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A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization
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Combinatorial protein engineering identifies potent CRISPR activators with reduced toxicity.

Marla Giddins1,2,3, Alexander F Kratz2,4, Mark B De Los Santos2,5,6

  • 1Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY, USA.

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|November 20, 2025
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Summary

Researchers developed a new method to test thousands of CRISPR activators, discovering potent new tools (MHV and MMH) that outperform existing ones and revealing insights into activator toxicity and function.

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Area of Science:

  • Molecular Biology
  • Protein Engineering
  • Synthetic Biology

Background:

  • Current protein engineering methods limit the exploration of natural protein domains.
  • This restricts the development of advanced synthetic biological tools.

Purpose of the Study:

  • To develop a high-throughput approach for creating and testing chimeric proteins.
  • To explore the combinatorial landscape of multi-domain CRISPR activators and identify improved tools.

Main Methods:

  • Developed a novel protein engineering strategy to generate and screen over 15,000 multi-domain CRISPR activators.
  • Assessed cellular toxicity and gene expression regulation capacity of generated activators.
  • Analyzed biochemical features of activation domains and their combinatorial effects.

Main Results:

  • Identified significant cellular toxicity in many tested activators, independent of gene regulation function.
  • Characterized the impact of activation domain combinations on activator performance.
  • Discovered two highly potent CRISPR activators, MHV and MMH, exhibiting superior performance.

Conclusions:

  • The developed method enables extensive exploration of protein domain combinations.
  • MHV and MMH represent next-generation CRISPR activators with enhanced efficacy and broader applicability.
  • Understanding activator toxicity and biochemical interactions is crucial for designing effective synthetic tools.