Reprogrammable Gel Electrophoresis Detection Assay Using CRISPR-Cas12a and Hybridization Chain Reaction
Hybridization chain reaction (HCR) is a DNA-based target-induced cascade reaction. Due to its unique enzyme-free amplification feature, HCR is often employed for sensing applications. Much like DNA nanostructures that have been designed to respond to a specific stimulus, HCR employs nucleic acids th...
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| Veröffentlicht in: | Analytical chemistry (Washington) 2021-02, Vol.93 (4), p.1934-1938 |
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| container_title | Analytical chemistry (Washington) |
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| creator | Kachwala, Mahera J Smith, Christopher W Nandu, Nidhi Yigit, Mehmet V |
| description | Hybridization chain reaction (HCR) is a DNA-based target-induced cascade reaction. Due to its unique enzyme-free amplification feature, HCR is often employed for sensing applications. Much like DNA nanostructures that have been designed to respond to a specific stimulus, HCR employs nucleic acids that reconfigure and assemble in the presence of a specific trigger. Despite its standalone capabilities, HCR is highly modular; therefore, it can be advanced and repurposed when coupled with latest discoveries. To this effect, we have developed a gel electrophoresis-based detection approach which combines the signal amplification feature of HCR with the programmability and sensitivity of the CRISPR-Cas12a system. By incorporating CRISPR-Cas12a, we have achieved greater sensitivity and reversed the signal output from TURN OFF to TURN ON. CRISPR-Cas12a also enabled us to rapidly reprogram the assay for the detection of both ssDNA and dsDNA target sequences by replacing a single reaction component in the detection kit. Detection of conserved, both ssDNA and dsDNA, regions of tobacco curly shoot virus (TCSV) and hepatitis B virus (HepBV) genomes is demonstrated with this methodology. This low-cost gel electrophoresis assay can detect as little as 1.5 fmol of the target without any additional target amplification steps and is about 100-fold more sensitive than HCR-alone approach. |
| doi_str_mv | 10.1021/acs.analchem.0c04949 |
| format | Article |
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Due to its unique enzyme-free amplification feature, HCR is often employed for sensing applications. Much like DNA nanostructures that have been designed to respond to a specific stimulus, HCR employs nucleic acids that reconfigure and assemble in the presence of a specific trigger. Despite its standalone capabilities, HCR is highly modular; therefore, it can be advanced and repurposed when coupled with latest discoveries. To this effect, we have developed a gel electrophoresis-based detection approach which combines the signal amplification feature of HCR with the programmability and sensitivity of the CRISPR-Cas12a system. By incorporating CRISPR-Cas12a, we have achieved greater sensitivity and reversed the signal output from TURN OFF to TURN ON. CRISPR-Cas12a also enabled us to rapidly reprogram the assay for the detection of both ssDNA and dsDNA target sequences by replacing a single reaction component in the detection kit. Detection of conserved, both ssDNA and dsDNA, regions of tobacco curly shoot virus (TCSV) and hepatitis B virus (HepBV) genomes is demonstrated with this methodology. This low-cost gel electrophoresis assay can detect as little as 1.5 fmol of the target without any additional target amplification steps and is about 100-fold more sensitive than HCR-alone approach.</description><identifier>ISSN: 0003-2700</identifier><identifier>ISSN: 1520-6882</identifier><identifier>EISSN: 1520-6882</identifier><identifier>DOI: 10.1021/acs.analchem.0c04949</identifier><identifier>PMID: 33404234</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>Amplification ; Analytical chemistry ; Assaying ; Biosensing Techniques - methods ; Cascade chemical reactions ; Chemistry ; CRISPR ; CRISPR-Cas Systems ; Deoxyribonucleic acid ; DNA ; DNA - chemistry ; Electrophoresis ; Electrophoresis, Gel, Two-Dimensional - methods ; Gel electrophoresis ; Genomes ; Hepatitis ; Hepatitis B ; Hybridization ; Nucleic Acid Amplification Techniques - methods ; Nucleic Acid Hybridization - methods ; Nucleic acids ; Sensitivity ; Tobacco ; Viruses</subject><ispartof>Analytical chemistry (Washington), 2021-02, Vol.93 (4), p.1934-1938</ispartof><rights>2021 American Chemical Society</rights><rights>Copyright American Chemical Society Feb 2, 2021</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a477t-717fceee8281fb2df4e88f052a0f336570e8faee9f10ad1d8b3a788e363d7ab33</citedby><cites>FETCH-LOGICAL-a477t-717fceee8281fb2df4e88f052a0f336570e8faee9f10ad1d8b3a788e363d7ab33</cites><orcidid>0000-0002-4349-3701</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/acs.analchem.0c04949$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/acs.analchem.0c04949$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>230,314,780,784,885,2765,27076,27924,27925,56738,56788</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/33404234$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Kachwala, Mahera J</creatorcontrib><creatorcontrib>Smith, Christopher W</creatorcontrib><creatorcontrib>Nandu, Nidhi</creatorcontrib><creatorcontrib>Yigit, Mehmet V</creatorcontrib><title>Reprogrammable Gel Electrophoresis Detection Assay Using CRISPR-Cas12a and Hybridization Chain Reaction</title><title>Analytical chemistry (Washington)</title><addtitle>Anal. Chem</addtitle><description>Hybridization chain reaction (HCR) is a DNA-based target-induced cascade reaction. Due to its unique enzyme-free amplification feature, HCR is often employed for sensing applications. Much like DNA nanostructures that have been designed to respond to a specific stimulus, HCR employs nucleic acids that reconfigure and assemble in the presence of a specific trigger. Despite its standalone capabilities, HCR is highly modular; therefore, it can be advanced and repurposed when coupled with latest discoveries. To this effect, we have developed a gel electrophoresis-based detection approach which combines the signal amplification feature of HCR with the programmability and sensitivity of the CRISPR-Cas12a system. By incorporating CRISPR-Cas12a, we have achieved greater sensitivity and reversed the signal output from TURN OFF to TURN ON. CRISPR-Cas12a also enabled us to rapidly reprogram the assay for the detection of both ssDNA and dsDNA target sequences by replacing a single reaction component in the detection kit. Detection of conserved, both ssDNA and dsDNA, regions of tobacco curly shoot virus (TCSV) and hepatitis B virus (HepBV) genomes is demonstrated with this methodology. This low-cost gel electrophoresis assay can detect as little as 1.5 fmol of the target without any additional target amplification steps and is about 100-fold more sensitive than HCR-alone approach.</description><subject>Amplification</subject><subject>Analytical chemistry</subject><subject>Assaying</subject><subject>Biosensing Techniques - methods</subject><subject>Cascade chemical reactions</subject><subject>Chemistry</subject><subject>CRISPR</subject><subject>CRISPR-Cas Systems</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA - chemistry</subject><subject>Electrophoresis</subject><subject>Electrophoresis, Gel, Two-Dimensional - methods</subject><subject>Gel electrophoresis</subject><subject>Genomes</subject><subject>Hepatitis</subject><subject>Hepatitis B</subject><subject>Hybridization</subject><subject>Nucleic Acid Amplification Techniques - methods</subject><subject>Nucleic Acid Hybridization - methods</subject><subject>Nucleic acids</subject><subject>Sensitivity</subject><subject>Tobacco</subject><subject>Viruses</subject><issn>0003-2700</issn><issn>1520-6882</issn><issn>1520-6882</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2021</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kV1v0zAUhi0EYmXwDxCyxA03Kccfid0bpCmMbdIkUGHX1kly0npK4mKnk8qvJ127Crjgxpbs532PrYextwLmAqT4iHWa44BdvaZ-DjXohV48YzORS8gKa-VzNgMAlUkDcMZepXQPIASI4iU7U0qDlkrP2GpJmxhWEfseq474FXX8sqN6jGGzDpGST_wzjdOBDwO_SAl3_C75YcXL5c33b8usxCQkchwafr2rom_8L3xkyzX6gS8JH6Ov2YsWu0Rvjvs5u_ty-aO8zm6_Xt2UF7cZamPGzAjT1kRkpRVtJZtWk7Ut5BKhVarIDZBtkWjRCsBGNLZSaKwlVajGYKXUOft06N1sq56amoYxYuc20fcYdy6gd3_fDH7tVuHBWWGM0XYq-HAsiOHnltLoep9q6jocKGyTk9rkuTTTMqHv_0HvwzZOSvbUAmQhbCEnSh-oOoaUIrWnxwhwe5NuMumeTLqjySn27s-PnEJP6iYADsA-fhr8387flf-u8w</recordid><startdate>20210202</startdate><enddate>20210202</enddate><creator>Kachwala, Mahera J</creator><creator>Smith, Christopher W</creator><creator>Nandu, Nidhi</creator><creator>Yigit, Mehmet V</creator><general>American Chemical Society</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QF</scope><scope>7QO</scope><scope>7QQ</scope><scope>7SC</scope><scope>7SE</scope><scope>7SP</scope><scope>7SR</scope><scope>7TA</scope><scope>7TB</scope><scope>7TM</scope><scope>7U5</scope><scope>7U7</scope><scope>7U9</scope><scope>8BQ</scope><scope>8FD</scope><scope>C1K</scope><scope>F28</scope><scope>FR3</scope><scope>H8D</scope><scope>H8G</scope><scope>H94</scope><scope>JG9</scope><scope>JQ2</scope><scope>KR7</scope><scope>L7M</scope><scope>L~C</scope><scope>L~D</scope><scope>P64</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0002-4349-3701</orcidid></search><sort><creationdate>20210202</creationdate><title>Reprogrammable Gel Electrophoresis Detection Assay Using CRISPR-Cas12a and Hybridization Chain Reaction</title><author>Kachwala, Mahera J ; Smith, Christopher W ; Nandu, Nidhi ; Yigit, Mehmet V</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a477t-717fceee8281fb2df4e88f052a0f336570e8faee9f10ad1d8b3a788e363d7ab33</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2021</creationdate><topic>Amplification</topic><topic>Analytical chemistry</topic><topic>Assaying</topic><topic>Biosensing Techniques - methods</topic><topic>Cascade chemical reactions</topic><topic>Chemistry</topic><topic>CRISPR</topic><topic>CRISPR-Cas Systems</topic><topic>Deoxyribonucleic acid</topic><topic>DNA</topic><topic>DNA - chemistry</topic><topic>Electrophoresis</topic><topic>Electrophoresis, Gel, Two-Dimensional - methods</topic><topic>Gel electrophoresis</topic><topic>Genomes</topic><topic>Hepatitis</topic><topic>Hepatitis B</topic><topic>Hybridization</topic><topic>Nucleic Acid Amplification Techniques - methods</topic><topic>Nucleic Acid Hybridization - methods</topic><topic>Nucleic acids</topic><topic>Sensitivity</topic><topic>Tobacco</topic><topic>Viruses</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Kachwala, Mahera J</creatorcontrib><creatorcontrib>Smith, Christopher W</creatorcontrib><creatorcontrib>Nandu, Nidhi</creatorcontrib><creatorcontrib>Yigit, Mehmet V</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Aluminium Industry Abstracts</collection><collection>Biotechnology Research Abstracts</collection><collection>Ceramic Abstracts</collection><collection>Computer and Information Systems Abstracts</collection><collection>Corrosion Abstracts</collection><collection>Electronics & Communications Abstracts</collection><collection>Engineered Materials Abstracts</collection><collection>Materials Business File</collection><collection>Mechanical & Transportation Engineering Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Solid State and Superconductivity Abstracts</collection><collection>Toxicology Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>METADEX</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ANTE: Abstracts in New Technology & Engineering</collection><collection>Engineering Research Database</collection><collection>Aerospace Database</collection><collection>Copper Technical Reference Library</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Materials Research Database</collection><collection>ProQuest Computer Science Collection</collection><collection>Civil Engineering Abstracts</collection><collection>Advanced Technologies Database with Aerospace</collection><collection>Computer and Information Systems Abstracts Academic</collection><collection>Computer and Information Systems Abstracts Professional</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Analytical chemistry (Washington)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Kachwala, Mahera J</au><au>Smith, Christopher W</au><au>Nandu, Nidhi</au><au>Yigit, Mehmet V</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Reprogrammable Gel Electrophoresis Detection Assay Using CRISPR-Cas12a and Hybridization Chain Reaction</atitle><jtitle>Analytical chemistry (Washington)</jtitle><addtitle>Anal. Chem</addtitle><date>2021-02-02</date><risdate>2021</risdate><volume>93</volume><issue>4</issue><spage>1934</spage><epage>1938</epage><pages>1934-1938</pages><issn>0003-2700</issn><issn>1520-6882</issn><eissn>1520-6882</eissn><abstract>Hybridization chain reaction (HCR) is a DNA-based target-induced cascade reaction. Due to its unique enzyme-free amplification feature, HCR is often employed for sensing applications. Much like DNA nanostructures that have been designed to respond to a specific stimulus, HCR employs nucleic acids that reconfigure and assemble in the presence of a specific trigger. Despite its standalone capabilities, HCR is highly modular; therefore, it can be advanced and repurposed when coupled with latest discoveries. To this effect, we have developed a gel electrophoresis-based detection approach which combines the signal amplification feature of HCR with the programmability and sensitivity of the CRISPR-Cas12a system. By incorporating CRISPR-Cas12a, we have achieved greater sensitivity and reversed the signal output from TURN OFF to TURN ON. CRISPR-Cas12a also enabled us to rapidly reprogram the assay for the detection of both ssDNA and dsDNA target sequences by replacing a single reaction component in the detection kit. Detection of conserved, both ssDNA and dsDNA, regions of tobacco curly shoot virus (TCSV) and hepatitis B virus (HepBV) genomes is demonstrated with this methodology. This low-cost gel electrophoresis assay can detect as little as 1.5 fmol of the target without any additional target amplification steps and is about 100-fold more sensitive than HCR-alone approach.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>33404234</pmid><doi>10.1021/acs.analchem.0c04949</doi><tpages>5</tpages><orcidid>https://orcid.org/0000-0002-4349-3701</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Amplification Analytical chemistry Assaying Biosensing Techniques - methods Cascade chemical reactions Chemistry CRISPR CRISPR-Cas Systems Deoxyribonucleic acid DNA DNA - chemistry Electrophoresis Electrophoresis, Gel, Two-Dimensional - methods Gel electrophoresis Genomes Hepatitis Hepatitis B Hybridization Nucleic Acid Amplification Techniques - methods Nucleic Acid Hybridization - methods Nucleic acids Sensitivity Tobacco Viruses |
| title | Reprogrammable Gel Electrophoresis Detection Assay Using CRISPR-Cas12a and Hybridization Chain Reaction |
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