Universal donor plasmids for biallelic degron-tagging in mammalian cells and organoids
TL;DR
A series of universal donor vectors are generated to enable biallelic integration of different degron tags using different selection strategies (FACS, drug selection) and can be fused in-frame to endogenous protein C-termini via homology-independent repair following Cas9-induced double strand breaks.
Abstract
Degron tags enable rapid, tuneable and reversible control of protein expression, and have become a critical tool for target validation and elucidating biological mechanisms. Tag engineering uses genome editing to generate biallelic gene fusions, though this process remains laborious and requires new donor vectors to be generated for each locus. Here, we have generated a series of universal donor vectors to enable biallelic integration of different degron tags (AID, dTAG, BromoTag, SMASh) using different selection strategies (FACS, drug selection). Using this system, tags can be fused in-frame to endogenous protein C-termini via homology-independent repair following Cas9-induced double strand breaks, as shown here using human lymphoma cells, mouse embryonic stem cells and colonic tumour organoids. This workflow simplifies the genetic engineering of degron tags and is applicable in contexts where homology-dependent repair is challenging.
