CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide

ACS Chem Biol. 2018 Feb 16;13(2):467-474. doi: 10.1021/acschembio.7b00549. Epub 2017 Sep 21.

Abstract

Intracellular signaling pathways are mediated by changes in protein abundance and post-translational modifications. A common approach for investigating signaling mechanisms and the effects induced by synthetic compounds is through overexpression of recombinant reporter genes. Genome editing with CRISPR/Cas9 offers a means to better preserve native biology by appending reporters directly onto the endogenous genes. An optimal reporter for this purpose would be small to negligibly influence intracellular processes, be readily linked to the endogenous genes with minimal experimental effort, and be sensitive enough to detect low expressing proteins. HiBiT is a 1.3 kDa peptide (11 amino acids) capable of producing bright and quantitative luminescence through high affinity complementation (KD = 700 pM) with an 18 kDa subunit derived from NanoLuc (LgBiT). Using CRISPR/Cas9, we demonstrate that HiBiT can be rapidly and efficiently integrated into the genome to serve as a reporter tag for endogenous proteins. Without requiring clonal isolation of the edited cells, we were able to quantify changes in abundance of the hypoxia inducible factor 1A (HIF1α) and several of its downstream transcriptional targets in response to various stimuli. In combination with fluorescent antibodies, we further used HiBiT to directly correlate HIF1α levels with the hydroxyproline modification that mediates its degradation. These results demonstrate the ability to efficiently tag endogenous proteins with a small luminescent peptide, allowing sensitive quantitation of the response dynamics in their regulated expression and covalent modifications.

MeSH terms

  • Antibodies / chemistry
  • Bioluminescence Resonance Energy Transfer Techniques
  • CRISPR-Associated Protein 9 / genetics
  • CRISPR-Cas Systems / genetics*
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Early Growth Response Transcription Factors / genetics
  • Early Growth Response Transcription Factors / metabolism
  • Gene Editing / methods*
  • Genes, Reporter / genetics
  • HeLa Cells
  • Humans
  • Hypoxia-Inducible Factor 1, alpha Subunit / genetics
  • Hypoxia-Inducible Factor 1, alpha Subunit / metabolism
  • Kruppel-Like Transcription Factors / genetics
  • Kruppel-Like Transcription Factors / metabolism
  • Leupeptins / pharmacology
  • Luciferases / metabolism
  • Luminescence
  • Luminescent Proteins / chemistry
  • Luminescent Proteins / genetics*
  • Luminescent Proteins / metabolism
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Neoplasm Proteins / genetics
  • Neoplasm Proteins / metabolism
  • Oligopeptides / chemistry
  • Oligopeptides / genetics*
  • Oligopeptides / metabolism
  • Protein Processing, Post-Translational
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism
  • Streptococcus pyogenes / enzymology

Substances

  • Antibodies
  • BNIP3 protein, human
  • Carrier Proteins
  • Early Growth Response Transcription Factors
  • HIF1A protein, human
  • HILPDA protein, human
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • KLF10 protein, human
  • Kruppel-Like Transcription Factors
  • LRP2BP protein, human
  • Leupeptins
  • Luminescent Proteins
  • Membrane Proteins
  • Neoplasm Proteins
  • Oligopeptides
  • Proto-Oncogene Proteins
  • Luciferases
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • benzyloxycarbonylleucyl-leucyl-leucine aldehyde