title: RAC-tagging: Recombineering And Cas9-assisted targeting for protein tagging and conditional analyses.
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.
authors.affiliation: Stem Cell Engineering, Biotechnology Center, Technische Universität Dresden, BioInnovationsZentrum, Tatzberg 47, Dresden 01307, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Baker
authors.affiliation: Genomics, Biotechnology Center, Technische Universität Dresden, BioInnovationsZentrum, Tatzberg 47, Dresden 01307, Germany.
authors.forename: Ashish
authors.initials: A
authors.lastname: Gupta
authors.affiliation: Stem Cell Engineering, Biotechnology Center, Technische Universität Dresden, BioInnovationsZentrum, Tatzberg 47, Dresden 01307, Germany.|Genomics, Biotechnology Center, Technische Universität Dresden, BioInnovationsZentrum, Tatzberg 47, Dresden 01307, Germany.
authors.forename: Mandy
authors.initials: M
authors.lastname: Obst
authors.affiliation: Shandong University-Helmholtz Joint Institute of Biotechnology, State Key Laboratory of Microbial Technology, Shandong University, Shanda Nanlu 27, 250100 Jinan, People's Republic of China.
authors.forename: Youming
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Stem Cell Engineering, Biotechnology Center, Technische Universität Dresden, BioInnovationsZentrum, Tatzberg 47, Dresden 01307, Germany.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Anastassiadis
authors.affiliation: Genomics, Biotechnology Center, Technische Universität Dresden, BioInnovationsZentrum, Tatzberg 47, Dresden 01307, Germany.|Shandong University-Helmholtz Joint Institute of Biotechnology, State Key Laboratory of Microbial Technology, Shandong University, Shanda Nanlu 27, 250100 Jinan, People's Republic of China.
authors.forename: Jun
authors.initials: J
authors.lastname: Fu
authors.affiliation: Genomics, Biotechnology Center, Technische Universität Dresden, BioInnovationsZentrum, Tatzberg 47, Dresden 01307, Germany.
authors.forename: A Francis
authors.initials: AF
authors.lastname: Stewart
mesh_terms: D064113:CRISPR-Cas Systems; D015870:Gene Expression; D023061:Gene Order; D018390:Gene Targeting / Q000379:methods; D007210:Indoleacetic Acids / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D059748:Proteolysis / Q000187:drug effects; D057188:Workflow; D000094704:RNA, Guide, CRISPR-Cas Systems
chemical_list: D007210:Indoleacetic Acids; D000094704:RNA, Guide, CRISPR-Cas Systems