tetano
Editor, Senior Moderator
Nat Chem Biol
. 2021 Aug 5.
doi: 10.1038/s41589-021-00842-2. Online ahead of print.
Accelerated RNA detection using tandem CRISPR nucleases
Tina Y Liu[SUP] 1 2 [/SUP], Gavin J Knott[SUP] 1 2 3 [/SUP], Dylan C J Smock[SUP] 1 2 [/SUP], John J Desmarais[SUP] 1 [/SUP], Sungmin Son[SUP] 4 [/SUP], Abdul Bhuiya[SUP] 4 5 [/SUP], Shrutee Jakhanwal[SUP] 1 2 [/SUP], Noam Prywes[SUP] 2 [/SUP], Shreeya Agrawal[SUP] 2 4 [/SUP], María Díaz de León Derby[SUP] 4 5 [/SUP], Neil A Switz[SUP] 6 [/SUP], Maxim Armstrong[SUP] 7 [/SUP], Andrew R Harris[SUP] 4 [/SUP], Emeric J Charles[SUP] 1 [/SUP], Brittney W Thornton[SUP] 1 [/SUP], Parinaz Fozouni[SUP] 8 9 10 11 [/SUP], Jeffrey Shu[SUP] 8 11 [/SUP], Stephanie I Stephens[SUP] 8 11 [/SUP], G Renuka Kumar[SUP] 8 11 [/SUP], Chunyu Zhao[SUP] 8 12 [/SUP], Amanda Mok[SUP] 13 [/SUP], Anthony T Iavarone[SUP] 14 [/SUP], Arturo M Escajeda[SUP] 15 [/SUP], Roger McIntosh[SUP] 15 [/SUP], Shineui Kim[SUP] 1 2 [/SUP], Eli J Dugan[SUP] 1 2 [/SUP], IGI Testing Consortium; Katherine S Pollard[SUP] 8 12 16 [/SUP], Ming X Tan[SUP] 13 [/SUP], Melanie Ott[SUP] 8 11 [/SUP], Daniel A Fletcher[SUP] 4 5 12 17 [/SUP], Liana F Lareau[SUP] 2 4 [/SUP], Patrick D Hsu[SUP] 18 19 20 21 22 [/SUP], David F Savage[SUP] 23 24 [/SUP], Jennifer A Doudna[SUP] 25 26 27 28 29 30 31 [/SUP]
Collaborators, Affiliations
Abstract
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 RNA recognition that triggers cleavage and release of a fluorescent reporter molecule, but long reaction times hamper their detection sensitivity and speed. 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 molecules per µl of RNA in 20 min. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA extracted from respiratory swab samples with quantitative reverse transcriptase PCR (qRT-PCR)-derived cycle threshold (C[SUB]t[/SUB]) values up to 33, using a compact detector. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables sensitive, direct RNA detection in a format that is amenable to point-of-care infection diagnosis as well as to a wide range of other diagnostic or research applications.
. 2021 Aug 5.
doi: 10.1038/s41589-021-00842-2. Online ahead of print.
Accelerated RNA detection using tandem CRISPR nucleases
Tina Y Liu[SUP] 1 2 [/SUP], Gavin J Knott[SUP] 1 2 3 [/SUP], Dylan C J Smock[SUP] 1 2 [/SUP], John J Desmarais[SUP] 1 [/SUP], Sungmin Son[SUP] 4 [/SUP], Abdul Bhuiya[SUP] 4 5 [/SUP], Shrutee Jakhanwal[SUP] 1 2 [/SUP], Noam Prywes[SUP] 2 [/SUP], Shreeya Agrawal[SUP] 2 4 [/SUP], María Díaz de León Derby[SUP] 4 5 [/SUP], Neil A Switz[SUP] 6 [/SUP], Maxim Armstrong[SUP] 7 [/SUP], Andrew R Harris[SUP] 4 [/SUP], Emeric J Charles[SUP] 1 [/SUP], Brittney W Thornton[SUP] 1 [/SUP], Parinaz Fozouni[SUP] 8 9 10 11 [/SUP], Jeffrey Shu[SUP] 8 11 [/SUP], Stephanie I Stephens[SUP] 8 11 [/SUP], G Renuka Kumar[SUP] 8 11 [/SUP], Chunyu Zhao[SUP] 8 12 [/SUP], Amanda Mok[SUP] 13 [/SUP], Anthony T Iavarone[SUP] 14 [/SUP], Arturo M Escajeda[SUP] 15 [/SUP], Roger McIntosh[SUP] 15 [/SUP], Shineui Kim[SUP] 1 2 [/SUP], Eli J Dugan[SUP] 1 2 [/SUP], IGI Testing Consortium; Katherine S Pollard[SUP] 8 12 16 [/SUP], Ming X Tan[SUP] 13 [/SUP], Melanie Ott[SUP] 8 11 [/SUP], Daniel A Fletcher[SUP] 4 5 12 17 [/SUP], Liana F Lareau[SUP] 2 4 [/SUP], Patrick D Hsu[SUP] 18 19 20 21 22 [/SUP], David F Savage[SUP] 23 24 [/SUP], Jennifer A Doudna[SUP] 25 26 27 28 29 30 31 [/SUP]
Collaborators, Affiliations
- PMID: 34354262
- DOI: 10.1038/s41589-021-00842-2
Abstract
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 RNA recognition that triggers cleavage and release of a fluorescent reporter molecule, but long reaction times hamper their detection sensitivity and speed. 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 molecules per µl of RNA in 20 min. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA extracted from respiratory swab samples with quantitative reverse transcriptase PCR (qRT-PCR)-derived cycle threshold (C[SUB]t[/SUB]) values up to 33, using a compact detector. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables sensitive, direct RNA detection in a format that is amenable to point-of-care infection diagnosis as well as to a wide range of other diagnostic or research applications.