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
, 12
(12)
pp. 1065-1075.
10.1080/15592294.2017.1395121.
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Generation of an arrayed CRISPR Cas9 library targeting epigenetic regulators from high content screens to in vivo assays.pdf - Published Version Download (1MB) | Preview |
Abstract
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.
| Type: | Article |
|---|---|
| Title: | Generation of an arrayed CRISPR-Cas9 library targeting epigenetic regulators: from high-content screens to in vivo assays |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1080/15592294.2017.1395121 |
| Publisher version: | http://doi.org/10.1080/15592294.2017.1395121 |
| Language: | English |
| Additional information: | Copyright © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
| Keywords: | epigenetics; CRISPR; arrayed library; sgRNA; high-content; DNA methylation; cancer biology; chromatin; chromatin remodeling; histone modifications |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10046603 |
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