High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells

Zhou, Yan; Zhu, Shifan; Cai, Changzu; Yuan, Peng; Li, Chunmei; Huang, Yanyi; Wei, Wensheng

doi:10.1038/nature13166

Cited by 628 publications

(526 citation statements)

References 26 publications

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“…In eukaryotes, the DSBs are more commonly repaired by the mechanism of error-prone non-homologous end joining (NHEJ), therefore generating sequence changes, for instance insertions and deletions (indels), around the DSBs . Owing to the simplicity of manipulation and versatility, the CRISPR/Cas9 system has been utilized as an attractive tool for various applications, such as genome-wide screening (Shalem et al, 2014;Zhou et al, 2014), gene repression and activation (Cheng et al, 2013;Doench et al, 2014;Gilbert et al, 2014), targeted fluorescence imaging (Tanenbaum et al, 2014) and novel approaches against pathogens including hepatitis B virus (Lin et al, 2014a;Seeger & Sohn, 2014), human papillomavirus (Kennedy et al, 2014), Epstein-Barr virus (Wang & Quake, 2014;Yuen et al, 2015), malaria (Wagner et al, 2014) and HIV-1 (Ebina et al, 2013;Hu et al, 2014;Ye et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9

Guan

et al. 2015

Journal of General Virology

177

132

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CCR5 serves as an essential coreceptor for human immunodeficiency virus type 1 (HIV-1) entry, and individuals with a CCR5 D32 variant appear to be healthy, making CCR5 an attractive target for control of HIV-1 infection. The CRISPR/Cas9, which functions as a naturally existing adaptive immune system in prokaryotes, has been recently harnessed as a novel nuclease system for genome editing in mammalian cells. Although CRISPR/Cas9 can be readily delivered into cell lines, due to the large size of the Cas9 protein, efficient delivery of CCR5-targeting CRISPR/Cas9 components into primary cells, including CD4 + T-cells, the primary target for HIV-1 infection in vivo, remains a challenge. In the current study, following design of a panel of top-ranked single-guided RNAs (sgRNAs) targeting the ORF of CCR5, we demonstrate that CRISPR/Cas9 can efficiently mediate the editing of the CCR5 locus in cell lines, resulting in the knockout of CCR5 expression on the cell surface. Next-generation sequencing revealed that various mutations were introduced around the predicted cleavage site of CCR5. For each of the three most effective sgRNAs that we analysed, no significant off-target effects were detected at the 15 top-scoring potential sites. More importantly, by constructing chimeric Ad5F35 adenoviruses carrying CRISPR/Cas9 components, we efficiently transduced primary CD4 + Tlymphocytes and disrupted CCR5 expression, and the positively transduced cells were conferred with HIV-1 resistance. To our knowledge, this is the first study establishing HIV-1 resistance in primary CD4 + T-cells utilizing adenovirus-delivered CRISPR/Cas9.

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Section: Introductionmentioning

confidence: 99%

Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9

Guan

et al. 2015

Journal of General Virology

177

132

View full text Add to dashboard Cite

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“…CRISPR has also facilitated multiplexed metabolic pathway engineering in yeast at high efficiencies (Jakociunas et al 2015a,b), as well as random mutagenesis of yeast chromosomal DNA for phenotypic screening of desired mutants (Ryan et al 2014). Indeed, genome-wide CRISPR-based knockout screens hold tremendous potential for functional genomics (Hilton and Gersbach 2015), having facilitated the discovery of genomic loci that confer drug resistance to cells (Koike-Yusa et al 2014;Shalem et al 2014;Wang et al 2014a;Zhou et al 2014), uncovered how cells can control induction of the host immune response (Parnas et al 2015), provided new insights into the genetic basis of cellular fitness (Hart et al 2015;Wang et al 2015b), and even shed light on how certain viruses induce cell death . Genome-wide CRISPR screens can also facilitate the discovery of functional noncoding elements (Kim et al 2013b;Korkmaz et al 2016), and provide a means for studying the structure and evolution of the human genome.…”

Section: Synthetic Biology and Genome-scale Engineeringmentioning

confidence: 99%

Genome-Editing Technologies: Principles and Applications

Gaj

Sirk

Shui

et al. 2016

Cold Spring Harb Perspect Biol

226

126

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Targeted nucleases have provided researchers with the ability to manipulate virtually any genomic sequence, enabling the facile creation of isogenic cell lines and animal models for the study of human disease, and promoting exciting new possibilities for human gene therapy. Here we review three foundational technologies-clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs). We discuss the engineering advances that facilitated their development and highlight several achievements in genome engineering that were made possible by these tools. We also consider artificial transcription factors, illustrating how this technology can complement targeted nucleases for synthetic biology and gene therapy.

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“…For instance, a recent report developed a high-throughput assay in human cells using a CRISPR/cas-based method to knock out genes systematically to determine their function in bacterial toxicity [67]. Additionally, CRISPR/cas and TALENS have been shown to have low rates of off-targeting following whole-genome sequencing of their modified iPSCs [68].…”

Section: Figmentioning

confidence: 99%

Induced Pluripotent Stem Cell Models to Enable In Vitro Models for Screening in the Central Nervous System

Hunsberger

Efthymiou

Malik

et al. 2015

Stem Cells and Development

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There is great need to develop more predictive drug discovery tools to identify new therapies to treat diseases of the central nervous system (CNS). Current nonpluripotent stem cell-based models often utilize non-CNS immortalized cell lines and do not enable the development of personalized models of disease. In this review, we discuss why in vitro models are necessary for translational research and outline the unique advantages of induced pluripotent stem cell (iPSC)-based models over those of current systems. We suggest that iPSC-based models can be patient specific and isogenic lines can be differentiated into many neural cell types for detailed comparisons. iPSC-derived cells can be combined to form small organoids, or large panels of lines can be developed that enable new forms of analysis. iPSC and embryonic stem cell-derived cells can be readily engineered to develop reporters for lineage studies or mechanism of action experiments further extending the utility of iPSC-based systems. We conclude by describing novel technologies that include strategies for the development of diversity panels, novel genomic engineering tools, new three-dimensional organoid systems, and modified high-content screens that may bring toxicology into the 21st century. The strategic integration of these technologies with the advantages of iPSC-derived cell technology, we believe, will be a paradigm shift for toxicology and drug discovery efforts.Disease Modeling D iseases of the central nervous system (CNS) affect a large number of people, but therapeutic intervention is hampered by the lack of useful models for many of these diseases. Current research on human subjects, particularly for drug discovery for CNS diseases, is severely (and appropriately) limited by ethical guidelines. Therefore, surrogate models are needed that share important anatomical, physiological, and genetic features to advance new treatments and therapies for CNS diseases [1].Developing rapid and effective therapies for CNS diseases requires the availability of in vitro models that accurately recapitulate disease phenotypes and predict patient treatment response. A proper model must be both sensitive and predictive while reflecting both normal and disease processes. Equally important, these models should enable the investigation of genetic and environmental risk factors contributing to diseases in a rapid and economical way. Currently used models often do not reflect a typical human response [2-4], despite efforts underway to better characterize these models and increase their preclinical value in predicting safety and efficacy in the clinic [5,6]. Therefore, there is a great need to develop disease-and patient-specific models from cells directly affected in CNS disorders. These cellbased models, we envision, could either replace or supplement current animal models and enable the efficient translation of basic research into the clinical setting. Limitations with current CNS modelsCurrently, drug discovery relies on the use of animalbased or cell-based models, ...

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High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells

Cited by 628 publications

References 26 publications

Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9

Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9

Genome-Editing Technologies: Principles and Applications

Induced Pluripotent Stem Cell Models to Enable In Vitro Models for Screening in the Central Nervous System

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