cells. We identified MAP2K3 as a YAP regulator involved in inhibitory YAP phosphorylation induced by COPI subunit depletion. The endoplasmic reticulum stress response pathway activated by COPI malfunction appears to connect COPI and MAP2K3. In addition, we provide evidence that YAP inhibition by COPI disruption may contribute to transcriptional up-regulation of PTGS2 and proinflammatory cytokines. Our study offers a resource for investigating Hippo-independent YAP regulation as a therapeutic target for cancers and suggests a link between YAP and COPI-associated inflammatory diseases.

scholarly journals Genome-Scale CRISPR Screening Identifies Novel Human Pluripotent Gene Networks

2018 ◽  
Author(s):  
Robert J. Ihry ◽  
Max R. Salick ◽  
Daniel J. Ho ◽  
Marie Sondey ◽  
Sravya Kommineni ◽  
...  
Keyword(s):  
Gene Networks ◽  
Loss Of Function ◽  
Preclinical Research ◽  
Novel Genes ◽  
Cell Library ◽  
Fundamental Properties ◽  
Cell Clones ◽  
Genome Wide ◽  
Genome Scale ◽  
At Will

ABSTRACTHuman pluripotent stem cells (hPSCs) generate a wide variety of disease-relevant cells that can be used to improve the translation of preclinical research. Despite the potential of hPSCs, their use for genetic screening has been limited because of technical challenges. We developed a renewable Cas9/sgRNA-hPSC library where loss-of-function mutations can be induced at will. Our inducible-mutant hPSC library can be used for an unlimited number of genome-wide screens. We screened for novel genes involved in 3 of the fundamental properties of hPSCs: Their ability to self-renew/survive, their capacity to differentiate into somatic cells, and their inability to survive as single-cell clones. We identified a plethora of novel genes with unidentified roles in hPSCs. These results are available as a resource for the community to increase the understanding of both human development and genetics. In the future, our stem cell library approach will be a powerful tool to identify disease-modifying genes.VISUAL ABSTRACT

scholarly journals A genome-scale CRISPR interference guide library enables comprehensive phenotypic profiling in yeast

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Nicholas J. McGlincy ◽  
Zuriah A. Meacham ◽  
Kendra K. Reynaud ◽  
Ryan Muller ◽  
Rachel Baum ◽  
...  
Keyword(s):  
In Vitro Transcription ◽  
Essential Genes ◽  
Competitive Growth ◽  
Loss Of Function ◽  
Design Rules ◽  
Growth Defects ◽  
Genome Wide ◽  
A Genome ◽  
Genome Scale

Abstract Background CRISPR/Cas9-mediated transcriptional interference (CRISPRi) enables programmable gene knock-down, yielding loss-of-function phenotypes for nearly any gene. Effective, inducible CRISPRi has been demonstrated in budding yeast, and genome-scale guide libraries enable systematic, genome-wide genetic analysis. Results We present a comprehensive yeast CRISPRi library, based on empirical design rules, containing 10 distinct guides for most genes. Competitive growth after pooled transformation revealed strong fitness defects for most essential genes, verifying that the library provides comprehensive genome coverage. We used the relative growth defects caused by different guides targeting essential genes to further refine yeast CRISPRi design rules. In order to obtain more accurate and robust guide abundance measurements in pooled screens, we link guides with random nucleotide barcodes and carry out linear amplification by in vitro transcription. Conclusions Taken together, we demonstrate a broadly useful platform for comprehensive, high-precision CRISPRi screening in yeast.

scholarly journals Genome scale CRISPR Cas9a knockout screen reveals genes that controls glioblastoma susceptibility to the alkylating agent temozolomide

2021 ◽  
Author(s):  
Chidiebere U Awah ◽  
Jan Winter ◽  
Olorunseun Ogunwobi
Keyword(s):  
Overall Survival ◽  
Large Scale ◽  
Alkylating Agent ◽  
Cancer Resistance ◽  
Tumor Resistance ◽  
Loss Of Function ◽  
Mechanisms Of Resistance ◽  
Genome Wide ◽  
Genome Level ◽  
Genome Scale

Glioblastoma is the most fatal of all primary human brain tumors with 14 months survival, at best. The mainstay therapy for this tumor involves temozolomide, surgery, radiotherapy and tumor treating electric field. Cancer resistance to commonly available chemotherapeutics remains a major challenge in glioblastoma patients receiving treatment and unfavorably impact their overall survival and outcome. However, the lack of progress in this area could be attributed to lack of tools to probe unbiasedly at the genome wide level the coding and non-coding elements contribution on a large scale for factors that control resistance to chemotherapeutics. Understanding the mechanisms of resistance to chemotherapeutics will enable precision medicine in the treatment of cancer patients. CRISPR Cas9a has emerged as a functional genomics tool to study at genome level the factors that control cancer resistance to drugs. Recently, we used genome wide CRISPR-Cas9a screen to identify genes responsible for glioblastoma susceptibility to etoposide. We extended our inquiry to understand genes that control glioblastoma response to temozolomide by using genome scale CRISPR. This study shows that the unbiased genome-wide loss of function approach can be applied to discover genes that influence tumor resistance to chemotherapeutics and contribute to chemoresistance in glioblastoma.

scholarly journals Next-generation libraries for robust RNA interference-based genome-wide screens

2015 ◽  
Vol 112 (26) ◽  
pp. E3384-E3391 ◽  
Author(s):  
Martin Kampmann ◽  
Max A. Horlbeck ◽  
Yuwen Chen ◽  
Jordan C. Tsai ◽  
Michael C. Bassik ◽  
...  
Keyword(s):  
Rna Interference ◽  
Mammalian Cell Culture ◽  
High Sensitivity ◽  
High Specificity ◽  
Next Generation ◽  
Loss Of Function ◽  
Shrna Expression ◽  
Protein Coding ◽  
Genome Wide ◽  
Shrna Library

Genetic screening based on loss-of-function phenotypes is a powerful discovery tool in biology. Although the recent development of clustered regularly interspaced short palindromic repeats (CRISPR)-based screening approaches in mammalian cell culture has enormous potential, RNA interference (RNAi)-based screening remains the method of choice in several biological contexts. We previously demonstrated that ultracomplex pooled short-hairpin RNA (shRNA) libraries can largely overcome the problem of RNAi off-target effects in genome-wide screens. Here, we systematically optimize several aspects of our shRNA library, including the promoter and microRNA context for shRNA expression, selection of guide strands, and features relevant for postscreen sample preparation for deep sequencing. We present next-generation high-complexity libraries targeting human and mouse protein-coding genes, which we grouped into 12 sublibraries based on biological function. A pilot screen suggests that our next-generation RNAi library performs comparably to current CRISPR interference (CRISPRi)-based approaches and can yield complementary results with high sensitivity and high specificity.

137 Novel Genome-wide CRISPR Screen for Glioblastoma Invasion

Neurosurgery ◽  
2017 ◽  
Vol 64 (CN_suppl_1) ◽  
pp. 232-232
Author(s):  
Laura Marie Prolo ◽  
Amy Li ◽  
David Morgens ◽  
Richard Reimer ◽  
Michael Bassik ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Cell Populations ◽  
Boyden Chamber ◽  
Normal Brain ◽  
Loss Of Function ◽  
Glioma Invasion ◽  
Knock Out ◽  
Genome Wide ◽  
A Genome

Abstract INTRODUCTION Among high-grade pediatric brain tumors, glioblastoma multiforme (GBM) is particularly difficult to treat. Invasion of GBM cells into normal brain parenchyma renders malignant cells inaccessible to surgical intervention and poised to drive tumor recurrence. In order to better understand the molecular mechanisms and signaling pathways underlying pediatric GBM cell invasion we performed a genome-wide CRISPR/Cas9 loss of function screen for genes that inhibit invasion. METHODS CRISPR/Cas9 is an emerging technology for genome editing that can be used to knock out genes that are targeted by short RNA guide sequences (sgRNAs). First, we made an immortalized human glioma line that stably expresses the RNA-guided DNA endonuclease, Cas9, and showed that Cas9 is functional in these cells. We then used a pooled approach to knock-out individual genes on a genome-wide scale in the Cas9 GBM cell line. Pooled cells were subjected to a Boyden chamber invasion assay which separated the cell populations based on invasive phenotype. To determine which genes were enriched in the invasive cell fraction compared to the noninvasive cell population we used next generation sequencing of the sgRNAs isolated and amplified from each group. RESULTS >Analysis of sgRNA enrichment in invasive compared to noninvasive cell populations identified specific genes expected to be altered in migration, such as those involved with actin based cell projections, as well as novel candidate genes. We are currently testing the necessity and sufficiency of these genes for glioma invasion in vitro and in vivo using a xenograft mouse model. CONCLUSION In this study, we identified critical genes necessary for GBM invasion using a novel genome-wide knock-out approach. Once validated, the genes of interest may serve as prognostic surveillance markers or targets for therapeutic intervention in pediatric glioblastoma.

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