Sortase-mediated fluorescent labeling of CRISPR complexes

Methods Enzymol. 2019;616:43-59. doi: 10.1016/bs.mie.2018.10.031. Epub 2018 Dec 17.


Fluorescent labeling of proteins is a critical requirement for single-molecule imaging studies. Many protein labeling strategies require harsh conditions or large epitopes that can inactivate the target protein, either by decreasing the protein's enzymatic activity or by blocking protein-protein interactions. Here, we provide a detailed protocol to efficiently label CRISPR-Cas complexes with a small fluorescent peptide via sortase-mediated transpeptidation. The sortase tag consists of just a few amino acids that are specifically recognized at either the N- or the C-terminus, making this strategy advantageous when the protein is part of a larger complex. Sortase is active at high ionic strength, 4°C, and with a broad range of organic fluorophores. We discuss the design, optimization, and single-molecule fluorescent imaging of CRISPR-Cas complexes on DNA curtains. Sortase-mediated transpeptidation is a versatile addition to the protein labeling toolkit.

Keywords: Cas1–Cas2; Cas3; Cascade; DNA curtains; Fluorescence; Protein labeling.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Associated Proteins / analysis*
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Cysteine Endopeptidases / analysis*
  • Escherichia coli / chemistry*
  • Escherichia coli / cytology
  • Escherichia coli Proteins / analysis*
  • Fluorescent Dyes / analysis*
  • Models, Molecular
  • Optical Imaging / methods
  • Staining and Labeling / methods


  • CRISPR-Associated Proteins
  • Escherichia coli Proteins
  • Fluorescent Dyes
  • Cysteine Endopeptidases