Iterative crRNA design and a PAM-free strategy enabled an ultra-specific RPA-CRISPR/Cas12a detection platform
Abstract CRISPR/Cas12a is a highly promising detection tool. However, detecting single nucleotide variations (SNVs) remains challenging. Here, we elucidate Cas12a specificity through crRNA engineering and profiling of single- and double-base mismatch tolerance across three targets. Our findings indi...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2024-11-01
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| Series: | Communications Biology |
| Online Access: | https://doi.org/10.1038/s42003-024-07173-7 |
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| _version_ | 1850061912695373824 |
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| author | Xujian Mao Jian Xu Jingyi Jiang Qiong Li Ping Yao Jinyi Jiang Li Gong Yin Dong Bowen Tu Rong Wang Hongbing Tang Fang Yao Fengming Wang |
| author_facet | Xujian Mao Jian Xu Jingyi Jiang Qiong Li Ping Yao Jinyi Jiang Li Gong Yin Dong Bowen Tu Rong Wang Hongbing Tang Fang Yao Fengming Wang |
| author_sort | Xujian Mao |
| collection | DOAJ |
| description | Abstract CRISPR/Cas12a is a highly promising detection tool. However, detecting single nucleotide variations (SNVs) remains challenging. Here, we elucidate Cas12a specificity through crRNA engineering and profiling of single- and double-base mismatch tolerance across three targets. Our findings indicate that Cas12a specificity depends on the number, type, location, and distance of mismatches within the R-loop. We also find that introducing a wobble base pair at position 14 of the R-loop does not affect the free energy change when the spacer length is truncated to 17 bp. Therefore, we develop a new universal specificity enhancement strategy via iterative crRNA design, involving truncated spacers and a wobble base pair at position 14 of the R-loop, which tremendously increases specificity without sacrificing sensitivity. Additionally, we construct a PAM-free one-pot detection platform for SARS-CoV-2 variants, which effectively distinguishes SNV targets across various GC contents. In summary, our work reveals new insights into the specificity mechanism of Cas12a and demonstrates significant potential for in vitro diagnostics. |
| format | Article |
| id | doaj-art-d1ae9bdefa654066b72eea20171ab114 |
| institution | DOAJ |
| issn | 2399-3642 |
| language | English |
| publishDate | 2024-11-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Communications Biology |
| spelling | doaj-art-d1ae9bdefa654066b72eea20171ab1142025-08-20T02:50:04ZengNature PortfolioCommunications Biology2399-36422024-11-017111310.1038/s42003-024-07173-7Iterative crRNA design and a PAM-free strategy enabled an ultra-specific RPA-CRISPR/Cas12a detection platformXujian Mao0Jian Xu1Jingyi Jiang2Qiong Li3Ping Yao4Jinyi Jiang5Li Gong6Yin Dong7Bowen Tu8Rong Wang9Hongbing Tang10Fang Yao11Fengming Wang12Pathogen Inspection Center, Changzhou Center for Disease Control and PreventionPathogen Inspection Center, Changzhou Center for Disease Control and PreventionPathogen Inspection Center, Changzhou Center for Disease Control and PreventionPathogen Inspection Center, Changzhou Center for Disease Control and PreventionPathogen Inspection Center, Changzhou Center for Disease Control and PreventionPathogen Inspection Center, Changzhou Center for Disease Control and PreventionPathogen Inspection Center, Changzhou Center for Disease Control and PreventionPathogen Inspection Center, Changzhou Center for Disease Control and PreventionPathogen Inspection Center, Changzhou Center for Disease Control and PreventionChina School of Public Health, Nanjing Medical UniversityPathogen Inspection Center, Changzhou Center for Disease Control and PreventionPathogen Inspection Center, Changzhou Center for Disease Control and PreventionPathogen Inspection Center, Changzhou Center for Disease Control and PreventionAbstract CRISPR/Cas12a is a highly promising detection tool. However, detecting single nucleotide variations (SNVs) remains challenging. Here, we elucidate Cas12a specificity through crRNA engineering and profiling of single- and double-base mismatch tolerance across three targets. Our findings indicate that Cas12a specificity depends on the number, type, location, and distance of mismatches within the R-loop. We also find that introducing a wobble base pair at position 14 of the R-loop does not affect the free energy change when the spacer length is truncated to 17 bp. Therefore, we develop a new universal specificity enhancement strategy via iterative crRNA design, involving truncated spacers and a wobble base pair at position 14 of the R-loop, which tremendously increases specificity without sacrificing sensitivity. Additionally, we construct a PAM-free one-pot detection platform for SARS-CoV-2 variants, which effectively distinguishes SNV targets across various GC contents. In summary, our work reveals new insights into the specificity mechanism of Cas12a and demonstrates significant potential for in vitro diagnostics.https://doi.org/10.1038/s42003-024-07173-7 |
| spellingShingle | Xujian Mao Jian Xu Jingyi Jiang Qiong Li Ping Yao Jinyi Jiang Li Gong Yin Dong Bowen Tu Rong Wang Hongbing Tang Fang Yao Fengming Wang Iterative crRNA design and a PAM-free strategy enabled an ultra-specific RPA-CRISPR/Cas12a detection platform Communications Biology |
| title | Iterative crRNA design and a PAM-free strategy enabled an ultra-specific RPA-CRISPR/Cas12a detection platform |
| title_full | Iterative crRNA design and a PAM-free strategy enabled an ultra-specific RPA-CRISPR/Cas12a detection platform |
| title_fullStr | Iterative crRNA design and a PAM-free strategy enabled an ultra-specific RPA-CRISPR/Cas12a detection platform |
| title_full_unstemmed | Iterative crRNA design and a PAM-free strategy enabled an ultra-specific RPA-CRISPR/Cas12a detection platform |
| title_short | Iterative crRNA design and a PAM-free strategy enabled an ultra-specific RPA-CRISPR/Cas12a detection platform |
| title_sort | iterative crrna design and a pam free strategy enabled an ultra specific rpa crispr cas12a detection platform |
| url | https://doi.org/10.1038/s42003-024-07173-7 |
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