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Accelerated RNA detection using tandem CRISPR nucleases

Direct, amplification-free detection of RNA has the potential to transform molecular diagnostics by enabling simple on-site analysis of human or environmental samples. CRISPR-Cas nucleases offer programmable RNA-guided recognition of RNA that triggers cleavage and release of a fluorescent reporter m...

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Detalles Bibliográficos
Autores principales: Liu, Tina Y., Knott, Gavin J., Smock, Dylan C. J., Desmarais, John J., Son, Sungmin, Bhuiya, Abdul, Jakhanwal, Shrutee, Prywes, Noam, Agrawal, Shreeya, de León Derby, María Díaz, Switz, Neil A., Armstrong, Maxim, Harris, Andrew R., Charles, Emeric J., Thornton, Brittney W., Fozouni, Parinaz, Shu, Jeffrey, Stephens, Stephanie I., Kumar, G. Renuka, Zhao, Chunyu, Mok, Amanda, Iavarone, Anthony T., Escajeda, Arturo M., McIntosh, Roger, Kim, Shin E., Dugan, Eli J., Pollard, Katherine S., Tan, Ming X., Ott, Melanie, Fletcher, Daniel A., Lareau, Liana F., Hsu, Patrick D., Savage, David F., Doudna, Jennifer A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010768/
https://www.ncbi.nlm.nih.gov/pubmed/33791736
http://dx.doi.org/10.1101/2021.03.19.21253328
Descripción
Sumario:Direct, amplification-free detection of RNA has the potential to transform molecular diagnostics by enabling simple on-site analysis of human or environmental samples. CRISPR-Cas nucleases offer programmable RNA-guided recognition of RNA that triggers cleavage and release of a fluorescent reporter molecule(1,2), but long reaction times hamper sensitivity and speed when applied to point-of-care testing. Here we show that unrelated CRISPR nucleases can be deployed in tandem to provide both direct RNA sensing and rapid signal generation, thus enabling robust detection of ~30 RNA copies/microliter in 20 minutes. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that detected SARS-CoV-2 RNA from nasopharyngeal samples with PCR-derived Ct values up to 29 in microfluidic chips, using a compact imaging system. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables direct RNA detection in a format amenable to point-of-care infection diagnosis, as well as to a wide range of other diagnostic or research applications.