RAC-tagging: Recombineering and Cas9-assisted targeting for protein tagging and conditional analyses

Research output: Contribution to journalResearch articleContributedpeer-review

Contributors

Abstract

A fluent method for gene targeting to establish protein tagged and ligand inducible conditional loss-of-function alleles is described. We couple new recombineering applications for one-step cloning of gRNA oligonucleotides and rapid generation of short-arm (∼1 kb) targeting constructs with the power of Cas9-assisted targeting to establish protein tagged alleles in embryonic stem cells at high efficiency. RAC (Recombineering And Cas9)-tagging with Venus, BirM, APEX2 and the auxin degron is facilitated by a recombineering-ready plasmid series that permits the reuse of gene-specific reagents to insert different tags. Here we focus on protein tagging with the auxin degron because it is a ligand-regulated loss-of-function strategy that is rapid and reversible. Furthermore it includes the additional challenge of biallelic targeting. Despite high frequencies of monoallelic RAC-targeting, we found that simultaneous biallelic targeting benefits from long-arm (>4 kb) targeting constructs. Consequently an updated recombineering pipeline for fluent generation of long arm targeting constructs is also presented.

Details

Original languageEnglish
Article number25529
JournalScientific reports
Volume6
Publication statusPublished - 24 May 2016
Peer-reviewedYes

External IDs

PubMed 27216209
ORCID /0000-0002-4754-1707/work/142248091