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

Sci Rep. 2016 May 24:6:25529. doi: 10.1038/srep25529.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems*
  • Gene Expression
  • Gene Order
  • Gene Targeting* / methods
  • Indoleacetic Acids / pharmacology
  • Plasmids / genetics
  • Protein Engineering* / methods
  • Proteolysis / drug effects
  • RNA, Guide, CRISPR-Cas Systems
  • Workflow

Substances

  • Indoleacetic Acids