A microfluidic CRISPR-Cas13 platform for high-throughput multiplexed RNA quantification and kinetic analysis

qCARMEN is a high-throughput, CRISPR-based technology that rapidly and accurately quantifies many RNA targets at once using microfluidics, fluorescence, and mathematical modeling, making gene expression analysis faster, cheaper, and more scalable than traditional methods.

Background

RNA quantification tools are often either high-throughput or cost-effective, but rarely are they both​. Existing methods can profile the transcriptome at great expense or are limited to quantifying a handful of genes by labor constraints​. A technique that enables higher throughput at reduced costs would enable multi-gene kinetic studies, gene regulatory network analysis, and combinatorial genetic screens.

Technology

Princeton researchers have created qCARMEN: quantitative Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids. qCARMEN is a novel RNA quantitation technique that leverages the programmable RNA-targeting capabilities of CRISPR-Cas13 to address existing challenges by quantifying >4,500 gene-sample pairs in a single experiment​. Beyond probing host responses, qCARMEN can be applied to clinical applications such as predicting disease outcomes and monitoring the spread of infectious diseases​. qCARMEN is a simple and inexpensive technique that enhances the scalability of RNA quantitation for novel applications with performance similar to gold-standard methods.

Benefits

  • Enables high-throughput, multiplexed quantification of changes in host gene expression​
  • Significantly lowers labor and financial costs associated with highly multiplexed kinetics studies and gene regulatory network analyses ​

Applications

  • Immediate usage in high-throughput, multiplexed RNA expression studies​
  • Generation of large datasets for training machine learning models to predict clinical outcomes​
  • Active processing of hundreds of samples in a clinical setting to predict disease outcomes​
  • Production as a series of kits for detecting specific RNAs/genes of interest or custom panels/kits designed for users as a service​

Additional info

Experimental Validation:

qCARMEN underwent experimental validation through several key steps: ​

(1) Synthetic RNA targets ​

  • Dilutions quantified to demonstrate quantitative capabilities ​

(2) Cell culture samples​

  • Gene expression changes measured in virus-infected samples​
  • Results compared to gold-standard qPCR method​

(3) Additional RNA samples ​

  • Tested on interferon-stimulated and TGF-β-treated cells​

All reagents used for these experiments are readily available for examination​

Publication:

Kang, B., Zhang, J., Schwoerer, M. P., Nelson, A. N., Schoeman, E., Guo, A., Ploss, A., & Myhrvold, C. (2023). Highly multiplexed mRNA quantitation with CRISPR-Cas13.

bioRxiv : the preprint server for biology, 2023.08.16.553527. https://doi.org/10.1101/2023.08.16.553527​

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.
  • Brian Kang is a Ph.D. candidate at Stanford University. He was previously a Research Specialist in the Department of Molecular Biology at Princeton University and received his B.S.E. in Chemical and Biological Engineering at Princeton University
  • Alexander Ploss, Ph.D.​ is an Associate Professor in the Department of Molecular Biology at Princeton University. He completed his Ph.D. in Immunology at Memorial Sloan-Kettering Cancer Center and Cornell University and postdoctoral training at the Rockefeller University in New York. Dr. Ploss previously held faculty positions at the Rockefeller University before joining the faculty of the Department of Molecular Biology at Princeton University where he is currently an Associate Professor. In support and recognition of his work he received several awards including the including the Astella’s Young Investigator Award from the Infectious Disease Society of America