A simple and rapid CRISPR-Cas12a based detection test for diastatic Saccharomyces cerevisiae

Ida Uotila; Kristoffer Krogerus

doi:10.58430/jib.v129i2.21

Authors

Ida Uotila VTT Technical Research Centre of Finland
Kristoffer Krogerus VTT Technical Research Centre of Finland

DOI:

https://doi.org/10.58430/jib.v129i2.21

Keywords:

beer, contamination, diastatic yeast, detection, CRISPR-Cas12a

Abstract

Diastatic Saccharomyces cerevisiae is a common contaminant in the brewing industry. Currently available detection methods are either time consuming or require specialised equipment. The aim of this study was to develop a new rapid and simple assay for the detection of diastatic yeast from samples of beer and yeast. More specifically, the aim was to develop a simple and rapid assay that requires minimal laboratory equipment or training, and yields results as accurate as PCR-based methods. The assay consists of three main steps: DNA extraction, pre-amplification of DNA, and CRISPR-Cas12a based detection and visualisation. Different pre-amplification and visualisation techniques were compared, and the final assay involved a one-pot reaction where LAMP and Cas12a were consecutively used to pre-amplify and detect a fragment from the STA1 gene in a single tube. These reactions required a heat block, a pipette, and a centrifuge with the assay result visualised on a lateral flow strip. The assay was used to monitor an intentionally contaminated brewing fermentation and was shown to yield results as accurate as PCR with previously published primers. Furthermore, the assay yielded results in approximately 75 minutes. The developed assay offers reliable and rapid quality control for breweries of all sizes and can be performed without expensive laboratory equipment. It is suggested that the assay will be particularly useful for smaller breweries without well-equipped laboratories who are looking to implement better quality control.

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References

Ali Z, Aman R, Mahas A, Rao GS, Tehseen M, Marsic T, Salunke R, Subudhi AK, Hala SM, Hamdan SM, Pain A, Alofi FS, Alsomali A, Hashem AM, Khogeer A, Almontashiri NAM, Abedalthagafi M, Hassan N, Mahfouz MM. 2020. iSCAN: An RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2. Virus Res 288:198129. DOI: https://doi.org/10.1016/j.virusres.2020.198129

Bao Y, Jiang Y, Xiong E, Tian T, Zhang Z, Lv J, Li Y, Zhou X. 2020. CUT-LAMP: Contamination-free loop-mediated isothermal amplification based on the CRISPR/Cas9 cleavage. ACS Sensors 5:1082–1091. DOI: https://doi.org/10.1021/acssensors.0c00034

Blount BA, Driessen MRM, Ellis T. 2016. GC Preps: Fast and easy extraction of stable yeast genomic DNA. Sci Rep 6:26863. DOI: https://doi.org/10.1038/srep26863

Broughton JP, Deng X, Yu G, Fasching CL, Servellita V, Singh J, Miao X, Streithorst JA, Granados A, Sotomayor-Gonzalez A, Zorn K, Gopez A, Hsu E, Gu W, Miller S, Pan C-Y, Guevara H, Wadford DA, Chen JS, Chiu CY. 2020. CRISPR–Cas12-based detection of SARS-CoV-2. Nat Biotechnol 38:870–874. DOI: https://doi.org/10.1038/s41587-020-0513-4

Burns LT, Sislak CD, Gibbon NL, Saylor NR, Seymour MR, Shaner LM, Gibney PA. 2021. Improved functional assays and risk assessment for STA1+ strains of Saccharomyces cerevisiae. J Am Soc Brew Chem 79:167–180. DOI: https://doi.org/10.1080/03610470.2020.1796175

Chen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, Doudna JA. 2018. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science 360:436–439. DOI: https://doi.org/10.1126/science.aar6245

Dao Thi VL, Herbst K, Boerner K, Meurer M, Kremer LP, Kirrmaier D, Freistaedter A, Papagiannidis D, Galmozzi C, Stanifer ML, Boulant S, Klein S, Chlanda P, Khalid D, Barreto Miranda I, Schnitzler P, Kräusslich H-G, Knop M, Anders S. 2020. A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples. Sci Transl Med 12: DOI: https://doi.org/10.1126/scitranslmed.abc7075

Engel SR, Dietrich FS, Fisk DG, Binkley G, Balakrishnan R, Costanzo MC, Dwight SS, Hitz BC, Karra K, Nash RS, Weng S, Wong ED, Lloyd P, Skrzypek MS, Miyasato SR, Simison M, Cherry JM. 2014. The reference genome sequence of Saccharomyces cerevisiae: then and now. G3 (Bethesda) 4:389–98. DOI: https://doi.org/10.1534/g3.113.008995

Gallone B, Steensels J, Prahl T, Soriaga L, Saels V, Herrera-Malaver B, Merlevede A, Roncoroni M, Voordeckers K, Miraglia L, Teiling C, Steffy B, Taylor M, Schwartz A, Richardson T, White C, Baele G, Maere S, Verstrepen KJ. 2016. Domestication and divergence of Saccharomyces cerevisiae beer yeasts. Cell 166:1397-1410.e16. DOI: https://doi.org/10.1016/j.cell.2016.08.020

Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin E V., Hung DT, Sabeti PC, Collins JJ, Zhang F. 2017. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 356:438–442. DOI: https://doi.org/10.1126/science.aam9321

Hayashi N, Minato T, Kanai K, Ikushima S, Yoshida S, Tada S, Taguchi H, Ogawa Y. 2009. Differentiation of species belonging to Saccharomyces Sensu Stricto using a loop-mediated isothermal amplification method. J Am Soc Brew Chem 67:118–126. DOI: https://doi.org/10.1094/ASBCJ-2009-0309-01

Ho NRY, Lim GS, Sundah NR, Lim D, Loh TP, Shao H. 2018. Visual and modular detection of pathogen nucleic acids with enzyme–DNA molecular complexes. Nat Commun 9:3238. DOI: https://doi.org/10.1038/s41467-018-05733-0

Jia B, Li X, Liu W, Lu C, Lu X, Ma L, Li Y-Y, Wei C. 2019. GLAPD: Whole genome based LAMP primer design for a set of target genomes. Front Microbiol 10.3389. DOI: https://doi.org/10.3389/fmicb.2019.02860

Kellner MJ, Koob JG, Gootenberg JS, Abudayyeh OO, Zhang F. 2019. SHERLOCK: nucleic acid detection with CRISPR nucleases. Nat Protoc 14:2986–3012. DOI: https://doi.org/10.1038/s41596-019-0210-2

Krogerus K, Gibson B. (2020). A re-evaluation of diastatic Saccharomyces cerevisiae strains and their role in brewing. Appl Microbiol Biotechnol 104:3745–3756. DOI: https://doi.org/10.1007/s00253-020-10531-0

Krogerus K, Magalhães F, Kuivanen J, Gibson B. 2019. A deletion in the STA1 promoter determines maltotriose and starch utilization in STA1+ Saccharomyces cerevisiae strains. Appl Microbiol Biotechnol 103:7597–7615. DOI: https://doi.org/10.1007/s00253-019-10021-y

Li L, Li S, Wu N, Wu J, Wang G, Zhao G, Wang J. 2019. HOLMESv2: A CRISPR-Cas12b-assisted platform for nucleic acid detection and DNA methylation quantitation. ACS Synth Biol 8:2228–2237. DOI: https://doi.org/10.1021/acssynbio.9b00209

Li S-Y, Cheng Q-X, Wang J-M, Li X-Y, Zhang Z-L, Gao S, Cao R-B, Zhao G-P, Wang J. 2018. CRISPR-Cas12a-assisted nucleic acid detection. Cell Discov 4:20 . DOI: https://doi.org/10.1038/s41421-018-0028-z

Liu L, Zhao G, Li X, Xu Z, Lei H, Shen X. 2022. Development of rapid and easy detection of Salmonella in food matrics using RPA-CRISPR/Cas12a method. LWT 162:113443. DOI: https://doi.org/10.1016/j.lwt.2022.113443

Ma L, Peng L, Yin L, Liu G, Man S. 2021. CRISPR-Cas12a-powered dual-mode biosensor for ultrasensitive and cross-validating detection of pathogenic bacteria. ACS Sensors 6:2920–2927. DOI: https://doi.org/10.1021/acssensors.1c00686

Mahas A, Hassan N, Aman R, Marsic T, Wang Q, Ali Z, Mahfouz MM. 2021. LAMP-coupled CRISPR–Cas12a module for rapid and sensitive detection of plant DNA viruses. Viruses 13:466. DOI: https://doi.org/10.3390/v13030466

Meier-Dörnberg T, Jacob F, Michel M, Hutzler M. 2017. Incidence of Saccharomyces cerevisiae var. diastaticus in the beverage industry: cases of contamination, 2008–2017. Tech Q Master Brew Assoc Am 54:140–148.

Meier-Dörnberg T, Kory OI, Jacob F, Michel M, Hutzler M. 2018. Saccharomyces cerevisiae variety diastaticus friend or foe?—spoilage potential and brewing ability of different Saccharomyces cerevisiae variety diastaticus yeast isolates by genetic, phenotypic and physiological characterization. FEMS Yeast Res 18: foy023. DOI: https://doi.org/10.1093/femsyr/foy023

Meng Q, Yang H, Zhang G, Sun W, Ma P, Liu X, Dang L, Li G, Huang X, Wang X, Liu J, Leng Q. 2021. CRISPR/Cas12a-assisted rapid identification of key beer spoilage bacteria. Innov Food Sci Emerg Technol 74:102854. DOI: https://doi.org/10.1016/j.ifset.2021.102854

Michel M, Meier-Dörnberg T, Kleucker A, Jacob F, Hutzler M. 2016. A new approach for detecting spoilage yeast in pure bottom-fermenting and pure Torulaspora delbrueckii pitching yeast, propagation yeast, and finished beer. J Am Soc Brew Chem 74:200–205 . DOI: https://doi.org/10.1094/ASBCJ-2016-3148-01

Notomi T. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:63e – 63. DOI: https://doi.org/10.1093/nar/28.12.e63

Piepenburg O, Williams CH, Stemple DL, Armes NA. 2006. DNA detection using recombination proteins. PLoS Biol 4:e204. DOI: https://doi.org/10.1371/journal.pbio.0040204

Powell CD, Kerruish DWM. 2017. Beer-spoiling yeasts: genomics, detection, and control. p 289-327. In Bokulich NA, Bamforth CW (eds). Brewing Microbiology – Current Research, Omics and Microbial Ecology. Caister Academic Press, Norfolk, UK. DOI: https://doi.org/10.21775/9781910190616.11

Traynor S, Zhou R, Spence N, Preiss R. 2021. Open-source PCR and agar-based methods for cost-effective detection of diastatic yeast. Tech Q Master Brew Assoc Am 58:76-97. DOI: https://doi.org/10.1094/TQ-58-2-0611-01

Tsuchiya Y, Ogawa M, Nakakita Y, Nara Y, Kaneda H, Watari J, Minekawa H, Soejima T. 2007. Identification of beer-spoilage microorganisms using the loop-mediated isothermal amplification method. J Am Soc Brew Chem 65:77–80. DOI: https://doi.org/10.1094/ASBCJ-2007-0227-01

Wang B, Wang R, Wang D, Wu J, Li J, Wang J, Liu H, Wang Y. 2019. Cas12aVDet: A CRISPR/Cas12a-based platform for rapid and visual nucleic acid detection. Anal Chem 91:12156–12161. DOI: https://doi.org/10.1021/acs.analchem.9b01526

Wang D-G, Brewster J, Paul M, Tomasula P. 2015. Two methods for increased specificity and sensitivity in loop-mediated isothermal amplification. Molecules 20:6048–6059. DOI: https://doi.org/10.3390/molecules20046048

Wang R, Qian C, Pang Y, Li M, Yang Y, Ma H, Zhao M, Qian F, Yu H, Liu Z, Ni T, Zheng Y, Wang Y. 2021. opvCRISPR: One-pot visual RT-LAMP-CRISPR platform for SARS-cov-2 detection. Biosens Bioelectron 172:112766. DOI: https://doi.org/10.1016/j.bios.2020.112766

Wang Y, Ke Y, Liu W, Sun Y, Ding X. 2020. A one-pot toolbox based on Cas12a/crRNA enables rapid foodborne pathogen detection at attomolar level. ACS Sensors 5:1427–1435. DOI: https://doi.org/10.1021/acssensors.0c00320

Yamashita I, Nakamura M, Fukui S. 1987. Gene fusion is a possible mechanism underlying the evolution of STA1. J Bacteriol 169:2142–2149. DOI: https://doi.org/10.1128/jb.169.5.2142-2149.1987

Yamauchi H, Yamamoto H, Shibano Y, Amaya N, Saeki T. 1998. Rapid methods for detecting Saccharomyces diastaticus, a beer spoilage yeast, using the polymerase chain reaction. J Am Soc Brew Chem 56:58–63. DOI: https://doi.org/10.1094/ASBCJ-56-0058

Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL. 2012. Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13:134. DOI: https://doi.org/10.1186/1471-2105-13-134

Zhang M, Wang H, Wang H, Wang F, Li Z. 2021. CRISPR/Cas12a-assisted ligation-initiated loop-mediated isothermal amplification (CAL-LAMP) for highly specific detection of microRNAs. Anal Chem 93:7942–7948. DOI: https://doi.org/10.1021/acs.analchem.1c00686