Abstract

A Generic Strategy for CRISPR/Cas-mediated Gene Tagging

Daniel Lackner, Senior Scientist, Horizon Discovery

Genome engineering has been greatly enhanced by the availability of Cas9 endonuclease which can be targeted to almost any genomic locus using so called guide RNAs (gRNAs). However, the introduction of foreign DNA sequences, referred to as gene tagging, is still cumbersome as it requires the synthesis or cloning of homology templates. Here, we present a strategy that enables the tagging of endogenous loci using one generic donor template. The donor plasmid contains the tag of interest flanked by two gRNA recognition sites (tia11) that allow for the excision of the tag from the plasmid. Co-transfection of cells with Cas9, a guide RNA specifying the genomic locus of interest, the generic donor plasmid and a tia11- specific guide RNA triggers the insertion of the template by a homology-independent mechanism. We show that this strategy is efficient and delivers clones that display a predictable integration pattern. As showcases, we generated cell lines in which genes are endogenously tagged with NanoLuc luciferase or TurboGFP.