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Completely Free from PAM Limitations: Asymmetric RPA with CRISPR/Cas12a for Nucleic Acid Assays.
Cao, Gaihua; Yang, Nannan; Xiong, Yifan; Shi, Meimei; Wang, Lin; Nie, Fuping; Huo, Danqun; Hou, Changjun.
Afiliación
  • Cao G; Key Laboratory for Biorheological Science and Technology of Ministry of Education, Bioengineering College of Chongqing University, Chongqing 400044, PR China.
  • Yang N; State Key Laboratory of Cattle Diseases Detection (Chongqing) of Customs. Diagnosis and Testing Laboratory of Lumpy Skin Disease, Chongqing Customs Technology Center, Chongqing 400020, PR China.
  • Xiong Y; Key Laboratory for Biorheological Science and Technology of Ministry of Education, Bioengineering College of Chongqing University, Chongqing 400044, PR China.
  • Shi M; State Key Laboratory of Cattle Diseases Detection (Chongqing) of Customs. Diagnosis and Testing Laboratory of Lumpy Skin Disease, Chongqing Customs Technology Center, Chongqing 400020, PR China.
  • Wang L; Key Laboratory for Biorheological Science and Technology of Ministry of Education, Bioengineering College of Chongqing University, Chongqing 400044, PR China.
  • Nie F; State Key Laboratory of Cattle Diseases Detection (Chongqing) of Customs. Diagnosis and Testing Laboratory of Lumpy Skin Disease, Chongqing Customs Technology Center, Chongqing 400020, PR China.
  • Huo D; State Key Laboratory of Cattle Diseases Detection (Chongqing) of Customs. Diagnosis and Testing Laboratory of Lumpy Skin Disease, Chongqing Customs Technology Center, Chongqing 400020, PR China.
  • Hou C; Science and Technology Research Center of China Customs, Beijing 100730, PR China.
ACS Sens ; 8(12): 4655-4663, 2023 Dec 22.
Article en En | MEDLINE | ID: mdl-38010352
Experimentally, Cas12a can recognize multiple protospacer adjacent motif (PAM) sequences and is not restricted to the "TTTN". However, the application of the CRISPR/Cas12a system is still limited by the PAM for double-stranded DNA (dsDNA). Here, we developed asymmetric RPA (Asy-RPA) to completely break the limitations of PAM. Asy-RPA not only achieved efficient amplification but also converted dsDNA to single-stranded DNA (ssDNA) without complicated steps. The ssDNA products activated the trans-cleavage activity of Cas12a, outputting signals. The application of Asy-RPA completely freed Cas12a from the PAM, which can be more widely used in nucleic acid detection, such as lumpy skin disease virus, with an actual detection limit as low as 1.21 × 101 copies·µL-1. More importantly, Cas12a was intolerant to mutations on ssDNA. This provided technical support for the detection and identification of wild-type Mycobacterium tuberculosis (WT-TB) and rifampin-resistant mutant-type M. tuberculosis (MT-TB). The detection limit was as low as 1 fM for 1% mixed samples. The detection and availability of different treatment options for treatment-resistant and WT-TB were significant for the elimination of TB. In summary, the platform consisting of Asy-RPA and CRISPR/Cas12a was suitable for the detection of various viruses and bacteria and was a boon for the detection of dsDNA without recognizable PAM.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Sistemas CRISPR-Cas / Mycobacterium tuberculosis Límite: Animals Idioma: En Revista: ACS Sens Año: 2023 Tipo del documento: Article Pais de publicación: Estados Unidos
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Sistemas CRISPR-Cas / Mycobacterium tuberculosis Límite: Animals Idioma: En Revista: ACS Sens Año: 2023 Tipo del documento: Article Pais de publicación: Estados Unidos