Development of synthetic guide RNA libraries for CRISPR-mediated transcriptional activation screening for gain-of-function studies

Functional gene analysis studies have been empowered by development of CRISPR-Cas9 gene knockout tools, however the CRISPR-Cas9 system has also been adapted for inhibition or activation of gene transcription. A nuclease-deactivated Cas9 (dCas9) can be fused to various effector domains to produce an RNA-guided transcription factor for either inhibition (CRISPRi) or activation (CRISPRa) of target genes. For overexpression studies, CRISPRa holds significant advantages over traditional vector-based gene expression, because genes are upregulated from their native promoter and endogenous genomic context. The majority of CRISPRa research performed to date has utilized single guide RNA (sgRNA) expressed from a DNA vector, primarily in the context of pooled lentiviral screening approaches. Here we describe the development of CRISPRa synthetic guide RNAs for the use in arrayed screening, so that we combine this next-generation transcriptional activation method with the ability to support more complex assays in a one-gene-per well format. Considerations for arrayed activation screens will be shown. The combination of gain-of-function from CRISPRa with loss-of-function using RNAi or canonical CRISPR-Cas9 for gene knockout allows for robust characterization of gene mechanisms and pathways. Citation Format: Annaleen Vermeulen, Zaklina Strezoska, Sarah M. Dickerson, Maren M. Gross, Eldon T. Chou, Elena Maksimova, Matthew R. Perkett, Shawn McClelland, Anja V. Smith. Development of synthetic guide RNA libraries for CRISPR-mediated transcriptional activation screening for gain-of-function studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2761.