Molecular recordings by directed CRISPR spacer acquisition.

Area of Science:

• Microbiology
• Molecular Biology
• Synthetic Biology

Background:

• Stable recording of molecular events in cells is crucial for understanding cellular history.
• CRISPR-Cas systems offer potential for targeted DNA manipulation and recording.

Purpose of the Study:

• To engineer the type I-E CRISPR-Cas system of E. coli for recording synthetic DNA sequences.
• To develop a multimodal intracellular recording device based on CRISPR-Cas adaptation.

Main Methods:

• Utilized the type I-E CRISPR-Cas system of E. coli to mediate the acquisition of synthetic DNA.
• Applied directed evolution to modify protospacer adjacent motif recognition by the Cas1-Cas2 complex.
• Enabled simultaneous recording in two distinct modes.

Main Results:

• Demonstrated the ability to generate stable records of specific DNA sequences within bacterial genomes.
• Successfully altered protospacer adjacent motif recognition for enhanced recording capabilities.
• Revealed novel insights into the spacer acquisition process fundamental to CRISPR-Cas adaptation.

Conclusions:

• The engineered CRISPR-Cas system serves as a foundation for a multimodal intracellular recording device.
• This technology enables the examination of long cellular histories with potential applications in developmental biology and synthetic devices.
• The findings advance our understanding of CRISPR-Cas adaptation mechanisms.