Generation of an arrayed CRISPR-Cas9 library targeting epigenetic regulators: from high-content screens to in vivo assays
| Autor: | Henser-Brownhill, Tristan, Monserrat, Josep, Scaffidi, Paola |
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| Rok vydání: | 2017 |
| Předmět: |
Male
0301 basic medicine Cancer Research Genetic Vectors Mice SCID Computational biology Biology Chromatin remodeling chromatin remodeling Epigenesis Genetic Genome engineering Mice 03 medical and health sciences Mice Inbred NOD Animals Humans CRISPR Genomic library Epigenetics Guide RNA Cloning Molecular Molecular Biology Gene cancer biology Gene Library DNA methylation epigenetics histone modifications Lentivirus high-content Hep G2 Cells Research Papers arrayed library 030104 developmental biology chromatin sgRNA CRISPR-Cas Systems RNA Guide Kinetoplastida |
| Zdroj: | Epigenetics Henser-Brownhill, T, Monserrat, J & Scaffidi, P 2017, ' Generation of an arrayed CRISPR-Cas9 library targeting epigenetic regulators: from high-content screens to in vivo assays ', Epigenetics, vol. 12, no. 12, pp. 1065-1075 . https://doi.org/10.1080/15592294.2017.1395121 |
| ISSN: | 1559-2308 1559-2294 |
| DOI: | 10.1080/15592294.2017.1395121 |
| Popis: | The CRISPR-Cas9 system has revolutionized genome engineering, allowing precise modification of DNA in various organisms. The most popular method for conducting CRISPR-based functional screens involves the use of pooled lentiviral libraries in selection screens coupled with next-generation sequencing. Screens employing genome-scale pooled small guide RNA (sgRNA) libraries are demanding, particularly when complex assays are used. Furthermore, pooled libraries are not suitable for microscopy-based high-content screens or for systematic interrogation of protein function. To overcome these limitations and exploit CRISPR-based technologies to comprehensively investigate epigenetic mechanisms, we have generated a focused sgRNA library targeting 450 epigenetic regulators with multiple sgRNAs in human cells. The lentiviral library is available both in an arrayed and pooled format and allows temporally-controlled induction of gene knock-out. Characterization of the library showed high editing activity of most sgRNAs and efficient knock-out at the protein level in polyclonal populations. The sgRNA library can be used for both selection and high-content screens, as well as for targeted investigation of selected proteins without requiring isolation of knock-out clones. Using a variety of functional assays we show that the library is suitable for both in vitro and in vivo applications, representing a unique resource to study epigenetic mechanisms in physiological and pathological conditions. |
| Databáze: | OpenAIRE |
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