Generation of auxin inducible degron (AID) knock-in cell lines for targeted protein degradation in mammalian cells

• Published in: STAR protocols 2021-12, Vol.2 (4), p.100949-100949, Article 100949
• Main Authors: Adhikari, Bikash, Narain, Ashwin, Wolf, Elmar
• Format: Article
• Language: English
• Subjects: Animals; Cell Biology; Cell Line, Tumor; Cloning, Molecular - methods; CRISPR; CRISPR-Cas Systems - genetics; Gene Knock-In Techniques - methods; Humans; Molecular Biology; Plant Proteins - genetics; Proteolysis; Protocol; RNA, Guide, CRISPR-Cas Systems - genetics; Transfection
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2021.100949

Targeted protein degradation using degrons, such as the mini-Auxin-inducible degron (mAID), has an advantage over genetic silencing/knockout. However, the efficiency of sgRNA, homologous recombination, tedious expansion, and screening single clones makes the process of tagging endogenous proteins long and laborious. This protocol describes a practical and economical way to obtain AID-tagged endogenous proteins using CRISPR/Cas9-mediated homology-directed repair (HDR). We use the generation of endogenously AID-tagged SPT6 in U2OS cells as an example but provide sufficient details for usage in other cell types. For complete details on the use and execution of this protocol, please refer to Narain et al. (2021). [Display omitted] •An efficient protocol to obtain mammalian cells with endogenously AID-tagged protein•Detailed steps to design adjustable knock-in tag, sgRNA, HDR with SPT6-AID as example•A practical way to select, expand and characterize clones with homozygous knock-in Targeted protein degradation using degrons, such as the mini-Auxin-inducible degron (mAID), has an advantage over genetic silencing/knockout. However, the efficiency of sgRNA, homologous recombination, tedious expansion, and screening single clones makes the process of tagging endogenous proteins long and laborious. This protocol describes a practical and economical way to obtain AID-tagged endogenous proteins using CRISPR/Cas9-mediated homology-directed repair (HDR). We use the generation of endogenously AID-tagged SPT6 in U2OS cells as an example but provide sufficient details for usage in other cell types.