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Researchers engineered light-sensitive Cas9 using a LOV domain, creating a tool for controlling gene expression. Unexpectedly, this also yielded a temperature-sensitive Cas9 variant for precise genetic control.

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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.