A systematic screening assay identifies efficient small guide RNAs for CRISPR activation

Abstract

AbstractCRISPR-mediated gene activation (CRISPRa) encompasses a growing field of biotechnological approaches with exciting implications for gene therapy. However, there is a lack of experimental validation tools for selecting efficient sgRNAs for downstream applications. Here, we present a screening assay capable of identifying efficient single- and double sgRNAs through fluorescence quantification in vitro. In addition, we provide a tailored Golden Gate cloning workflow for streamlined incorporation of selected sgRNA candidates into lentiviral (LVs) or adeno-associated vectors (AAVs). The overall workflow was validated using therapeutically relevant genes for neurodegenerative diseases, such asTfeb,Adam17, andSirt1. The most efficient sgRNAs also demonstrated activation of endogenous gene expression at mRNA and protein levels. Further proof- of-principle assays usingTfebindicated that gene activation was accompanied by increased levels of Lc3b. This data demonstrates the potential of the screening assay to identify functionally efficient sgRNA candidates across multiple genes along with streamlined cloning of viral vectors and may assist in accelerating future developments of CRISPRa-focused applications.