Engineered LwaCas13a with enhanced collateral activity for nucleic acid detection
Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 13 (Cas13) has been rapidly developed for nucleic-acid-based diagnostics by using its characteristic collateral activity. Despite the recent progress in optimizing the Cas13 system for the detection of nucle...
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| Veröffentlicht in: | Nature chemical biology 2023-01, Vol.19 (1), p.45-54 |
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| creator | Yang, Jie Song, Yang Deng, Xiangyu Vanegas, Jeffrey A. You, Zheng Zhang, Yuxuan Weng, Zhengyan Avery, Lori Dieckhaus, Kevin D. Peddi, Advaith Gao, Yang Zhang, Yi Gao, Xue |
| description | Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 13 (Cas13) has been rapidly developed for nucleic-acid-based diagnostics by using its characteristic collateral activity. Despite the recent progress in optimizing the Cas13 system for the detection of nucleic acids, engineering Cas13 protein with enhanced collateral activity has been challenging, mostly because of its complex structural dynamics. Here we successfully employed a novel strategy to engineer the
Leptotrichia wadei
(Lwa)Cas13a by inserting different RNA-binding domains into a unique active-site-proximal loop within its higher eukaryotes and prokaryotes nucleotide-binding domain. Two LwaCas13a variants showed enhanced collateral activity and improved sensitivity over the wild type in various buffer conditions. By combining with an electrochemical method, our variants detected the SARS-CoV-2 genome at attomolar concentrations from both inactive viral and unextracted clinical samples, without target preamplification. Our engineered LwaCas13a enzymes with enhanced collateral activity are ready to be integrated into other Cas13a-based platforms for ultrasensitive detection of nucleic acids.
By inserting RNA-binding domains to an active-site-proximal loop amidst CRISPR–Cas, Yang, Song et al. generate variants with enhanced collateral activity for ultrasensitive and amplification-free RNA detection when coupled with electrochemical sensing platforms. |
| doi_str_mv | 10.1038/s41589-022-01135-y |
| format | Article |
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Leptotrichia wadei
(Lwa)Cas13a by inserting different RNA-binding domains into a unique active-site-proximal loop within its higher eukaryotes and prokaryotes nucleotide-binding domain. Two LwaCas13a variants showed enhanced collateral activity and improved sensitivity over the wild type in various buffer conditions. By combining with an electrochemical method, our variants detected the SARS-CoV-2 genome at attomolar concentrations from both inactive viral and unextracted clinical samples, without target preamplification. Our engineered LwaCas13a enzymes with enhanced collateral activity are ready to be integrated into other Cas13a-based platforms for ultrasensitive detection of nucleic acids.
By inserting RNA-binding domains to an active-site-proximal loop amidst CRISPR–Cas, Yang, Song et al. generate variants with enhanced collateral activity for ultrasensitive and amplification-free RNA detection when coupled with electrochemical sensing platforms.</description><identifier>ISSN: 1552-4450</identifier><identifier>EISSN: 1552-4469</identifier><identifier>DOI: 10.1038/s41589-022-01135-y</identifier><identifier>PMID: 36138140</identifier><language>eng</language><publisher>New York: Nature Publishing Group US</publisher><subject>631/45 ; 631/92 ; Binding ; Biochemical Engineering ; Biochemistry ; Bioorganic Chemistry ; Cell Biology ; Chemistry ; Chemistry and Materials Science ; Chemistry/Food Science ; COVID-19 ; CRISPR ; CRISPR-Cas Systems - genetics ; Domains ; Electrochemistry ; Eukaryotes ; Genome ; Genomes ; Humans ; Nucleic acids ; Nucleic Acids - genetics ; Nucleotides ; Platforms ; Prokaryotes ; Protein engineering ; Proteins ; Ribonucleic acid ; RNA ; SARS-CoV-2 - genetics ; Severe acute respiratory syndrome coronavirus 2</subject><ispartof>Nature chemical biology, 2023-01, Vol.19 (1), p.45-54</ispartof><rights>The Author(s), under exclusive licence to Springer Nature America, Inc. 2022. Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.</rights><rights>2022. The Author(s), under exclusive licence to Springer Nature America, Inc.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c419t-b08f738ad5345573e4eccf965230761e8e897463a44f1000fe8bfdb8523a3bf43</citedby><cites>FETCH-LOGICAL-c419t-b08f738ad5345573e4eccf965230761e8e897463a44f1000fe8bfdb8523a3bf43</cites><orcidid>0000-0003-4399-2532 ; 0000-0003-3170-2980 ; 0000-0002-4037-0431 ; 0000-0003-4426-5636 ; 0000-0003-0626-0850 ; 0000-0003-3213-9704 ; 0000-0002-1604-4206 ; 0000-0002-0907-663X</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>315,781,785,27929,27930</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/36138140$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Yang, Jie</creatorcontrib><creatorcontrib>Song, Yang</creatorcontrib><creatorcontrib>Deng, Xiangyu</creatorcontrib><creatorcontrib>Vanegas, Jeffrey A.</creatorcontrib><creatorcontrib>You, Zheng</creatorcontrib><creatorcontrib>Zhang, Yuxuan</creatorcontrib><creatorcontrib>Weng, Zhengyan</creatorcontrib><creatorcontrib>Avery, Lori</creatorcontrib><creatorcontrib>Dieckhaus, Kevin D.</creatorcontrib><creatorcontrib>Peddi, Advaith</creatorcontrib><creatorcontrib>Gao, Yang</creatorcontrib><creatorcontrib>Zhang, Yi</creatorcontrib><creatorcontrib>Gao, Xue</creatorcontrib><title>Engineered LwaCas13a with enhanced collateral activity for nucleic acid detection</title><title>Nature chemical biology</title><addtitle>Nat Chem Biol</addtitle><addtitle>Nat Chem Biol</addtitle><description>Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 13 (Cas13) has been rapidly developed for nucleic-acid-based diagnostics by using its characteristic collateral activity. Despite the recent progress in optimizing the Cas13 system for the detection of nucleic acids, engineering Cas13 protein with enhanced collateral activity has been challenging, mostly because of its complex structural dynamics. Here we successfully employed a novel strategy to engineer the
Leptotrichia wadei
(Lwa)Cas13a by inserting different RNA-binding domains into a unique active-site-proximal loop within its higher eukaryotes and prokaryotes nucleotide-binding domain. Two LwaCas13a variants showed enhanced collateral activity and improved sensitivity over the wild type in various buffer conditions. By combining with an electrochemical method, our variants detected the SARS-CoV-2 genome at attomolar concentrations from both inactive viral and unextracted clinical samples, without target preamplification. Our engineered LwaCas13a enzymes with enhanced collateral activity are ready to be integrated into other Cas13a-based platforms for ultrasensitive detection of nucleic acids.
By inserting RNA-binding domains to an active-site-proximal loop amidst CRISPR–Cas, Yang, Song et al. generate variants with enhanced collateral activity for ultrasensitive and amplification-free RNA detection when coupled with electrochemical sensing platforms.</description><subject>631/45</subject><subject>631/92</subject><subject>Binding</subject><subject>Biochemical Engineering</subject><subject>Biochemistry</subject><subject>Bioorganic Chemistry</subject><subject>Cell Biology</subject><subject>Chemistry</subject><subject>Chemistry and Materials Science</subject><subject>Chemistry/Food Science</subject><subject>COVID-19</subject><subject>CRISPR</subject><subject>CRISPR-Cas Systems - genetics</subject><subject>Domains</subject><subject>Electrochemistry</subject><subject>Eukaryotes</subject><subject>Genome</subject><subject>Genomes</subject><subject>Humans</subject><subject>Nucleic acids</subject><subject>Nucleic Acids - genetics</subject><subject>Nucleotides</subject><subject>Platforms</subject><subject>Prokaryotes</subject><subject>Protein engineering</subject><subject>Proteins</subject><subject>Ribonucleic acid</subject><subject>RNA</subject><subject>SARS-CoV-2 - 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Academic</collection><jtitle>Nature chemical biology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Yang, Jie</au><au>Song, Yang</au><au>Deng, Xiangyu</au><au>Vanegas, Jeffrey A.</au><au>You, Zheng</au><au>Zhang, Yuxuan</au><au>Weng, Zhengyan</au><au>Avery, Lori</au><au>Dieckhaus, Kevin D.</au><au>Peddi, Advaith</au><au>Gao, Yang</au><au>Zhang, Yi</au><au>Gao, Xue</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Engineered LwaCas13a with enhanced collateral activity for nucleic acid detection</atitle><jtitle>Nature chemical biology</jtitle><stitle>Nat Chem Biol</stitle><addtitle>Nat Chem Biol</addtitle><date>2023-01-01</date><risdate>2023</risdate><volume>19</volume><issue>1</issue><spage>45</spage><epage>54</epage><pages>45-54</pages><issn>1552-4450</issn><eissn>1552-4469</eissn><abstract>Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 13 (Cas13) has been rapidly developed for nucleic-acid-based diagnostics by using its characteristic collateral activity. Despite the recent progress in optimizing the Cas13 system for the detection of nucleic acids, engineering Cas13 protein with enhanced collateral activity has been challenging, mostly because of its complex structural dynamics. Here we successfully employed a novel strategy to engineer the
Leptotrichia wadei
(Lwa)Cas13a by inserting different RNA-binding domains into a unique active-site-proximal loop within its higher eukaryotes and prokaryotes nucleotide-binding domain. Two LwaCas13a variants showed enhanced collateral activity and improved sensitivity over the wild type in various buffer conditions. By combining with an electrochemical method, our variants detected the SARS-CoV-2 genome at attomolar concentrations from both inactive viral and unextracted clinical samples, without target preamplification. Our engineered LwaCas13a enzymes with enhanced collateral activity are ready to be integrated into other Cas13a-based platforms for ultrasensitive detection of nucleic acids.
By inserting RNA-binding domains to an active-site-proximal loop amidst CRISPR–Cas, Yang, Song et al. generate variants with enhanced collateral activity for ultrasensitive and amplification-free RNA detection when coupled with electrochemical sensing platforms.</abstract><cop>New York</cop><pub>Nature Publishing Group US</pub><pmid>36138140</pmid><doi>10.1038/s41589-022-01135-y</doi><tpages>10</tpages><orcidid>https://orcid.org/0000-0003-4399-2532</orcidid><orcidid>https://orcid.org/0000-0003-3170-2980</orcidid><orcidid>https://orcid.org/0000-0002-4037-0431</orcidid><orcidid>https://orcid.org/0000-0003-4426-5636</orcidid><orcidid>https://orcid.org/0000-0003-0626-0850</orcidid><orcidid>https://orcid.org/0000-0003-3213-9704</orcidid><orcidid>https://orcid.org/0000-0002-1604-4206</orcidid><orcidid>https://orcid.org/0000-0002-0907-663X</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | 631/45 631/92 Binding Biochemical Engineering Biochemistry Bioorganic Chemistry Cell Biology Chemistry Chemistry and Materials Science Chemistry/Food Science COVID-19 CRISPR CRISPR-Cas Systems - genetics Domains Electrochemistry Eukaryotes Genome Genomes Humans Nucleic acids Nucleic Acids - genetics Nucleotides Platforms Prokaryotes Protein engineering Proteins Ribonucleic acid RNA SARS-CoV-2 - genetics Severe acute respiratory syndrome coronavirus 2 |
| title | Engineered LwaCas13a with enhanced collateral activity for nucleic acid detection |
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