• Je něco špatně v tomto záznamu ?

A thermostable Cas12b from Brevibacillus leverages one-pot discrimination of SARS-CoV-2 variants of concern

LT. Nguyen, NC. Macaluso, BLM. Pizzano, MN. Cash, J. Spacek, J. Karasek, MR. Miller, JA. Lednicky, RR. Dinglasan, M. Salemi, PK. Jain

. 2022 ; 77 (-) : 103926. [pub] 20220313

Jazyk angličtina Země Nizozemsko

Typ dokumentu časopisecké články

Perzistentní odkaz   https://www.medvik.cz/link/bmc22010836

Grantová podpora
U01 GH002338 CGH CDC HHS - United States

BACKGROUND: Current SARS-CoV-2 detection platforms lack the ability to differentiate among variants of concern (VOCs) in an efficient manner. CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) based detection systems have the potential to transform the landscape of COVID-19 diagnostics due to their programmability; however, most of these methods are reliant on either a multi-step process involving amplification or elaborate guide RNA designs. METHODS: Three Cas12b proteins from Alicyclobacillus acidoterrestris (AacCas12b), Alicyclobacillus acidiphilus (AapCas12b), and Brevibacillus sp. SYP-B805 (BrCas12b) were expressed and purified, and their thermostability was characterised by differential scanning fluorimetry, cis-, and trans-cleavage activities over a range of temperatures. The BrCas12b was then incorporated into a reverse transcription loop-mediated isothermal amplification (RT-LAMP)-based one-pot reaction system, coined CRISPR-SPADE (CRISPR Single Pot Assay for Detecting Emerging VOCs). FINDINGS: Here we describe a complete one-pot detection reaction using a thermostable Cas12b effector endonuclease from Brevibacillus sp. to overcome these challenges detecting and discriminating SARS-CoV-2 VOCs in clinical samples. CRISPR-SPADE was then applied for discriminating SARS-CoV-2 VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) and validated in 208 clinical samples. CRISPR-SPADE achieved 92·8% sensitivity, 99·4% specificity, and 96·7% accuracy within 10-30 min for discriminating the SARS-CoV-2 VOCs, in agreement with S gene sequencing, achieving a positive and negative predictive value of 99·1% and 95·1%, respectively. Interestingly, for samples with high viral load (Ct value ≤ 30), 100% accuracy and sensitivity were attained. To facilitate dissemination and global implementation of the assay, a lyophilised version of one-pot CRISPR-SPADE reagents was developed and combined with an in-house portable multiplexing device capable of interpreting two orthogonal fluorescence signals. INTERPRETATION: This technology enables real-time monitoring of RT-LAMP-mediated amplification and CRISPR-based reactions at a fraction of the cost of a qPCR system. The thermostable Brevibacillus sp. Cas12b offers relaxed primer design for accurately detecting SARS-CoV-2 VOCs in a simple and robust one-pot assay. The lyophilised reagents and simple instrumentation further enable rapid deployable point-of-care diagnostics that can be easily expanded beyond COVID-19. FUNDING: This project was funded in part by the United States-India Science & Technology Endowment Fund- COVIDI/247/2020 (P.K.J.), Florida Breast Cancer Foundation- AGR00018466 (P.K.J.), National Institutes of Health- NIAID 1R21AI156321-01 (P.K.J.), Centers for Disease Control and Prevention- U01GH002338 (R.R.D., J.A.L., & P.K.J.), University of Florida, Herbert Wertheim College of Engineering (P.K.J.), University of Florida Vice President Office of Research and CTSI seed funds (M.S.), and University of Florida College of Veterinary Medicine and Emerging Pathogens Institute (R.R.D.).

Citace poskytuje Crossref.org

000      
00000naa a2200000 a 4500
001      
bmc22010836
003      
CZ-PrNML
005      
20220506131356.0
007      
ta
008      
220425s2022 ne f 000 0|eng||
009      
AR
024    7_
$a 10.1016/j.ebiom.2022.103926 $2 doi
035    __
$a (PubMed)35290826
040    __
$a ABA008 $b cze $d ABA008 $e AACR2
041    0_
$a eng
044    __
$a ne
100    1_
$a Nguyen, Long T $u Department of Chemical Engineering, Herbert Wertheim College of Engineering, University of Florida, Gainesville, FL, USA
245    12
$a A thermostable Cas12b from Brevibacillus leverages one-pot discrimination of SARS-CoV-2 variants of concern / $c LT. Nguyen, NC. Macaluso, BLM. Pizzano, MN. Cash, J. Spacek, J. Karasek, MR. Miller, JA. Lednicky, RR. Dinglasan, M. Salemi, PK. Jain
520    9_
$a BACKGROUND: Current SARS-CoV-2 detection platforms lack the ability to differentiate among variants of concern (VOCs) in an efficient manner. CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) based detection systems have the potential to transform the landscape of COVID-19 diagnostics due to their programmability; however, most of these methods are reliant on either a multi-step process involving amplification or elaborate guide RNA designs. METHODS: Three Cas12b proteins from Alicyclobacillus acidoterrestris (AacCas12b), Alicyclobacillus acidiphilus (AapCas12b), and Brevibacillus sp. SYP-B805 (BrCas12b) were expressed and purified, and their thermostability was characterised by differential scanning fluorimetry, cis-, and trans-cleavage activities over a range of temperatures. The BrCas12b was then incorporated into a reverse transcription loop-mediated isothermal amplification (RT-LAMP)-based one-pot reaction system, coined CRISPR-SPADE (CRISPR Single Pot Assay for Detecting Emerging VOCs). FINDINGS: Here we describe a complete one-pot detection reaction using a thermostable Cas12b effector endonuclease from Brevibacillus sp. to overcome these challenges detecting and discriminating SARS-CoV-2 VOCs in clinical samples. CRISPR-SPADE was then applied for discriminating SARS-CoV-2 VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) and validated in 208 clinical samples. CRISPR-SPADE achieved 92·8% sensitivity, 99·4% specificity, and 96·7% accuracy within 10-30 min for discriminating the SARS-CoV-2 VOCs, in agreement with S gene sequencing, achieving a positive and negative predictive value of 99·1% and 95·1%, respectively. Interestingly, for samples with high viral load (Ct value ≤ 30), 100% accuracy and sensitivity were attained. To facilitate dissemination and global implementation of the assay, a lyophilised version of one-pot CRISPR-SPADE reagents was developed and combined with an in-house portable multiplexing device capable of interpreting two orthogonal fluorescence signals. INTERPRETATION: This technology enables real-time monitoring of RT-LAMP-mediated amplification and CRISPR-based reactions at a fraction of the cost of a qPCR system. The thermostable Brevibacillus sp. Cas12b offers relaxed primer design for accurately detecting SARS-CoV-2 VOCs in a simple and robust one-pot assay. The lyophilised reagents and simple instrumentation further enable rapid deployable point-of-care diagnostics that can be easily expanded beyond COVID-19. FUNDING: This project was funded in part by the United States-India Science & Technology Endowment Fund- COVIDI/247/2020 (P.K.J.), Florida Breast Cancer Foundation- AGR00018466 (P.K.J.), National Institutes of Health- NIAID 1R21AI156321-01 (P.K.J.), Centers for Disease Control and Prevention- U01GH002338 (R.R.D., J.A.L., & P.K.J.), University of Florida, Herbert Wertheim College of Engineering (P.K.J.), University of Florida Vice President Office of Research and CTSI seed funds (M.S.), and University of Florida College of Veterinary Medicine and Emerging Pathogens Institute (R.R.D.).
650    12
$a Brevibacillus $x genetika $7 D058675
650    12
$a COVID-19 $x diagnóza $7 D000086382
650    _2
$a lidé $7 D006801
650    _2
$a guide RNA, Kinetoplastida $7 D017394
650    _2
$a SARS-CoV-2 $x genetika $7 D000086402
655    _2
$a časopisecké články $7 D016428
700    1_
$a Macaluso, Nicolas C $u Department of Chemical Engineering, Herbert Wertheim College of Engineering, University of Florida, Gainesville, FL, USA
700    1_
$a Pizzano, Brianna L M $u Department of Agricultural and Biological Engineering, College of Agricultural and Life Sciences, University of Florida, Gainesville, FL, USA
700    1_
$a Cash, Melanie N $u Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL, USA; Emerging Pathogens Institute, University of Florida, Gainesville, FL, USA
700    1_
$a Spacek, Jan $u Sparsek S.R.O., Příkop 838/6, Zábrdovice, Brno 60200, Czech Republic
700    1_
$a Karasek, Jan $u SCIERING S.R.O., Příkop 838/6, Zábrdovice, Brno 62100, Czech Republic
700    1_
$a Miller, Megan R $u Emerging Pathogens Institute, University of Florida, Gainesville, FL, USA; Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA
700    1_
$a Lednicky, John A $u Emerging Pathogens Institute, University of Florida, Gainesville, FL, USA; Department of Environmental and Global Health, College of Public Health and Health Professions, University of Florida, Gainesville, FL, USA
700    1_
$a Dinglasan, Rhoel R $u Emerging Pathogens Institute, University of Florida, Gainesville, FL, USA; Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA
700    1_
$a Salemi, Marco $u Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL, USA; Emerging Pathogens Institute, University of Florida, Gainesville, FL, USA
700    1_
$a Jain, Piyush K $u Department of Chemical Engineering, Herbert Wertheim College of Engineering, University of Florida, Gainesville, FL, USA; UF Health Cancer Center, University of Florida, Gainesville, FL, USA. Electronic address: Jainp@ufl.edu
773    0_
$w MED00190061 $t EBioMedicine $x 2352-3964 $g Roč. 77, č. - (2022), s. 103926
856    41
$u https://pubmed.ncbi.nlm.nih.gov/35290826 $y Pubmed
910    __
$a ABA008 $b sig $c sign $y p $z 0
990    __
$a 20220425 $b ABA008
991    __
$a 20220506131349 $b ABA008
999    __
$a ok $b bmc $g 1788787 $s 1162034
BAS    __
$a 3
BAS    __
$a PreBMC
BMC    __
$a 2022 $b 77 $c - $d 103926 $e 20220313 $i 2352-3964 $m EBioMedicine $n EBioMedicine $x MED00190061
GRA    __
$a U01 GH002338 $p CGH CDC HHS $2 United States
LZP    __
$a Pubmed-20220425

Najít záznam

Citační ukazatele

    Možnosti archivace