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Sortase-mediated fluorescent labeling of CRISPR complexes

Kaylee E Dillard1, Jeffrey M Schaub1, Maxwell W Brown1

  • 1Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, United States.

Methods in Enzymology
|January 30, 2019
PubMed

Insights

This study presents a new method for fluorescently labeling CRISPR-Cas complexes using sortase-mediated transpeptidation. This efficient technique preserves protein function for advanced single-molecule imaging studies.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biophysics

Background:

  • Fluorescent labeling is essential for single-molecule imaging.
  • Existing methods often use harsh conditions or large tags, risking protein inactivation.

Purpose of the Study:

  • To develop an efficient and gentle protocol for fluorescently labeling CRISPR-Cas complexes.
  • To enable high-resolution single-molecule imaging of these complexes.

Main Methods:

  • Utilized sortase-mediated transpeptidation for labeling.
  • Employed a small fluorescent peptide tag recognized by sortase.
  • Optimized labeling conditions for CRISPR-Cas complexes.

Main Results:

  • Successfully labeled CRISPR-Cas complexes with high efficiency.
  • Demonstrated preservation of protein enzymatic activity and interaction capabilities.
  • Achieved successful single-molecule fluorescent imaging on DNA curtains.

Conclusions:

  • Sortase-mediated transpeptidation offers a versatile and effective tool for protein labeling.
  • This method is advantageous for studying large protein complexes like CRISPR-Cas.
  • The protocol supports advanced applications in single-molecule biophysics.
Keywords:
Cas1–Cas2Cas3CascadeDNA curtainsFluorescenceProtein labeling

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