A One-Pot CRISPR/Cas9-Typing PCR for DNA Detection and Genotyping

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease Cas9 (Cas9) has high specificity to its target DNA as a gene editing tool. This characteristic makes it useful for detection. Combining the advantages of CRISPR/Cas9 and PCR, this study establishes novel CRISPR/Cas9-based detection method, named CRISPR/Cas9-typing PCR version 4.0 (ctPCR4.0). method can detect in one pot with sensitivity. In homogenous reaction, is first cleaved by pair Cas9– single-guide RNA complexes thus releases two single strands free 3? ends, allowing oligonucleotides anneal strands. The annealed provide templates polymerization from ends. A universal primer annealing site produced at end then amplified using primer. was verified accurately detecting cloned L1 fragments 10 genotypes high-risk human papilloma viruses (HPVs). validated highest-risk HPVs, HPV 16 18, genomic HPV-positive cervical carcinoma cells, HeLa SiHa. Finally, further HPVs 30 clinical samples. widely known have sensitivity due exponential amplification DNA. Therefore, been an indispensable tool various life-related areas. For example, RT-PCR used SARS-CoV-2 plays critical role diagnosis current COVID-19 pandemic. However, found such defects, which advanced sustainable development technique. Until now, three types techniques developed, including traditional PCR,1Mullis K.B. unusual origin polymerase chain-reaction.Sci Am. 1990; 262: 56-61Crossref PubMed Scopus (499) Google Scholar real-time quantitative (qPCR),2Arya M. Shergill I.S. Williamson Gommersall L. Arya N. Patel H.R.H. Basic principles PCR.Expert Rev Mol Diagn. 2005; 5: 209-219Crossref (311) digital PCR.3Cao Cui X. Hu J. Li Z. Choi J.R. Yang Q. Lin Hui L.Y. Xu F. Advances chain reaction (dPCR) emerging biomedical applications.Biosens Bioelectron. 2017; 90: 459-474Crossref (127) Although these are different techniques, basic mechanism underlying remains unchanged. That is, specific primers be designed amplifying design PCR. some cases, difficult find optimal DNA, especially highly homologous members family viruses. Primer become more problematic multiplex required possess both identical temperature. inevitable mismatch temperature (typically 58°C) often leads nonspecific false-positive results, always challenges PCR-based diagnosis. how simplify or even remove tedious desired operators. (CRISPR) immune system bacteria against phages.4Barrangou R. Fremaux C. Deveau H. Richards Boyaval P. Moineau S. Romero D.A. Horvath CRISPR provides acquired resistance prokaryotes.Science. 2007; 315: 1709-1712Crossref (3292) It immediately developed into new type tool.5Jinek East A. Cheng Ma E. Doudna RNA-programmed genome cells.Elife. 2013; 2: e00471Crossref (1320) Scholar,6Cong Ran F.A. Cox D. Barretto Habib Hsu P.D. Wu Jiang W. Marraffini L.A. Zhang Multiplex engineering CRISPR/Cas systems.Science. 339: 819-823Crossref (8602) Cas proteins, CRISPR-associated II extensively characterized explored most simplicity.7Sander J.D. Joung J.K. CRISPR-Cas systems editing, regulating targeting genomes.Nat Biotechnol. 2014; 32: 347-355Crossref (1895) alone function sequence-specific associating CRISPR-related (crRNA) transactivated crRNA, were simplified integrating crRNA (sgRNA).8Jinek Chylinski K. Fonfara I. Hauer J.A. Charpentier programmable dual-RNA-guided adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref (7584) Because Cas9-sgRNA interact natural double-stranded (dsDNA) sequence specificity, gradually applied other than editing. early Zika viruses.9Pardee Green A.A. Takahashi M.K. Braff Lambert G. Lee J.W. Ferrante T. Donghia Fan Daringer N.M. Bosch Dudley D.M. O'Connor D.H. Gehrke Collins J.J. Rapid, low-cost virus biomolecular components.Cell. 2016; 165: 1255-1266Abstract Full Text PDF (545) recent years, Cas-related methods rapidly typical cleavage activity dsDNA, nickase activity, catalytically deactivated (dCas9), CRISPR-mediated, ultrasensitive Target DNA-polymerase (CUT-PCR),10Lee S.H. Yu Hwang G.-H. Kim H.S. Ye Park D.Y. Cho Y.-K. J.-S. Bae CUT-PCR: PCR.Oncogene. 36: 6823-6829Crossref (45) DNA-loop-mediated isothermal (CUT-LAMP),11Bao Y. Xiong Tian Lv Zhou CUT-LAMP: contamination-free loop-mediated based on cleavage.ACS Sens. 2020; 1082-1091Crossref (25) finding low abundance sequences hybridization (FLASH),12Quan Langelier Kuchta Batson Teyssier Lyden Caldera McGeever Dimitrov B. King Wilheim Murphy Ares L.P. Travisano K.A. Sit Amato Mumbengegwi D.R. Smith J.L. Bennett Gosling Mourani P.M. Calfee C.S. Neff N.F. Chow E.D. P.S. Greenhouse DeRisi Crawford FLASH: next-generation diagnostic multiplexed antimicrobial sequences.Nucleic Acids Res. 2019; 47: e83Crossref (57) nickase-based (Cas9nAR),13Wang Liu Sun H.-H. Yin B.-C. An RNA-guided amplification.Angew Chem Int Ed Engl. 58: 5382-5386Crossref (34) CRISPR-Cas9-triggered nicking endonuclease-mediated strand displacement (CRISDA),14Zhou Ying Zhao Chu P.K. X.-F. detection.Nat Commun. 2018; 9: 5012Crossref (103) CRISPR-Chip,15Hajian Balderston Tran deBoer Etienne Sandhu Wauford N.A. Chung J.-Y. Nokes Athaiya Paredes Peytavi Goldsmith Murthy Conboy I.M. Aran Detection unamplified genes via CRISPR-Cas9 immobilized graphene field-effect transistor.Nat Biomed Eng. 3: 427-437Crossref (170) paired dCas9 proteins linked split halves luciferase.16Zhang Qian Wei Wang Dong H.M. Ouyang Lou Paired systematic platform featured nucleic acid pathogenic strains.ACS Synth Biol. 6: 211-216Crossref (46) These studies indicated great potential not easily grafted directly onto platform, allow easy, automatic, high-throughput Essentially, interaction dsDNA protein-assisted RNA–DNA naturally temperature, differs hybridization, occurs artificially latter forms basis Southern Northern blots, microarray chips. methods, sample undergo high-temperature denaturation, creating complicated single-stranded environment probes their targets. If controlled given, all possible occur limited true targets multitude mismatch. control critical. Even so, inevitable, ubiquitous bands spots hybridization. nondenatured state may avoid considerable that seriously methods. Clearly, Cas9-assisted sgRNA-DNA (37°C) good chance addressing problem, solve nonspecificity results 58°C). therefore focused developing combine aimed overcome key limitation determined primers. tried develop CRISPR/Cas9-dependent no longer primers, but rather CRISPR/Cas9. By Cas9-sgRNA, CRISPR-typing (ctPCR) developed.17Wang Long (ctPCR), Cas9-based method.Sci Rep. 8: 14126Crossref (21) Scholar, 18Zhang Xia Detecting typing technique.Anal Biochem. 561-562: 37-46Crossref (16) 19Zhang Shui Cas9/sgRNAs-associated reverse (CARP) Bioanal Chem. 410: 2889-2900Crossref (22) ctPCR (ctPCR1.0),17Wang sgRNA cut Cas9-sgRNA. released fragment ligated adaptor universal-specific second (ctPCR2.0),19Zhang detected cleaving ligating intramolecular circular intermolecular concatenated linear finally reverse-oriented clear ctPCR1.0 -2.0 still multiple-step ligase-needed nonhomogeneous third (ctPCR3.0),18Zhang qPCR reactions, containing not. typed comparing cycle threshold (CT) values reactions. reactions performed parallel obtain final CT To remaining shortcomings only necessary functional ingredients needed. Any enzymes ligase adaptors removed. significant advantage one-pot method. homogeneous contains sample, insert oligos, primer, ingredients. whole process finished instruments without tube opening. feasibility, reliability, testing papillomavirus (hrHPVs) plasmids (gDNA) cancer cells cell lines, SiHa, HeLa, C-33A, purchased Type Culture Collection Chinese Academy Sciences, Shanghai, China. C33-A cultured Dulbecco's modified Eagle's medium (Gibco, Thermo Fisher Scientific, Waltham, MA). SiHa (Gibco). All media supplemented 10% fetal bovine serum (HyClone Laboratories, Logan, UT), 100 units/mL penicillin (Thermo Scientific), ?g/mL streptomycin Scientific). Cells incubated 37°C humidified incubator 5% CO2. Thirty samples provided Yixing Tumor Hospital (Yixing, China). collected brush cervix patients screening. SgRNA online software CHOPCHOP 3.0.0 (http://chopchop.cbu.uib.no), hg19 reference genome. hrHPVs shown Table 1. Each had sgRNAs designed, sgRNAa sgRNAb, respectively (Table 1). According sgRNA, 2) synthesized three-round fusion protocol.19Zhang brief, PCR1 done F1 R 2). PCR2 product F2 Sg-R PCR3 F3 1) T7 promoter template preparing vitro transcription previously described.19Zhang extracted TRIzol (Invitrogen; Scientific) according manufacturer's protocol. quality prepared analyzed NanoDrop 2000 spectrophotometer agarose gel electrophoresis.Table 1sgRNAHPVGenesgRNASequenceSize bpHPV 16L1sgRNA16a5?-TGTCATAACGTCTGCAGTTA-3?101HPV 16L1sgRNA16b5?-GCACACTAGAAGATACTTAT-3?HPV 18L1sgRNA18a5?-TGCTGCACCGGCTGAAAATA-3?256HPV 18L1sgRNA18b5?-GCATCATATTGCCCAGGTAC-3?HPV 33L1sgRNA33a5?-CTGAGAGGTAACAAACCTAT-3?64HPV 33L1sgRNA33b5?-AAGGAAAAGGAAGACCCCTT-3?HPV 35L1sgRNA35a5?-ACCGCCTTCTGGTACCTTAG-3?224HPV 35L1sgRNA35b5?-ACACAGACATATTTGTACTA-3?HPV 45L1sgRNA45a5?-ACCACCTACTACCAGTTTGG-3?228HPV 45L1sgRNA45b5?-TAATGTTAAATTAGTACTGC-3?HPV 51L1sgRNA51a5?-AGTTACTTGGAGTAAATGTT-3?258HPV 51L1sgRNA51b5?-ACAGGCTAAGCCAGATCCTT-3?HPV 52L1sgRNA52a5?-GGAATACCTTCGTCATGGCG-3?84HPV 52L1sgRNA52b5?-AGTCATGTTAGTGCTACGAG-3?HPV 56L1sgRNA56a5?-TATTGGGTTATCCCCGCCAG-3?131HPV 56L1sgRNA56b5?-CATATTCCTCCACATGTCTA-3?HPV 58L1sgRNA58a5?-GCTACGAGTGGTATCAACCA-3?174HPV 58L1sgRNA58b5?-AATGACATATATACATACTA-3?HPV 59L1sgRNA59a5?-CTGGTAGGTGTGTATACATT-3?197HPV 59L1sgRNA59b5?-GGCCTGTAGATCTTAAGGAA-3?HPV, virus; RNA; sgRNAa, forward; reverse. Open table tab 2Oligonucleotides Used Prepare Transcriptional Template PCRNameSequence16-3-F25?-TGTCATAACGTCTGCAGTTAGTTTTAGAGCTAGAAATAGCAAG-3?16-3-F35?-TTCTAATACGACTCACTATAGTGTCATAACGTCTGCAGTTAG-3?-3?16-5-F25?-GCACACTAGAAGATACTTATGTTTTAGAGCTAGAAATAGCAAG-3?16-5-F35?-TTCTAATACGACTCACTATAGGCACACTAGAAGATACTTATG-3?18-11-F25?-TGCTGCACCGGCTGAAAATAGTTTTAGAGCTAGAAATAGCAAG-3?18-11-F35?-TTCTAATACGACTCACTATAGTGCTGCACCGGCTGAAAATAG-3?18-18-F25?-GCATCATATTGCCCAGGTACGTTTTAGAGCTAGAAATAGCAAG-3?18-18-F35?-TTCTAATACGACTCACTATAGGCATCATATTGCCCAGGTACG-3?33-3-F25?-CTGAGAGGTAACAAACCTATGTTTTAGAGCTAGAAATAGCAAG-3?33-3-F35?-TTCTAATACGACTCACTATAGCTGAGAGGTAACAAACCTATG-3?33-10-F25?-AAGGAAAAGGAAGACCCCTTGTTTTAGAGCTAGAAATAGCAAG-3?33-10-F35?-TTCTAATACGACTCACTATAGAAGGAAAAGGAAGACCCCTTG-3?35-3-F25?-ACCGCCTTCTGGTACCTTAGGTTTTAGAGCTAGAAATAGCAAG-3?35-3-F35?-TTCTAATACGACTCACTATAGACACAGACATATTTGTACTAG-3?45-1-F25?-ACCACCTACTACCAGTTTGGGTTTTAGAGCTAGAAATAGCAAG-3?45-1-F35?-TTCTAATACGACTCACTATAGACCACCTACTACCAGTTTGGG-3?45-3-F25?-TAATGTTAAATTAGTACTGCGTTTTAGAGCTAGAAATAGCAAG-3?45-3-F35?-TTCTAATACGACTCACTATAGTAATGTTAAATTAGTACTGCG-3?51-7-F25?-AGTTACTTGGAGTAAATGTTGTTTTAGAGCTAGAAATAGCAAG-3?51-7-F35?-TTCTAATACGACTCACTATAGAGTTACTTGGAGTAAATGTTG-3?51-13-F25?-ACAGGCTAAGCCAGATCCTTGTTTTAGAGCTAGAAATAGCAAG-3?51-13-F35?-TTCTAATACGACTCACTATAGACAGGCTAAGCCAGATCCTTG-3?52-1-F25?-GGAATACCTTCGTCATGGCGGTTTTAGAGCTAGAAATAGCAAG-3?52-1-F35?-TTCTAATACGACTCACTATAGGGAATACCTTCGTCATGGCGG-3?52-2-F25?-AGTCATGTTAGTGCTACGAGGTTTTAGAGCTAGAAATAGCAAG-3?52-2-F35?-TTCTAATACGACTCACTATAGAGTCATGTTAGTGCTACGAGG-3?56-1-F25?-TATTGGGTTATCCCCGCCAGGTTTTAGAGCTAGAAATAGCAAG-3?56-1-F35?-TTCTAATACGACTCACTATAGTATTGGGTTATCCCCGCCAGG-3?56-13-F25?-CATATTCCTCCACATGTCTAGTTTTAGAGCTAGAAATAGCAAG-3?56-13-F35?-TTCTAATACGACTCACTATAGCATATTCCTCCACATGTCTAG-3?58-3-F25?-GCTACGAGTGGTATCAACCAGTTTTAGAGCTAGAAATAGCAAG-3?58-3-F35?-TTCTAATACGACTCACTATAGGCTACGAGTGGTATCAACCAG-3?58-28-F25?-AATGACATATATACATACTAGTTTTAGAGCTAGAAATAGCAAG-3?58-28-F35?-TTCTAATACGACTCACTATAGAATGACATATATACATACTAG-3?59-5-F25?-CTGGTAGGTGTGTATACATTGTTTTAGAGCTAGAAATAGCAAG-3?59-5-F35?-TTCTAATACGACTCACTATAGCTGGTAGGTGTGTATACATTG-3?59-6-F25?-GGCCTGTAGATCTTAAGGAAGTTTTAGAGCTAGAAATAGCAAG-3?59-6-F35?-TTCTAATACGACTCACTATAGGGCCTGTAGATCTTAAGGAAG-3?F15?-GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG-3?R5?-AAAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCC-3?sgR5?-AAAAAAAAGCACCGACTCGGTGCCACTTTTTC-3?F, R, reverse; RNA. HPV, F, Ten clones (pHPV16, pHPV18, pHPV33, pHPV35, pHPV45, pHPV51, pHPV52, pHPV56, pHPV58, pHPV59) constructed cloning full-length plasmids. plasmid purified Escherichia coli DH5? (CB101; Tiangen Biotech, Beijing, China) mini-kit (CW0502S; CWBIO, Nanjing, gDNA (SiHa, C-33A) blood/cell/tissue extraction kit (DP304; Tiangen). electrophoresis. instructions protein (New England Biolabs, Ipswich, assembled 30-?L 1× buffer, nmol/L nuclease. kept minutes, 200 ng added reaction. 15 minutes. products 1% 50-?L normal contained PrimeSTAR HS (R040A; Takara, Dalian, China), 1), sgRNAb nuclease, 50 each insertion [upstream (UPS) downstream (DNS)] 3), (oJW102) 25 SYBR Master Mix (YEASEN, (UPS DNS) run machine (ABI StepOnePlus; program 72°C 95°C 40 cycles seconds, 58°C 45 5 ctPCR4.0 detection, used. 3Insertion Oligonucleotides Universal CRISPR-Typing Version (ctPCR4.0)NameSequenceHPV16-UPS5?-GCGGTGACCCGGGAGATCTGAATTCTCTGCAGACGTTATGACA-[NH2]-3?HPV16-DNS5?-GCGGTGACCCGGGAGATCTGAATTCTAGTATCTTCTAGTGTGC-[NH2]-3?HPV18-DNS5?-GCGGTGACCCGGGAGATCTGAATTCTTTTCAGCCGGTGCAGCA-[NH2]-3?HPV18-UPS5?-GCGGTGACCCGGGAGATCTGAATTCTCCTGGGCAATATGATGC-[NH2]-3?HPV33-UPS5?-GCGGTGACCCGGGAGATCTGAATTCTGGTTTGTTACCTCTCAG-[NH2]-3?HPV33-DNS5?-GCGGTGACCCGGGAGATCTGAATTCTGGGTCTTCCTTTTCCTT-[NH2]-3?HPV35-DNS5?-GCGGTGACCCGGGAGATCTGAATTCTAGGTACCAGAAGGCGGT-[NH2]-3?HPV35-UPS5?-GCGGTGACCCGGGAGATCTGAATTCTTACAAATATGTCTGTGT-[NH2]-3?HPV45-DNS5?-GCGGTGACCCGGGAGATCTGAATTCTAACTGGTAGTAGGTGGT-[NH2]-3?HPV45-UPS5?-GCGGTGACCCGGGAGATCTGAATTCTGTACTAATTTAACATTA-[NH2]-3?HPV51-DNS5?-GCGGTGACCCGGGAGATCTGAATTCTGATCTGGCTTAGCCTGT-[NH2]-3?HPV51-UPS5?-GCGGTGACCCGGGAGATCTGAATTCTATTTACTCCAAGTAACT-[NH2]-3?HPV52-DNS5?-GCGGTGACCCGGGAGATCTGAATTCTCATGACGAAGGTATTCC-[NH2]-3?HPV52-UPS5?-GCGGTGACCCGGGAGATCTGAATTCTGTAGCACTAACATGACT-[NH2]-3?HPV56-DNS5?-GCGGTGACCCGGGAGATCTGAATTCTGCGGGGATAACCCAATA-[NH2]-3?HPV56-UPS5?-GCGGTGACCCGGGAGATCTGAATTCTACATGTGGAGGAATATG-[NH2]-3?HPV58-UPS5?-GCGGTGACCCGGGAGATCTGAATTCTTTGATACCACTCGTAGC-[NH2]-3?HPV58-DNS5?-GCGGTGACCCGGGAGATCTGAATTCTTATGTATATATGTCATT-[NH2]-3?HPV59-UPS5?-GCGGTGACCCGGGAGATCTGAATTCTGTATACACACCTACCAG-[NH2]-3?HPV59-DNS5?-GCGGTGACCCGGGAGATCTGAATTCTCTTAAGATCTACAGGCC-[NH2]-3?oJW1025?-GCGGTGACCCGGGAGATCTGAATTCT-3?DNS, downstream; UPS, upstream. DNS, MY09 (5?-CGTCCMARRGGAWACTGATC-3?) MY11 (5?-GCMCAGGGWCATAAYAATGG-3?) gDNA, MY09, MY11, HS. 9600; 3 minutes; 35 seconds; sequenced Sanger sequencing (Sangon aligned Vevtor NTI 11.5 NCBI BLAST database (Basic Local Alignment Search Tool, https://blast.ncbi.nlm.nih.gov/Blast.cgi) identify sequences. study, ctPCR4.0, (Figure homogeneously pot. At start protein, (sgRNAa sgRNAb), inserting oligos (IOUPS IODNS), mixed together form running consisting (95°C minutes). During process, (inserting oligos) (Supplemental Figure S1 purity concentration spectrophotometer. Their electrophoresis S2). sequencing, identified Figures S3 S4). showed 20 quality. order evaluate plasmids, associated sgRNA18a sgRNA18b, sgRNA33a sgRNA33b, sgRNA58a sgRNA58b, respectively. almost completely corresponding 2), indicating efficiency cleavage. confirm feasibility (HPV 16, 33, 35, 45, 51, 52, 56, 58, 59) tested ctPCR4.0. when Cas9, genotypes, included could 3). No component, components also blank controls tests 3) <30; however, negative over 30. melting curves unimodal S5), amplifications. subsequent expected size we