CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification.

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• CRISPR-Cas9 is a powerful genome editing tool but has limitations for protein fusions and cellular activation.
• Engineering natural proteins is crucial for future biological applications.

Purpose of the Study:

• To develop an advanced platform for RNA-guided genome modification and protection using a rearranged CRISPR-Cas9 scaffold.
• To create novel single-molecule effectors with programmable inputs and outputs for sensing cellular activities.

Main Methods:

• Circular permutation of the Cas9 protein to create a new topological arrangement.
• Systematic interrogation of protein termini positioning relative to bound DNA.
• Development and characterization of protease-sensing Cas9s (ProCas9s).

Main Results:

• Circularly permuted Cas9 provides a platform for strategic functional domain fusions.
• ProCas9s were developed as single-molecule effectors capable of sensing various proteases.
• ProCas9 demonstrated the ability to trigger a cellular response to pathogen-associated protease activity.

Conclusions:

• Circular permutation of Cas9 offers a safer and more efficient genome-modifying enzyme platform.
• This technology advances molecular recorders and precision genome engineering in research, agriculture, and biomedicine.