Engineering of temperature- and light-switchable Cas9 variants

Area of Science:

• Molecular Biology
• Synthetic Biology
• Biotechnology

Background:

• Photoreceptors enable precise control of biological processes.
• Engineering new light-regulated protein actuators remains challenging.
• The Rhodobacter sphaeroides light-oxygen-voltage (LOV) domain dissociates upon blue-light exposure.

Purpose of the Study:

• To develop a general strategy for creating light-regulated protein actuators.
• To engineer light-sensitive Cas9 using the RsLOV domain.
• To explore the potential of RsLOV for controlling effector protein function.

Main Methods:

• Constructed a library of RsLOV inserted Cas9 variants.
• Screened variants using a high-throughput transcriptional repression assay in E. coli.
• Isolated and characterized light- and temperature-sensitive Cas9 variants (paRC9 and tsRC9).

Main Results:

• Identified paRC9, a moderately light-activatable Cas9 variant.
• Isolated tsRC9, the first temperature-sensitive Cas9 variant, inactive at 37°C but active at 29°C.
• Demonstrated temperature-dependent transcriptional control using tsRC9 in E. coli.

Conclusions:

• The RsLOV domain can confer light sensitivity to unrelated effector proteins like Cas9.
• The RsLOV domain unexpectedly imparted significant temperature sensitivity to Cas9.
• Engineered Cas9 variants offer new tools for spatiotemporal control of gene expression.