Light-Inducible CRISPR Labeling.

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• CRISPR labeling visualizes chromatin architecture in live cells using a deactivated Cas9 protein.
• Current CRISPR labeling lacks temporal control, as Cas9 binds DNA immediately upon expression, potentially affecting genome regulation and architecture.
• Controlling Cas9 DNA binding is crucial for precise chromatin studies and analyzing Cas9 binding kinetics.

Purpose of the Study:

• To develop a light-inducible CRISPR labeling method for temporal control over Cas9 DNA binding.
• To enable precise interrogation of chromatin spatial organization and dynamics.
• To facilitate direct study of Cas9 DNA binding kinetics in living human cells.

Main Methods:

• Utilized CASANOVA, an optogenetic anti-CRISPR protein, to trap Streptococcus pyogenes (Spy)Cas9 in an inactive state in the dark.
• Developed a protocol for light-inducible CRISPR labeling by using blue light to permit Cas9 DNA targeting.
• Employed telomeres as target loci and detailed experimental steps for inducible labeling and automated microscopy data analysis.

Main Results:

• Demonstrated successful light-inducible CRISPR labeling of telomeres in live cells.
• Showcased CASANOVA's ability to reversibly inhibit and activate SpyCas9 DNA binding upon light exposure.
• Established a protocol for precise temporal control over CRISPR labeling experiments.

Conclusions:

• Light-inducible CRISPR labeling with CASANOVA offers temporal control over chromatin visualization.
• This technique enhances the study of chromatin dynamics and Cas9-DNA interactions in living cells.
• The protocol provides a robust method for precise and controlled genomic locus labeling.