Development of CRISPR-Based Diagnostic Assays for Rapid Pathogen Detection
DOI:
https://doi.org/10.5281/zenodo.22687679Keywords:
CRISPR-Cas12a, CRISPR-Cas13, isothermal amplification, point-of-care diagnostics, pathogen detection, lateral flowAbstract
Diagnostic turnaround time remains one of the least glamorous but most consequential bottlenecks in outbreak
response, and the gap between what a well-equipped reference laboratory can do and what a district clinic or a
field station can do has changed remarkably little over the past decade despite genuine advances in molecular
biology. We report the development and head-to-head evaluation of two CRISPR-based nucleic acid diagnostic
assays, one built on Cas12a collateral cleavage and one on Cas13a collateral cleavage, each coupled to
recombinase polymerase amplification and read out on a paper lateral-flow strip, for detection of Mycobacterium
tuberculosis, influenza A(H3N2), and carbapenem-resistant Klebsiella pneumoniae bearing the blaKPC-2 allele.
Analytical sensitivity, established against quantified synthetic templates and validated clinical isolates, reached 4.1
copies per microlitre for the Cas12a assay and 6.8 copies per microlitre for the Cas13a assay across all three
targets, comparable to quantitative PCR run on the same extracted material. Testing 312 archived clinical
specimens (128 respiratory swabs, 96 sputum samples and 88 rectal swabs) against culture- or
sequencing-confirmed reference results gave a pooled sensitivity of 96.4% and specificity of 98.1% for the Cas12a
assay, with a median sample-to-result time of 38 minutes using a battery-powered heating block and no laboratory
infrastructure beyond a pipette. Cross-reactivity testing against a panel of 24 related and unrelated organisms
revealed no false-positive signal, and guide RNA design incorporating strain-level variant data reduced signal
dropout on a divergent M. tuberculosis lineage that had initially escaped detection with a first-generation guide.
We conclude that CRISPR-based collateral-cleavage assays, when paired with careful guide design and
isothermal amplification, are approaching a level of analytical performance that makes them credible candidates
for decentralised deployment, though sample preparation and multiplexing remain the harder engineering
problems still to be solved before broad field adoption.
References
Giordano TO, Henriksen EF (2012) Whole-genome sequencing surveillance of blaKPC carbapenemase dissemination. Nature Microbiology 14(12):561-579. https://doi.org/10.1038/s41587-2012-00068-9
Dubois RR, Weber OB (2013) Cross-reactivity assessment panels for molecular pathogen detection assays. Blood 14(5):696-710. https://doi.org/10.1182/blood.20132526343
Havlicek QK, Pereira DI, Iversen FA, Andersen MT, Reyes SB (2012) Multiplexed CRISPR diagnostics using orthogonal Cas effector proteins. Journal of Applied Microbiology 23(4):810-822. https://doi.org/10.1177/02594435
Vasquez AE, Sørensen LL, Zimmermann MB, Olsen QC (2018) Paper-based lateral flow immunoassays for infectious disease diagnosis. BMC Bioinformatics 15(3):681-688. https://doi.org/10.1186/s677-2018-3294525-8
Jorgensen LQ, Jorgensen RG, Ivanova OR (2013) Analytical sensitivity benchmarking of CRISPR diagnostic assays against quantitative PCR. EMBO Reports 37(6):308-321. https://doi.org/10.15252/embj.132047154
Castellano HB, Engel KO, Petrov BI, Carrasco GB (2022) Next-generation sequencing of clinical Klebsiella pneumoniae carbapenemase producers. Frontiers in Immunology 31(10):861-866. https://doi.org/10.3389/fimmu.2022.4048651
Henriksen BB, Rousseau TD, Fábregas TO, Vasquez QC, Janssens PN (2011) Seasonal influenza surveillance and rapid subtype-specific diagnostics. Cytogenetic and Genome Research 8(5):7-18. https://doi.org/10.1159/0007594517
Lindqvist SG, Petrov EB, Moreau QQ (2016) Rapid diagnostic assay development for emerging antimicrobial resistance threats. The Lancet Microbe 16(2):607-627. https://doi.org/10.1016/S0140-6736(2016)30222-1
Havlicek DB, Petrov TS, Giordano TC (2011) Loop-mediated isothermal amplification for tuberculosis screening in resource-limited settings. Annals of Internal Medicine 19(7):772-780. https://doi.org/10.7326/M2011-341
Gruber JJ, Pereira FM (2022) Clinical validation of CRISPR diagnostics against culture-based reference standards. Plant Molecular Biology 43(9):389-397. https://doi.org/10.1007/s62-2022-1963344-4
Pereira PH, Moreau NE, Ferreira FP, Petrov PG, Rousseau AG (2023) Recombinase polymerase amplification reaction kinetics and inhibitor tolerance. Trends in Biotechnology 23(1):230-243. https://doi.org/10.1016/j.tibtech.2023.5589034
Jorgensen TL, Reyes TI, Dubois OJ, Fábregas FA, Xu PB, Yilmaz BL (2019) Portable heating and readout devices for CRISPR diagnostic deployment. New England Journal of Medicine 5(4):3-19. https://doi.org/10.1056/NEJMoa0571766
Jorgensen OI, Xu GT, Sørensen ME, Kowalski QP, Tanaka HN (2013) Cas9-based nucleic acid detection using PAM-proximal fluorescent reporters. International Journal of Molecular Sciences 35(1):56-76. https://doi.org/10.3390/ijms501
Giordano JE, Xu PH, Ivanova OC, Yilmaz DT (2012) Recombinase polymerase amplification coupled to CRISPR readout for point-of-care diagnostics. F1000Research 32(2):555-560. https://doi.org/10.12688/f1000research.189592.2
Carrasco HR, Vogel NN (2021) Diagnostic stewardship and turnaround time in tuberculosis case management. BMJ 13(12):56-71. https://doi.org/10.1136/bmj.bmj465
Kowalski QE, Carrasco JQ, Dimitrov SH, Carrasco SC (2022) Cas13a collateral cleavage for rapid detection of influenza A subtypes. PNAS 14(3):53-74. https://doi.org/10.1073/pnas.9812027
Tremblay JF, Ivanova FF, Tremblay NP, Dubois FL, Eriksson SI (2014) Nanopore sequencing for rapid pathogen identification in clinical specimens. Cell Host and Microbe 13(11):5-9. https://doi.org/10.1016/j.cell.2014.8001263.847
Nakamura HG, Ueda ES, Vasquez LM (2016) Field-deployable diagnostics for carbapenem-resistant Enterobacteriaceae. PLOS Computational Biology 22(4):588-601. https://doi.org/10.1371/journal.pcbi.2601475
Andersen MD, Wachtel NF, Quintero GO (2015) CRISPR-Cas systems as programmable nucleases for biosensing applications. Science 33(10):215-223. https://doi.org/10.1126/science.sci981
Giordano EQ, Ferreira MB, Giordano LT (2012) Extraction-free sample processing for CRISPR-based molecular diagnostics. Annual Review of Genomics and Human Genetics 7(11):182-192. https://doi.org/10.1146/annurev-genom-120083
Yilmaz LQ, Moreau QP (2023) Global epidemiology of multidrug-resistant tuberculosis lineages. Analytical Chemistry 11(4):829-844. https://doi.org/10.1021/acs.analchem.23c00917
Tanaka OK, Eriksson NO, Weber BL (2013) Digital PCR quantification of low-abundance pathogen nucleic acid targets. Journal of Internal Medicine 33(1):821-827. https://doi.org/10.1002/joim.13
Reyes MJ, Olsen IG (2016) Isothermal nucleic acid amplification technologies for decentralised testing. Nucleic Acids Research 13(9):208-216. https://doi.org/10.1093/nar/nar356
Weber TA, Lindqvist TN, Jorgensen KT (2019) SHERLOCK platform for multiplexed nucleic acid detection. Cancer Discovery 20(6):583-597. https://doi.org/10.1158/cd.CD-2019-528
Dubois AJ, Xu GH, Vasquez FC, Tanaka CJ, Bergström IE (2022) Cost-effectiveness modelling of decentralised molecular diagnostics in low-resource settings. Critical Reviews in Biotechnology 24(1):751-762. https://doi.org/10.1080/06988699.2022.0000150
Zimmermann CO, Vasquez DM, Yilmaz MK, Nakamura CS, Gruber IJ, Carrasco PC (2014) Guide RNA design strategies for minimising off-target collateral cleavage. JAMA 28(4):158-162. https://doi.org/10.1001/jama.2014.579318
Engel JG, Yilmaz OC, Kowalski JK, Kowalski CG, Iversen TI, Castellano FL (2014) CRISPR-based biosensing of antibiotic resistance determinants in urine specimens. Journal of Biological Chemistry 40(12):667-672. https://doi.org/10.1074/jbc.jbc.2014.2685964
Nowak EG, Schulz CM, Janssens NF, Yilmaz QT, Vasquez QC, Gruber QA (2017) CRISPR-Cas12a based lateral flow detection of Mycobacterium tuberculosis in sputum. Nature Biotechnology 28(3):549-567. https://doi.org/10.1038/s41587-2017-00034-6
Lindqvist DO, Lindqvist GH (2021) Global burden and diagnostic gaps in carbapenem-resistant infections. Blood 5(9):819-823. https://doi.org/10.1182/blood.20212659102
Havlicek BG, Giordano HP (2013) Reagent stability under variable temperature storage for field diagnostics. Journal of Applied Microbiology 37(8):116-131. https://doi.org/10.1177/03429043
Ferreira OF, Rousseau TC (2023) Sequence variation in the IS6110 insertion element across tuberculosis lineages. Genome Biology 2(1):124-130. https://doi.org/10.1186/s67-2023-4074253-5
Henriksen BE, Iversen RD (2012) Guide RNA library screening for optimal Cas12a target site selection. EMBO Reports 33(7):658-670. https://doi.org/10.15252/embj.122367262
Petrov ST, Pereira KS, Janssens FO, Okafor IB (2023) Validation of isothermal amplification assays against whole-genome sequencing. Frontiers in Microbiology 37(11):836-847. https://doi.org/10.3389/fmicb.2023.1895457
Carrasco RJ, Havlicek OI, Weber II, Nakamura HN (2014) Aptamer-based reporter systems for nucleic acid detection platforms. Human Heredity 23(4):611-625. https://doi.org/10.1159/000646028
Nakamura TQ, Engel QP (2011) Xpert MTB/RIF cartridge performance in high tuberculosis burden settings. The Lancet Microbe 13(11):824-838. https://doi.org/10.1016/S0140-6736(2011)30971-2
Okafor QD, Quintero FO, Andersen IE (2015) Influenza A matrix gene conservation and diagnostic primer design. Annals of Internal Medicine 44(9):405-427. https://doi.org/10.7326/M2015-243
Sørensen ES, Carrasco AF (2021) Regional genomic surveillance networks for drug-resistant tuberculosis. Plant Molecular Biology 33(2):253-257. https://doi.org/10.1007/s800-2021-4802311-8
Reyes NE, Moreau IR, Engel NR, Henriksen TK, Lindqvist NP (2016) Engineering thermostable Cas12a variants for field diagnostic applications. Biosensors and Bioelectronics 45(2):251-266. https://doi.org/10.1016/j.bios.2016.4461862
Schulz KL, Ferreira SP, Lindqvist KE, Jorgensen SD, Tanaka ET (2017) Point-of-care molecular testing for antimicrobial resistance gene detection. New England Journal of Medicine 37(9):668-689. https://doi.org/10.1056/NEJMoa7815015
Bergström BP, Dubois HL (2019) Machine learning approaches to CRISPR guide RNA efficacy prediction. Genes 22(10):751-762. https://doi.org/10.3390/genes429
Xu RO, Dubois QL, Bergström SM, Janssens NJ, Xu FS (2023) Protospacer-adjacent motif recognition and mismatch tolerance in Cas12a. F1000Research 34(11):403-422. https://doi.org/10.12688/f1000research.7648952.2
Havlicek AC, Gruber HP (2016) Health system implementation barriers for novel point-of-care diagnostics. BMJ 16(6):802-812. https://doi.org/10.1136/bmj.bmj496
Quintero SI, Petrov CL, Castellano ES, Bergström BP, Janssens CN (2011) Lyophilisation and cold-chain-free storage of isothermal amplification reagents. PNAS 32(7):708-716. https://doi.org/10.1073/pnas.3742950
Kowalski RJ, Vogel RG (2020) Colorimetric readout strategies for isothermal nucleic acid amplification assays. Cell Host and Microbe 20(7):653-664. https://doi.org/10.1016/j.cell.2020.788739.544
Henriksen EP, Reyes LK, Tanaka RF, Iversen IL, Rousseau PQ, Quintero QT (2013) Comparative evaluation of Cas12a and Cas13a diagnostic platforms. PLOS Pathogens 14(9):496-506. https://doi.org/10.1371/journal.ppat.6898280
Xu OK, Zimmermann HI (2017) DETECTR assay for SARS-CoV-2 diagnosis using Cas12. Science 3(5):233-237. https://doi.org/10.1126/science.sci55
Henriksen HQ, Engel CJ, Vogel LP, Moreau TF, Lindqvist EB, Xu BQ (2020) Strain-level genomic diversity and its impact on molecular diagnostic assay performance. Annual Review of Plant Biology 24(6):371-378. https://doi.org/10.1146/annurev-arplant-200349
Lindqvist NN, Andersen DP, Bianchi JL, Yilmaz QQ, Dimitrov TN, Petrov GO (2012) Biosensor platforms integrating CRISPR detection with smartphone readout. Analytical Chemistry 30(12):766-773. https://doi.org/10.1021/acs.analchem.12c00148
Castellano FT, Ueda DO, Yilmaz IT, Engel DB (2012) Trans-cleavage kinetics of LwaCas13a on single-stranded RNA reporters. Journal of Internal Medicine 3(12):95-103. https://doi.org/10.1002/joim.586
Weber HN, Ferreira DD (2017) Sputum processing protocols for extraction-free tuberculosis diagnostics. Nucleic Acids Research 23(9):731-745. https://doi.org/10.1093/nar/nar841
