biGMamAct: efficient CRISPR/Cas9-mediated docking of large functional DNA cargoes at theACTBlocus

Abstract

Recent advances in molecular and cell biology and imaging have unprecedentedly enabled multi-scale structure-functional studies of entire metabolic pathways from atomic to micrometer resolution, and the visualization of macromolecular complexes in situ, especially if these molecules are expressed with appropriately-engineered and easily-detectable tags. However, genome editing in eukaryotic cells is challenging when generating stable cell lines loaded with large DNA cargoes. To address this limitation, here, we have conceived biGMamAct, a system that allows the straightforward assembly of a multitude of genetic modules and their subsequent integration in the genome at the ACTB locus with high efficacy, through standardized cloning steps. Our technology encompasses a set of modular plasmids for mammalian expression, which can be efficiently docked into the genome in tandem with a validated Cas9/sgRNA pair through homologous-independent targeted insertion (HITI). As a proof of concept, we have generated a stable cell line loaded with an 18.3-kilobase-long DNA cargo to express 6 fluorescently-tagged proteins and simultaneously visualize 5 different subcellular compartments. Our protocol leads from the in-silico design to the genetic and functional characterization of single clones within 6 weeks and can be implemented by any researcher with familiarity with molecular biology and access to mammalian cell culturing infrastructure.