An emulsion-based high-throughput screening platform for engineering and profiling Cas13 variant activities

Background

Cas13 is the only member of the Class 2 CRISPR-Cas systems that exclusively targets single-stranded RNA (ssRNA), enabling gene expression manipulation without permanent changes to the genome​. Cas13 exhibits highly efficient on-target “cis” RNA cleavage activity, requiring near-perfect complementarity between the guide RNA and target RNA for activation. Once activated, Cas13 can also engage in off-target “trans” cleavage of nearby non-targeted RNAs; this variation is not well- understood due to limited comprehensive screening or directed evolution studies for Cas12 and Cas13​. There is an enduring need for methods to engineer novel nuclease variants with selectively enhanced or decreased cis- or trans-nuclease activity​

Technology

This technology provides a high-throughput platform for engineering and screening large libraries of Cas13 protein variants to identify those with altered RNA cleavage activities. The process begins by generating a diverse library of Cas13 variants through mutagenesis, with each DNA variant attached to microbeads that also display CRISPR RNA. These microbeads are encapsulated within water-in-oil-in-water emulsions, and an in vitro transcription/translation system is used to express the Cas13 variants inside the droplets. After initial expression, the emulsion is broken and reformed to introduce fluorescent reporter constructs that enable simultaneous measurement of both cis (on-target) and trans (collateral) cleavage activities. Flow cytometry is then used to sort droplets based on fluorescence signals, and next-generation or long-read sequencing is employed to identify the mutations present in variants with desirable properties.

What differentiates this technology is its ability to simultaneously screen tens to hundreds of thousands of Cas13 variants in a single experiment, a scale not achievable with traditional methods. The use of emulsion-based compartmentalization ensures that each variant is individually expressed and assayed, preventing cross-contamination and enabling accurate linkage between genotype and phenotype. Furthermore, the system’s design allows for the parallel assessment of both cis and trans cleavage activities, providing a comprehensive functional profile for each variant. The integration of microbead tethering, advanced sequencing, and iterative screening rounds enables rapid identification and optimization of Cas13 proteins with tailored properties for diagnostic or therapeutic applications, making this platform a powerful tool for advancing CRISPR-based technologies.

Benefits

• Enables high-throughput screening of tens to hundreds of thousands of Cas13 variants simultaneously.
• Allows simultaneous evaluation of both cis and trans cleavage activities of Cas13 variants.
• Facilitates identification of mutations that enhance or reduce Cas13 cleavage activity.
• Supports iterative screening to optimize Cas13 variants under different conditions.
• Integrates flow cytometry sorting with next-generation or long-read sequencing for precise mutation analysis.
• Improves development of sensitive and specific CRISPR-based diagnostic and therapeutic tools.
• Utilizes an emulsion-based in vitro transcription/translation system for efficient variant expression and testing.

Applications

• Method for engineering the CRISPR effector protein Cas13 to enhance or reduce its cleavage activity​
• Screening for nucleases such as Cas12 and Cas13 orthologs (i.e., variants with same basic function but unique properties)​
• Enhancement of Cas13-based diagnostics (i.e., ability to target or cleave RNA in cells) ​
• Usage in therapeutic products that cleave human or viral RNAs

Additional info

Experimental verification in progress, with the following validation completed: ​

1. Construction of a library of Cas13 variants
2. Proper expression of Cas13 in transcription/translation systems
3. Tethering of Cas13 to beads using the CRISPR RNA. ​

Publication:

Siddiqui, S.M., Welch, N.L., Nguyen, T.G. et al. Bead-based approaches for increased sensitivity and multiplexing of CRISPR diagnostics. Nat. Biomed. Eng (2025). https://doi.org/10.1038/s41551-025-01498-2​ 

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
• Gaeun Kim, M.S.​ is currently a Ph.D. candidate at Harvard University. Prior, she was a Scientific Associate at D.E. Shaw Research and a lab assistant in the Myhrvold Lab at Princeton University. At Princeton University, she worked on Cas13, COVID-19 related projects in collaboration with the Broad Institute, and assisted with management duties. She has also worked as a Research Associate in the Sabti Lab at the Broad Institute of MIT and Harvard. She obtained her M.S. in Bioengineering and BSH (Bachelor of Science with Honors) in Bioengineering and Mathematics from Stanford University
• Jon Arizti-Sanz, Ph.D.​ is currently a postdoctoral researcher at Stanford Univeristy. Dr. Arizti-Sanz completed his Ph.D. in Medical Engineering from the Health Sciences & Technology (HST) department, a collaborative program between Massachusetts Institute of Technology (MIT) and Harvard Medical School. Prior to his doctoral studies, Dr. Arizti-Sanz earned his degree in Materials Science and Engineering at Imperial College, London
• Ryan McNulty​ is currently a Ph.D. candidate in Quantitative & Computational Biology at Princeton University. He received a bachelor’s in chemical engineering from the University of Delaware, followed by two years working within the Nutrition & Biosciences division of DuPont (now IFF)