Cas12a-based one-pot SNP detection with high accuracy
CRISPR-Cas12a based one-pot detection system has been used in nucleic acid detection and diagnosis. However, it is not sensitive enough to distinguish single nucleotide polymorphisms (SNP), which has greatly restricted its application. To overcome these limitations, we engineered a LbCas12a variant...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2023-04-01
|
| Series: | Cell Insight |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2772892723000044 |
| _version_ | 1828014485344878592 |
|---|---|
| author | Hong-Xia Zhang Caixiang Zhang Shuhan Lu Xiaohan Tong Kun Zhang Hao Yin Ying Zhang |
| author_facet | Hong-Xia Zhang Caixiang Zhang Shuhan Lu Xiaohan Tong Kun Zhang Hao Yin Ying Zhang |
| author_sort | Hong-Xia Zhang |
| collection | DOAJ |
| description | CRISPR-Cas12a based one-pot detection system has been used in nucleic acid detection and diagnosis. However, it is not sensitive enough to distinguish single nucleotide polymorphisms (SNP), which has greatly restricted its application. To overcome these limitations, we engineered a LbCas12a variant with enhanced sensitivity against SNP, named seCas12a (sensitive Cas12a). SeCas12a-based one-pot SNP detection system is a versatile platform that could use both canonical and non-canonical PAM, and was almost not limited by mutation types to distinguish SNPs located between position 1 to 17. The use of truncated crRNA further improved SNP specificity of seCas12a. Mechanistically, we found only when the cis-cleavage was at low level between 0.01min−1 and 0.0006 min−1, a good signal-to-noise ratio can be achieved in one-pot test. SeCas12a-based one-pot SNP detection system was applied to detect pharmacogenomic SNPs in human clinical samples. Of thirteen donors tested in two different SNPs, the seCas12a mediated one-pot system could faithfully detect the SNPs in 30 min with 100% accuracy. |
| first_indexed | 2024-04-10T10:03:27Z |
| format | Article |
| id | doaj.art-0a9c40724ef14cef90a4e0c49830c68f |
| institution | Directory Open Access Journal |
| issn | 2772-8927 |
| language | English |
| last_indexed | 2024-04-10T10:03:27Z |
| publishDate | 2023-04-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Cell Insight |
| spelling | doaj.art-0a9c40724ef14cef90a4e0c49830c68f2023-02-16T04:19:01ZengElsevierCell Insight2772-89272023-04-0122100080Cas12a-based one-pot SNP detection with high accuracyHong-Xia Zhang0Caixiang Zhang1Shuhan Lu2Xiaohan Tong3Kun Zhang4Hao Yin5Ying Zhang6Department of Rheumatology and Immunology, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, ChinaDepartment of Rheumatology and Immunology, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, ChinaDepartment of Rheumatology and Immunology, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China; Department of Pathology, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, ChinaDepartment of Rheumatology and Immunology, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China; Department of Pathology, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, ChinaDepartment of Rheumatology and Immunology, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China; Department of Pathology, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, ChinaDepartment of Rheumatology and Immunology, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China; Department of Pathology, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China; Corresponding author. Department of Rheumatology and Immunology, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China.Department of Rheumatology and Immunology, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China; Corresponding author.CRISPR-Cas12a based one-pot detection system has been used in nucleic acid detection and diagnosis. However, it is not sensitive enough to distinguish single nucleotide polymorphisms (SNP), which has greatly restricted its application. To overcome these limitations, we engineered a LbCas12a variant with enhanced sensitivity against SNP, named seCas12a (sensitive Cas12a). SeCas12a-based one-pot SNP detection system is a versatile platform that could use both canonical and non-canonical PAM, and was almost not limited by mutation types to distinguish SNPs located between position 1 to 17. The use of truncated crRNA further improved SNP specificity of seCas12a. Mechanistically, we found only when the cis-cleavage was at low level between 0.01min−1 and 0.0006 min−1, a good signal-to-noise ratio can be achieved in one-pot test. SeCas12a-based one-pot SNP detection system was applied to detect pharmacogenomic SNPs in human clinical samples. Of thirteen donors tested in two different SNPs, the seCas12a mediated one-pot system could faithfully detect the SNPs in 30 min with 100% accuracy.http://www.sciencedirect.com/science/article/pii/S2772892723000044 |
| spellingShingle | Hong-Xia Zhang Caixiang Zhang Shuhan Lu Xiaohan Tong Kun Zhang Hao Yin Ying Zhang Cas12a-based one-pot SNP detection with high accuracy Cell Insight |
| title | Cas12a-based one-pot SNP detection with high accuracy |
| title_full | Cas12a-based one-pot SNP detection with high accuracy |
| title_fullStr | Cas12a-based one-pot SNP detection with high accuracy |
| title_full_unstemmed | Cas12a-based one-pot SNP detection with high accuracy |
| title_short | Cas12a-based one-pot SNP detection with high accuracy |
| title_sort | cas12a based one pot snp detection with high accuracy |
| url | http://www.sciencedirect.com/science/article/pii/S2772892723000044 |
| work_keys_str_mv | AT hongxiazhang cas12abasedonepotsnpdetectionwithhighaccuracy AT caixiangzhang cas12abasedonepotsnpdetectionwithhighaccuracy AT shuhanlu cas12abasedonepotsnpdetectionwithhighaccuracy AT xiaohantong cas12abasedonepotsnpdetectionwithhighaccuracy AT kunzhang cas12abasedonepotsnpdetectionwithhighaccuracy AT haoyin cas12abasedonepotsnpdetectionwithhighaccuracy AT yingzhang cas12abasedonepotsnpdetectionwithhighaccuracy |