Journal
EPIGENETICS
Volume 12, Issue 12, Pages 1065-1075Publisher
TAYLOR & FRANCIS INC
DOI: 10.1080/15592294.2017.1395121
Keywords
epigenetics; CRISPR; arrayed library; sgRNA; high-content; DNA methylation; cancer biology; chromatin; chromatin remodeling; histone modifications
Funding
- Cancer Research UK [C50796/A19448, FC001152]
- Medical Research Council (MRC) [FC001152]
- Wellcome Trust [FC001152]
- Cancer Research UK [19448] Funding Source: researchfish
- The Francis Crick Institute [10152, C50796/A19448] Funding Source: researchfish
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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.
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