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Related Experiment Videos

Reprogramming MHC specificity by CRISPR-Cas9-assisted cassette exchange.

William Kelton1, Ann Cathrin Waindok1, Theresa Pesch1

  • 1Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland.

Scientific Reports
|April 5, 2017
PubMed
Summary

This study demonstrates CRISPR-Cas9 gene editing to reprogram MHC specificity in immune cells, enabling potential solutions for transplantation mismatches. This advance offers a new strategy for cellular transplantation by correcting MHC incompatibilities.

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Area of Science:

  • Immunology
  • Genetics
  • Biotechnology

Background:

  • Programmable nucleases like CRISPR-Cas9 are advancing genome engineering for cellular immunotherapy.
  • Current applications primarily focus on gene knock-out/knock-in, with limited scope for scarless allele exchange.
  • Reprogramming the Major Histocompatibility Complex (MHC) locus is crucial for matched donor creation in allogeneic transplantation.

Purpose of the Study:

  • To demonstrate a proof-of-concept for reprogramming MHC specificity using CRISPR-Cas9-assisted cassette exchange.
  • To assess the functionality of MHC-reprogrammed cells in presenting antigens and activating T cells.
  • To optimize donor template formats for efficient MHC allele exchange.

Main Methods:

  • CRISPR-Cas9-mediated double-stranded breaks were introduced flanking the native MHC-I H2-Kd locus in murine macrophage cell lines (RAW264.7).

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  • Cassette exchange was performed using an orthogonal H2-Kb allele as a donor template.
  • Reprogrammed cells were selected via flow cytometry based on MHC surface expression and functional T cell activation assays were conducted.

    • Main Results:

    • Successful exchange of the native H2-Kd allele with the H2-Kb allele was achieved.
    • MHC surface expression facilitated straightforward selection of reprogrammed cells without additional markers.
    • Reprogrammed cells maintained functionality, presenting H2-Kd-restricted peptides and activating cognate T cells.
    • In situ linearization of donor templates significantly enhanced MHC-reprogramming efficiency.

    Conclusions:

    • CRISPR-Cas9-assisted cassette exchange provides a viable method for reprogramming MHC specificity.
    • This technique holds promise for addressing MHC mismatches in cellular transplantation and allogeneic cell therapy.
    • Optimized donor template design, particularly in situ linearization, is key to maximizing reprogramming efficiency.