Skip to main content
2024 BMES Annual Meeting Main banner
Icon Legend
This session is not in your schedule.
This session is in your schedule. Click again to remove it.

Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session C

Poster V8 - PAM-free CRISPR/Cas12 systems as next-generation diagnostics

Friday, October 25, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction: CRISPR-based DNA diagnostics have long been constrained by the dependency on Protospacer Adjacent Motif (PAM) sites, a challenge that has persisted despite over 1000 publications on type V CRISPR/Cas12 systems. Our groundbreaking method, PICNIC (PAM-Independent CRISPR Nucleic acid Identification Circuit), revolutionizes this field by completely bypassing the requirement for PAM sites. This novel approach leverages the unique properties of type V Cas12 enzymes, enabling them to bind to single-stranded DNA (ssDNA) without the need for PAM sequences.

Materials and

Methods: PICNIC involves a straightforward 10-minute modification to existing CRISPR protocols and employs readily available reagents, making it universally accessible for any laboratory. By converting double-stranded DNA (dsDNA) to ssDNA using heat and high pH, PICNIC enables Cas12 enzymes to detect virtually any target sequence across the genome, overcoming the limitations posed by the low occurrence (~1.17%) of common PAM motifs like TTTV.

Results, Conclusions, and Discussions: We validated PICNIC across 13 different Cas12a orthologs, including LbCas12a and AsCas12a, and extended its application to other Cas12 subtypes such as Cas12b and Cas12i. Our method facilitates the detection of non-canonical PAM sequences and clinically significant single-nucleotide polymorphisms (SNPs), demonstrated by identifying a drug-resistant HIV-1 variant in a PAM-less genomic region. Additionally, using a truncated guide RNA with a 15-nt spacer, we achieved precise SNP discrimination.

Furthermore, PICNIC's efficacy was showcased in the detection and genotyping of HCV-1a and HCV-1b from patient serum samples, achieving 100% detection accuracy and 95% specificity, respectively. These results highlight PICNIC's potential for rapid, accurate diagnostics and genotyping, making it a powerful tool for personalized medicine and pathogen surveillance.

Our study presents PICNIC as a transformative platform technology, poised to significantly broaden the scope of CRISPR-based diagnostics and meet the demands of diverse biomedical applications.

Acknowledgements (Optional): We extend our gratitude to the HCV-TARGET consortium, particularly Dr. David Nelson and Lauren Morelli, for their expert guidance and provision of clinical samples. We also thank the members of the Jain and Wang labs for their valuable discussions, and we acknowledge the support from the University of Florida (UF) Health Cancer Center. This work was financially supported by:

University of Florida, UF Herbert Wertheim College of Engineering,
Shah Foundation Endowment Funds,
NIH-NIAID R21AI156321,
NIH-NIAID R21AI168795, and
NIH-NIGMS R35GM147788.

The funding sources did not influence the design of the study, nor the collection, analysis, or interpretation of the data.