CRISPR/Cas9-Based Lateral Flow and Fluorescence Diagnostics
Abstract
:1. Introduction
2. Materials and Methods
2.1. Nucleic Acids
2.2. Amplification
2.3. Soak DNA Oligonucleotides
2.4. Guide RNAs
2.5. Biotinylated Cas9 and Lateral Flow
2.6. CRISPR/Cas9 Fluorescence Assay
2.7. RT-PCR
2.8. Genome Analysis
2.9. Graphing and Statistics
2.10. Images
3. Results
3.1. Rapid Nucleic Amplification and Lateral Flow Detection
3.2. Cas9 Allows for Specific SARS-Co-V2 ORF8a Sequence Detection
3.3. Cas9-Nuclease-Based Diagnostics for Single and Multiplexed Targets
3.4. LFA and Fluorescence Assay Validation with SARS-CoV-2 Genomic RNA
3.5. Cas9 Analysis of a SARS-CoV-2 Variant
4. Discussion
5. Conclusions
6. Patents
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Identification | Sequence (5′-3′) | Function |
---|---|---|
SARS-Co-V2 FAM Forward | /56-FAM/GAATTGTGCGTGGATGAGGCTGG | FAM/FITC-labeled forward primer for SARS-Co-V2 amplification from synthetic or viral genome templates. |
SARS-Co-V2 Biotinylated Reverse | /5Biosg/CAACACGAACGTCATGATACTC | Biotinylated-labeled reverse primer for SARS-Co-V2 amplification from synthetic template. |
SARS-Co-V2 Unlabeled Reverse | CAACACGAACGTCATGATACTC | Reverse primer for SARS-Co-V2 amplification from synthetic template. |
SARS-Co-V2 Genome Reverse | TTAGATGAAATCTAAAACAACACG | Reverse primer for SARS-Co-V2 amplification from genomic RNA template. |
SARS-Co-V2 T SNP DNA PCR template. | GAATTGTGCGTGGATGAG GCTGGTTCTAAATCACCCA TTCAGTACATCGATATCGGT AATTATACAGTTTCCTGTTTA CCTTTTACAATTAATTGCCAG GAACCTAAATTGGGTAGTCTT GTAGTGCGTTGTTCGTTCTATG AAGACTTTTTAGAGTATCATG ACGTTCGTGTTG | Synthetic SARS-CoV-2 PCR template containing a thymine SNP at position 28144. |
SARS-Co-V2 C SNP DNA PCR template. | GAATTGTGCGTGGATGAG GCTGGTTCTAAATCACCC ATTCAGTACATCGATATC GGTAATTATACAGTTTCC TGTTCACCTTTTACAATTA ATTGCCAGGAACCTAAAT TGGGTAGTCTTGTAGTGCG TTGTTCGTTCTATGAAGACT TTTTAGAGTATCATGACGTTCGTGTTG | Synthetic SARS-CoV-2 PCR template containing a cytosine SNP at position 28144. |
Influenza A Forward | CAAGACCAATCYTGTCACCTCTGAC | Forward primer for Influenza A amplification from synthetic DNA. |
Influenza A Reverse | GCATTYTGGACAAAVCGTCTACG | Reverse primer for Influenza A amplification from synthetic DNA. |
Synthetic Influenza A DNA PCR template | GCTCTCATGGAATGGCTAAAGACA AGACCAATCTTGTCACCTCTGACTA AGGGGATTTTAGGATTTGTGTTCAC GCTCACCGTGCCCAGTGAGCGAGG ACTGCAGCGTAGACGCTTTGTCCAA AATGCCCTAAATGGGAATGGGGAC CCGAACAACATGG | Synthetic PCR template to generate Influenza A DNA amplicons. |
Influenza B Forward | TCCTCAAYTCACTCTTCGAGCG | Forward primer for Influenza B amplification from synthetic DNA. |
Influenza B Reverse | CGGTGCTCTTGACCAAATTGG | Reverse primer for Influenza B amplification from synthetic DNA. |
Synthetic Influenza B DNA PCR template | TACAGTGGAGGATGAAGAAGATGG CCATCGGATCCTCAATTCACTCTTC GAGCGTCTTAATGAAGGACATTCA AAGCCAATTCGAGCAGCTGAAACT GCGGTGGGAGTCTTATCCCAATTTG GTCAAGAGCACCGACTATCACCAG AAGAGGGAGACAAT | Synthetic PCR template to generate Influenza B DNA amplicons. |
RSV Forward | GGCAAATATGGAAACATACGTGAA | Forward primer for RSV amplification from synthetic DNA. |
RSV Reverse | CATGGGCACCCATATTGTAA | Reverse primer for RSV from synthetic DNA. |
Synthetic RSV DNA PCR template | GGCAAATATGGAAACATACGTG AACAAGCTTCACGAAGGCTCCA CATACACAGCTGCTGTTCAATAC AATGTCCTAGAAAAAGACGATG ACCCTGCATCACTTACAATATGG GTGCCCATGTTCCAATCA | Synthetic PCR template to generate RSV DNA amplicons. |
Identification | Sequence (5′-3′) | Function |
---|---|---|
COVID T Probe: | /56-FAM/TCCTGTTTACCTTTTA CAATTAATTGCCAGGA/3IABkFQ/ | FAM fluorescent probe with Iowa Black quencher that recognizes SARS-CoV-2 with a thymine at position 28144. |
COVID C Probe: | /56-FAM/TCCTGTTCACCTTTTA CAATTAATTGCCAGGA/3IABkFQ | FAM fluorescent probe with Iowa Black quencher that recognizes SARS-CoV-2 with a cytosine at position 28144. |
Influenza A Probe: | /5TexRd-XN/CAGTCCTCGCTCACT GGGCACGGTGAGCGTGA/3IAbRQSp/ | Texas Red fluorescent probe with Iowa Black quencher that recognizes Influenza A sequence. |
Influenza B Probe: | /5YAkYel/TCCCACCGCAGTTTCAG CTGCTCGAATTGGCT/3IABkFQ/ | Yakima Yellow fluorescent probe with Iowa Black quencher that recognizes Influenza B sequence. |
RSV Probe: | /5Cy3/GCTCCACATACACAGCTG CTGTTCAATACAAT/3IAbRQSp/ | Cy3 fluorescent probe with Iowa Black quencher that recognizes RSV sequence. |
RSV Probe: | /56-TAMN/GCTCCACATACACAGCTG CTGTTCAATACAAT/3IAbRQSp/ | TAMRA fluorescent probe with Iowa Black quencher that recognizes RSV sequence. |
Identification | Sequence (5′-3′) | Function |
---|---|---|
PAM Soak Forward: | CGGGAGGGTGGGCGGGAGGGTGGG CGGGAGGGTGGGCGGGAGGGTGGG | PAM-rich ODN that acts as a bait for promiscuous Cas9 binding; sense/top/forward/strand. |
PAM Soak Reverse: | CCCACCCTCCCGCCCACCCTCCCG CCCACCCTCCCGCCCACCCTCCCG | PAM-rich ODN that acts as a bait for promiscuous Cas9 binding; anti-sense/bottom/reverse/strand. |
C Soak Forward: | GGAGGGTGGGGATTAATTGTAAA AGGTGAACGGGCGGGAGGGTGG | Bait ODN that contains SARS-CoV-2 sequences and a cytosine at position 28144; sense/top/forward/strand. In yellow is the anti-parralel guanine. |
C Soak Reverse: | CCACCCTCCCGCCCGTTCACCTTT TACAATTAATCCCCACCCTCC | Bait ODN that contains SARS-CoV-2 sequences and a cytosine at position 28144; anti-sense/bottom/reverse/strand. |
T Soak Forward: | GGAGGGTGGGGATTAATTGTAAA AGGTAAACGGGCGGGAGGGTGG | Bait ODN that contains SARS-CoV-2 sequences and a thymine at position 28144; sense/top/forward/strand. In yellow is the anti-parralel adenine. |
T Soak Reverse: | CCACCCTCCCGCCCGTTTACCTTT TACAATTAATCCCCACCCTCC | Bait ODN that contains SARS-CoV-2 sequences and a thymine at position 28144; anti-sense/bottom/reverse/strand. |
Identification. | Sequence (5′-3′) | Function |
---|---|---|
COVID-19 T sgRNA: | AUUAAUUGUAAAAGGUAAAC | Recognizes ORF8a that has a thymine at nucelotide position 28144. |
COVID-19 C sgRNA: | AUUAAUUGUAAAAGGUGAAC | Recognizes ORF8a that has a cytosine at nucelotide position 28144. |
Influenza A sgRNA: | CUCACCGUGCCCAGUGAGCG | Recognizes the influenza A amplicon. |
Influenza B sgRNA: | AAUUCGAGCAGCUGAAACUG | Recognizes the influenza B amplicon. |
RSV sgRNA: | UUGAACAGCAGCUGUGUAUG | Recognizes the influenza RSV amplicon. |
Control sgRNA: | CACUGGCUGUCGCUUCUCAA | Irrelevant control sgRNA that has no homology to viral genomes. |
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Osborn, M.J.; Bhardwaj, A.; Bingea, S.P.; Knipping, F.; Feser, C.J.; Lees, C.J.; Collins, D.P.; Steer, C.J.; Blazar, B.R.; Tolar, J. CRISPR/Cas9-Based Lateral Flow and Fluorescence Diagnostics. Bioengineering 2021, 8, 23. https://doi.org/10.3390/bioengineering8020023
Osborn MJ, Bhardwaj A, Bingea SP, Knipping F, Feser CJ, Lees CJ, Collins DP, Steer CJ, Blazar BR, Tolar J. CRISPR/Cas9-Based Lateral Flow and Fluorescence Diagnostics. Bioengineering. 2021; 8(2):23. https://doi.org/10.3390/bioengineering8020023
Chicago/Turabian StyleOsborn, Mark J., Akshay Bhardwaj, Samuel P. Bingea, Friederike Knipping, Colby J. Feser, Christopher J. Lees, Daniel P. Collins, Clifford J. Steer, Bruce R. Blazar, and Jakub Tolar. 2021. "CRISPR/Cas9-Based Lateral Flow and Fluorescence Diagnostics" Bioengineering 8, no. 2: 23. https://doi.org/10.3390/bioengineering8020023