Generation of an arrayed CRISPR-Cas9 library targeting epigenetic regulators: from high-content screens to in vivo assays
journal contributionposted on 20.08.2020, 11:19 authored by Tristan Henser-Brownhill, Josep Monserrat, Paola Scaffidi
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.
CRISPRDNA methylationarrayed librarycancer biologychromatinchromatin remodelingepigeneticshigh-contenthistone modificationssgRNAAnimalsCRISPR-Cas SystemsCloning, MolecularEpigenesis, GeneticGene LibraryGenetic VectorsHep G2 CellsHumansLentivirusMaleMiceMice, Inbred NODMice, SCIDRNA, GuideScaffidi FC001152GEP-ackHTS-ack0604 Genetics0601 Biochemistry and Cell Biology1101 Medical Biochemistry and MetabolomicsDevelopmental Biology