Engineered CRISPR-Cas13d Systems for Precise, Reversible, and Inducible RNA Knockdown in Living Cells

This technology enables precise, reversible control of RNA targeting in living cells using engineered CRISPR-Cas13d proteins that can be switched on or off by light or small molecules, allowing dynamic and programmable gene regulation.

Background

CRISPR-Cas13 allows precise RNA targeting but current systems remain continuously active after expression, lacking spatial and temporal control for dynamic experiments​. Researchers require reversible, selective RNA knockdown tools to target transcripts at specific times or in defined cell populations​.

Technology

Princeton researchers have developed engineered RfxCas13d (CRISPR) proteins that can be switched on or off by external stimuli: blue light (optogenetic control) or small molecule addition (chemical control)​.

Researchers screened a Cas13d-AsLOV2 fusion protein library and identified variants with distinct activity patterns:​

(1) OptoCas13d-off: Active in the dark to cleave RNA; switched off by blue light​

(2) OptoCas13d-on: Remains off in darkness, activated by blue light for RNA knockdown​

(3) ChemoCas13d: Activated specifically by the addition of rapamycin analogs

Domain insertion did not disrupt crRNA binding, and only slightly affected target RNA binding, indicating that activity control was achieved allosterically via the engineered domain​.

Benefits

  • Versatile and programmable RNA modification capabilities (i.e., cleavage, detection, editing imaging) ​
  • Inducible and fine-tuned system allows researcher to mitigate collateral effects and reduce cytotoxicity in cells​
  • Spatiotemporal control enables more precise investigations into RNA function and regulation with high-resolution ​

Applications

General application: high spatial and temporal precision for cellular RNA modulation, creating new opportunities for controlled studies of gene expression and function​

Examples of potential therapeutic and research uses: ​

  • Controlling the differentiation of stem cells for tissue patterning and engineering​ 
  • T-cell engineering​
  • Studying the dynamics of the innate immune response to viral infection​
  • Studying perturbation of essential genes​
  • Studying RNAs that are spatially localized within cells (e.g. subcellular RNA perturbation)

Additional info

Experimental Validation:

  • Experimental tests: knockdown of a GFP reporter, as well as several endogenous genes in mammalian cells​

Publication:

  • Multimodal control of Cas13 activity through domain insertion at an allosteric hotspot. Liyuan Zhu, Long T. Nguyen, Alexandra G. Bell, Kara M. Gillmann, Harrison Oatman, Jack Hariri, Cameron Myhrvold, Jared E. Toettcher. bioRxiv 2025.04.21.649818; doi:https://doi.org/10.1101/2025.04.21.649818​

Inventors:

  • Cameron Myhrvold, Ph.D.​ is an Assistant Professor in the Department of Molecular Biology at Princeton University. He completed his Ph.D. in Systems Biology at Harvard University as a Hertz Fellow and postdoctoral training at the Broad Institute of MIT and Harvard. His research focuses on developing CRISPR-based technologies for studying RNA, particularly RNAs involved in host-pathogen interactions. In support and recognition of his work, he has received many awards and honors, including the STAT Magazine Wunderkind Award, Forbes 30 Under 30 (Healthcare category), ASTMH Robert E. Shope, MD, Fellowship in Infectious Diseases, and Broad Institute Shark Tank Program
  • Jared Toettcher, Ph.D.​ is an Associate Professor of Molecular Biology and Bioengineering at Princeton University, as well as Deputy Director, Omenn-Darling Bioengineering Institute. He completed his B.S. in Bioengineering at UC Berkeley, graduate studies at MIT in Biological Engineering and a Cancer Research Institute postdoctoral fellowship at UCSF. Dr. Toettcher’s research focuses on dissecting how signaling pathways work together to orchestrate complex cell decision-making. Dr. Toettcher’s honors and awards include a Cancer Research Institute fellowship, NIH Ruth Kirschstein postdoctoral fellowship, an MIT Presidential Fellowship and a UC Berkeley Regents’ Scholarship
  • Liyuan Zhu, Ph.D.​ is currently a Scientist I (Protein Engineering) at Life Edit Therapeutics. Dr. Zhu was previously a graduate student in the Toettcher Lab at Princeton University, where he successfully developed light-responsive transcription factors and controllable RNA-guided RNA endonucleases (Cas13) using a combination of high-throughput selections and rational design. Dr. Zhu received his B.Sc. in Chemical Biology from Peking University
  • Long Nguyen, Ph.D.​ is currently a Princeton Bioengineering Institute - Innovators (PBI2) postdoctoral fellow in the Bioengineering Initiative Program. Dr. Nguyen is co-advised by Jared Toettcher and Cameron Myhrvold. He received his B.S. and Ph.D. in Chemical and Biomolecular Engineering at Georgia Institute of Technology and University of Florida, respectively. During his Ph.D., his primary focus was engineering the next-generation CRISPR-Cas systems for genome editing and diagnostics. Dr. Nguyen is interested in developing new optogenetic tools for CRISPR-Cas systems and cellular engineering