A platform using engineered RNA structures for highly specific and sensitive Cas13-based nucleic acid detection

This technology uses specially designed DNA or RNA strands to temporarily block Cas13 enzyme recognition, improving its ability to detect rare genetic mutations and distinguish single-nucleotide differences in RNA with much greater accuracy and sensitivity.

Background

Cas13, an RNA-targeting CRISPR nuclease, has become a valuable tool for various applications, including nucleic acid detection and RNA imaging. Cas13 is activated when a CRISPR RNA (crRNA) hybridizes with a complementary single-stranded RNA (ssRNA) protospacer in a target RNA. ​

Paradoxically, while Cas13 is not activated by double-stranded RNA (dsRNA) in vitro, it effectively targets RNA in highly structured environments. The mechanism by which Cas13 binds structured RNAs remains elusive, yet understanding this process is crucial for enhancing its RNA detection and perturbation capabilities​.

Technology

Princeton researchers have discovered that there are two distinct sequence-independent modes by which secondary structure affects Cas13 activity: (1) structure in the protospacer competes with crRNA binding via a strand-displacement mechanism, while (2) structure in the region 3’ to the protospacer allosterically inhibits activity​.

By exploiting the kinetics of the strand displacement process, Princeton researchers have improved Cas13-based RNA detection and enhanced mismatch discrimination by up to 50-fold, enabling sequence-agnostic mutation identification at low (<1%) allele frequencies​.

This novel work sets a new standard for CRISPR-based nucleic acid detection, allowing for intelligent secondary-structure-guided target selection and expanding the range of RNAs available for targeting with Cas13​.

Benefits

  • Robust, comprehensive, and sequence-agnostic improvement in mismatch detection and target specificity​
  • Compatibility with a wide range of existing Cas13-based techniques ​
  • Easy to implement experimentally ​(Only requires annealing a DNA oligo to the crRNA prior to mixing with target RNA; no toehold required​)
  • Cost-efficient approach​

Applications

  • Method for enhanced nucleic acid detection to detect infectious diseases (e.g., viruses, bacteria, other pathogens), human mutations (e.g., linked with genetic diseases), or other diseases (e.g., cancer)​
  • Novel detection reactions consisting of occluded crRNAs or target RNAs ​
  • Potential incorporation into a CRISPR-based diagnostic assay

Additional info

Experimental Validation:

  • Successful quantification of Cas13 activity reduction due to target RNA occlusion​
  • Improvement of Cas13’s mismatch specificity by an order of magnitude​

Innovative method performs robustly in a wide range of different target sequences, as well as mutation positions and types.

Publication:

Kimchi, O., Larsen, B. B., Dunkley, O. R. S., Te Velthuis, A. J. W., & Myhrvold, C. (2023). RNA structure modulates Cas13 activity and

enables mismatch detection. bioRxiv : the preprint server for biology, 2023.10.05.560533. https://doi.org/10.1101/2023.10.05.560533​ 

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.
  • Aartjan te Velthius, Ph.D.​ is an Assistant Professor of Molecular Biology at Princeton University. He received his Ph.D. cum laude for his work on the replication of the SARS coronavirus from the University of Leiden, the Netherlands. Dr. te Velthius subsequently joined the University of Oxford as Marie Curie/Rubicon fellow and Henry Welcome fellow to study influenza virus replication in the laboratories of Prof Ervin Fodor and Prof Achilles Kapanidis. He also taught at Lincoln College, Oxford, as Kemp Research fellow. In 2017, he started his own research group at the University of Cambridge with a Henry Dale fellowship from the Wellcome Trust and Royal Society. In 2021, he moved his lab to Princeton University, where his research focuses on how RNA viruses replicate and induce innate immune responses
  • Benjamin Larsen​ is currently a Ph.D. candidate in the Department of Molecular Biology at Princeton University. He is a graduate student in both the Myhrvold and te Velthius Labs. He received his B.S. in Biology and Biotechnology at Tufts University
  • Ofer Kimchi, Ph.D.​ is currently a Branco Weiss Fellow and Lewis-Sigler Theory Scholar at Princeton University. He is also a Fellow at the Center for the Physics of Biological Function at CUNY and Princeton University. He received his Ph.D. in Biophysics from Harvard University, advised by Michael Brenner.
  • Owen Dunkley​ is currently a Ph.D. candidate in Bioengineering at Stanford University. He was previously a Research Specialist in the Myhrvold Lab at Princeton University and received his M.Sc. in Experimental Medicine at McGill University and B.Sc. in Microbiology and Immunology at McGill University​