Miniature engineered Cas13 enzymes for efficient programmable RNA targeting, editing, and in vivo delivery

Background

CRISPR-Cas13 systems are powerful tools for programmable, sequence-specific RNA targeting​. Broader use has been limited by practical challenges, such as large protein size (~1000 amino acids) that complicates packaging​ and size constraints that result in difficult delivery into cells. There is a need for smaller, efficient Cas13 variants that maintain or improve RNA editing performance​.

Technology

Princeton researchers have developed a compact Cas13 variant (~400 amino acids) with substantially enhanced RNA editing efficiency​.

Experimental Process: 1) Validated top thermostabilizing mutations before applying predictive modeling​

2) After five engineering cycles, achieved 72% editing efficiency (vs. 5.5% in wild-type)​

3) For endogenous transcripts, showed 2.5–3x higher efficiency compared to optimized wild-type system

Iterative Engineering Strategy:

1) Relocalization of guide RNA to the cytosol​

2) Protein thermostability optimization using ΔΔG computational predictions​

3) Machine learning-guided refinement to iteratively evolve the protein

Result: a compact, delivery-compatible Cas13 enzyme that demonstrates a robust combination of structure-based design and data-driven evolution, enabling broader RNA-targeting applications​

Benefits

• Significantly smaller Cas13 enzyme (~421-450 amino acids) enabling packaging into single AAV vectors for efficient in vivo delivery
• Substantially enhanced RNA editing efficiency, improving from ~5.5% to up to 72% in reporter cells and 2.5-3 fold for endogenous transcripts
• Versatile programmable RNA targeting allowing diverse modifications including A-to-G and C-to-U base editing, acetylation, and live cell imaging
• Advanced protein engineering combining guide RNA relocalization, computational thermostability optimization, and machine learning-guided refinement for superior performance
• Fusion capability with various functional protein domains without exceeding delivery size constraints
• Potential for mutation-agnostic gene therapy applications, especially for haploinsufficient genetic diseases
• Validated robust RNA binding with reduced cleavage activity, supporting precise RNA editing without unwanted RNA degradation

Applications

RNA Editing for Gene Therapy: 

• Fusion of a catalytically inactive version of engineered ancestral Cas13 to RNA editing protein (e.g., ADAR) or other RNA-modifying enzymes (e.g., methylases, acetylases)​
• Given that engineered ancestral Cas13 enzymes are much smaller than other Cas13 orthologs, they can be fused to other enzymes and still fit within a single AAV vector, greatly facilitating in vivo delivery

Enhancement of RNA Stability: 

• Fusion of ancestral Cas13 ortholog to an RNA-stabilizing protein that binds to RNA in a mutation-agnostic fashion​
• Potential usage as treatment for haplo-insufficient genetic diseases

Synthetic Biology & Research Tool: 

• Compact, efficient RNA modulation system for controlling gene expression, studying RNA biology, and engineering cell behavior in research or bio-industrial contexts

Additional info

Experimental Validation:

• Experimentally demonstrated that the engineered miniature Cas13 can be used to perform A-to-G RNA base editing in a reporter cell line as well as A-to-G and C-to-U RNA base editing at several endogenous mRNA transcripts in mammalian cells​

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
• 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
• Qinhao Cao is currently a Ph.D. candidate in the Department of Molecular Biology at Princeton University, conducting research in the Toettcher Lab