Combinatorial protein engineering identifies potent CRISPR activators with reduced toxicity

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.