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Arrayed synthetic CRISPR libraries are a powerful resource for gene knockout or gene activation screening
Arrayed synthetic CRISPR libraries are a powerful resource for gene knockout or gene activation screening
Submitted on 22 Jun 2018

Žaklina Strezoska, Hidevaldo Machado, Matthew Perkett, Eldon Chou, Elena Maksimova, Emily Anderson, Maren Gross, Anja Smith
Dharmacon, part of Horizon Discovery Group
This poster was presented at Keystone Symposia on Mol. and Cell. Biology: Exosomes/Microvesicles
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Poster Abstract
Originally discovered as an adaptive immune system defense against foreign nucleic acid in bacteria and archaea, CRISPR-Cas9 technology has revolutionized the field of biology as a tool for genome engineering. Particularly, the Cas9 nuclease from S. pyogenes has been successfully used to produce gene knockouts (CRISPRko). In addition, this system can also be repurposed for target gene activation (CRISPRa) by fusing functional transcriptional activator domains to a nuclease-deactivated Cas9 (dCas9), enabling gain-of-function experiments. Here we demonstrate strategies to screen multiple genes with CRISPRko or CRISPRa using synthetic crRNA. A synthetic crRNA approach is amenable to screening in an arrayed, well-by-well fashion and expands the types of phenotypic readouts that can be used, including high-content and morphology-based assays. For CRISPRko, we used a cell cycle reporter cell line to perform an arrayed screen targeting 169 genes with four crRNAs per gene using high content analysis (HCA) to identify genes that regulate the cell cycle. Multiple parameters were used to classify cells into different cellular states and phases of the cell cycle: apoptotic cells, cells with irregularly shaped nuclei, cells in G1 phase, cells either in S or G2 phase, cells in mitosis or with condensed chromatin, and cells with multinuclear DNA component. Most hits had three to four positive crRNAs per target gene, enabling identification of target genes with high confidence, and demonstrating the power of combining synthetic crRNAs libraries with HCA assays in screening for complex cellular phenotypes in an arrayed format. For CRISPRa, we examined the functionality and advantages of using the synthetic approach in multiple cell lines. We show how the transcriptional activation leads to a significant increase in the target protein level, which in turn causes phenotypic effects by inhibiting or activating downstream genes. The arrayed crRNA library screening methods presented are broadly applicable as a strategy to inactivate or up-regulate any gene for systematic functional gene analysis.

Ž. Strezoska, M.R. Perkett, E.T. Chou, E. Maksimova, E.M. Anderson, S. McClelland, M.L. Kelley, A. Vermeulen and A. van Brabant Smith. High-content analysis screening for cell cycle regulators using arrayed synthetic crRNA libraries. Journal of Biotechnology 251, 189-200 (2017).Report abuse »
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