Validation of Synthetic CRISPR Reagents as a Tool for Arrayed Functional Genomic Screening

Tan, Jenille; Martin, Scott E.

doi:10.1371/journal.pone.0168968

Cited by 38 publications

(25 citation statements)

References 34 publications

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“…For this reason, an important prerequisite for these screens would be to be able to transfect low number of cells with minimal cytotoxicity. We have observed that with liquid transfection some cell lines such as RPE‐1 can be efficiently transfected when low number of cells was seeded (2,500 cells/well) as previously reported in the literature for other cell lines (Tan & Martin, ; Strezoska et al , ; de Groot et al , ). However, some cell lines such as NCI‐H358, NCI‐N87, or HEK293T that also express doxycycline‐inducible Cas9 (Fig EV1) suffer from increased cytotoxicity and high variability under the same conditions.…”

Section: Resultssupporting

confidence: 74%

A solid‐phase transfection platform for arrayed CRISPR screens

Serçin

Reither

Roidos

et al. 2019

Molecular Systems Biology

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Arrayed CRISPR‐based screens emerge as a powerful alternative to pooled screens making it possible to investigate a wide range of cellular phenotypes that are typically not amenable to pooled screens. Here, we describe a solid‐phase transfection platform that enables CRISPR‐based genetic screens in arrayed format with flexible readouts. We demonstrate efficient gene knockout upon delivery of guide RNAs and Cas9/guide RNA ribonucleoprotein complexes into untransformed and cancer cell lines. In addition, we provide evidence that our platform can be easily adapted to high‐throughput screens and we use this approach to study oncogene addiction in tumor cells. Finally demonstrating that the human primary cells can also be edited using this method, we pave the way for rapid testing of potential targeted therapies.

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Section: Resultssupporting

confidence: 74%

A solid‐phase transfection platform for arrayed CRISPR screens

Serçin

Reither

Roidos

et al. 2019

Molecular Systems Biology

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“…Some groups have previously attempted to develop arrayed CRISPR screens (Hultquist et al 2016;McCleland et al 2016;Tan and Martin 2016;Datlinger et al 2017;Strezoska et al 2017). These studies were based on guide RNA libraries consisting of individual lentiviral sgRNAs for single genes or synthesized CRISPR RNA (crRNA) in a manner similar to siRNA screens.…”

mentioning

confidence: 99%

Arrayed CRISPR screen with image-based assay reliably uncovers host genes required for coxsackievirus infection

Lee

Kim

et al. 2018

Genome Res.

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Pooled CRISPR screens based on lentiviral systems have been widely applied to identify the effect of gene knockout on cellular phenotype. Although many screens were successful, they also have the limitation that genes conferring mild phenotypes or those essential for growth can be overlooked, as every genetic perturbation is incorporated in the same population. Arrayed screens, on the other hand, incorporate a single genetic perturbation in each well and could overcome these limitations. However, arrayed screens based on siRNA-mediated knockdown were recently criticized for low reproducibility caused by incomplete inhibition of gene expression. To overcome these limitations, we developed a novel arrayed CRISPR screen based on a plasmid library expressing a single guide RNA (sgRNA) and disrupted 1514 genes, encoding kinases, proteins related to endocytosis, and Golgi-localized proteins, individually using 4542 sgRNAs (three sgRNAs per gene). This screen revealed host factors required for infection by coxsackievirus B3 (CVB3) from Picornaviridae, which includes human pathogens causing diverse diseases. Many host factors that had been overlooked in a conventional pooled screen were identified for CVB3 infection, including entry-related factors, translational initiation factors, and several replication factors with different functions, demonstrating the advantage of the arrayed screen. This screen was quite reliable and reproducible, as most genes identified in the primary screen were confirmed in secondary screens. Moreover, ACBD3, whose phenotype was not affected by siRNA-mediated knockdown, was reliably identified. We propose that arrayed CRISPR screens based on sgRNA plasmid libraries are powerful tools for arrayed genetic screening and applicable to larger-scale screens.

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“…However, correlation analysis between KIAA1377 expression versus clinicopathological variables revealed that there was a significant correlation of KIAA1377 expression with lymph nodes metastases, which leads to the suggestion that KIAA1377 might be involved in the lymph node metastasis of ESCC cells. The slight discrepancy between in vivo and in vitro prompted us to rework the biological roles of KIAA1377 implicated in the proliferation and motility of SCC cells by using the alternative CRISPR‐Cas9 technique (Shalem et al, ), which is distinctively different from the shRNA system in the case of silencing efficiency (Tan and Martin, ; Wang et al, ). In the current observation, knock‐out of KIAA1377 mediated by the CRISPR‐Cas9 system turns out to be able to markedly slow down the growth and motility of KYSE‐150 and HeLa cells, preliminarily defining the oncogenic roles of KIAA1377 in SCC cells.…”

Section: Discussionmentioning

confidence: 99%

Let‐7b‐5p inhibits proliferation and motility in squamous cell carcinoma cells through negative modulation of KIAA1377

Liu

Tan

et al. 2019

Cell Biology International

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KIAA1377 has been found to be linked with lymph node metastasis in esophageal squamous cell carcinoma (SCC) in our previous study; however, the regulation of KIAA1377 remains far from understood. Herein, to understand the regulation of KIAA1377 from the angle of microRNA (miRNA)–messenger RNA (mRNA) modulation in the setting of SCC cells, the basal level of KIAA1377 was determined by quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blot analysis in KYSE‐150 and HeLa cells; biological roles of KIAA1377 contributing in the proliferation, migration, and invasion were evaluated using 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT), wound‐healing and Transwell assays, respectively, after KIAA1377 was knocked out mediated by the CRISPR‐Cas9 system. Bioinformatic prediction revealed that let‐7b‐5p was a putative miRNA regulating KIAA1377, which was ensuingly validated by the luciferase reporter assay; after which, variation of KIAA1377 expression was further verified by qRT‐PCR and western blot analysis. Moreover, the biological roles of let‐7b‐5p in proliferation, migration, and invasion of KYSE‐150 and HeLa cells were also evaluated. It was exhibited that KIAA1377 was able to promote the proliferation and motility of both KYSE‐150 and HeLa cells, which can be reverted by re‐expression of let‐7b‐5p. The luciferase reporter assay verified that let‐7b‐5p can diametrically target KIAA1377. Collectively, our data demonstrated that let‐7b‐5p can directly but negatively regulate KIAA1377 in SCC cell lines, Ecal109, and HeLa cells.

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Validation of Synthetic CRISPR Reagents as a Tool for Arrayed Functional Genomic Screening

Cited by 38 publications

References 34 publications

A solid‐phase transfection platform for arrayed CRISPR screens

A solid‐phase transfection platform for arrayed CRISPR screens

Arrayed CRISPR screen with image-based assay reliably uncovers host genes required for coxsackievirus infection

Let‐7b‐5p inhibits proliferation and motility in squamous cell carcinoma cells through negative modulation of KIAA1377

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