Abstract
AbstractEndogenous tagging makes it possible to study a protein’s localization, dynamics, and function within its native regulatory context. This is typically accomplished by using CRISPR to introduce a sequence encoding a functional tag into the reading frame of a gene; however, the efficiency of the process is often limited. Here, we introduce the “quickTAG,” or qTAG system, a versatile collection of optimized repair cassettes designed to make CRISPR-mediated tagging more accessible. By partitioning the cassette to include both a desired tag sequence linked with a selectable marker, integrations can be quickly isolated post-editing. These constructs include several key features that enhance flexibility and ease of use, such as: cassette designs for N– and C-terminus tagging; standardized cloning adaptors to simplify the incorporation of homology arms for HDR or MMEJ-based repairs; restriction sites next to each genetic element within the cassette for easy switching of tags and selectable markers; and the inclusion of lox sites flanking the selectable marker to allow for marker gene removal following integration. Our library of ∼120 plasmids is available on Addgene and includes ready-to-use constructs targeting frequently overexpressed genes as well as ready-to-clone cassettes to tag your own genes of interest. These initial cassettes were engineered to harbor a variety of tags for a range of applications, including fluorescence imaging, proximity-dependent biotinylation, epitope tagging, and targeted protein degradation. This protocol can yield a selected population of edited cells within ∼2-3 weeks, and a clonal-validated cell line within ∼5 weeks. Alongside this protocol, the qTAG system will provide a framework to streamline endogenous tagging in addition to serving as an open resource for researchers to adapt and tailor for their own purposes.
Publisher
Cold Spring Harbor Laboratory