Systematic mapping of chimera-permissive sites by CRISPR-guided PAM scanning

Area of Science:

• Protein Engineering
• Synthetic Biology
• Molecular Biology

Background:

• Designing protein chimeras with new functions is challenging due to context-dependent insertion constraints.
• Preserving host protein function is critical when engineering protein chimeras.

Purpose of the Study:

• To develop a method for mapping chimera-permissive sites in living cells.
• To apply this method to peptide and reporter insertions for functional analysis.
• To establish a scalable framework for protein chimera engineering and in vivo accessibility studies.

Main Methods:

• Utilized CRISPR-guided protospacer adjacent motif (PAM) scanning in yeast.
• Generated insertion chimeras encoding protease cleavage sequences in a GPCR pathway.
• Scanned insertion sites in yeast Ste2 and human A2A and MTNR1A receptors.

Main Results:

• Identified 63% of insertion sites in a GPCR pathway that retain signaling function.
• Mapped site-resolved in-cell accessibility, distinguishing protected and exposed protein regions.
• Engineered bi-functional chimeras retaining native function and reporter activity.

Conclusions:

• PAM scanning is a scalable, protein-agnostic framework for mapping insertion tolerance.
• This approach enables interrogation of protein accessibility in vivo.
• Facilitates ground-truth benchmarking for predictive chimera engineering.