Multiple RNA Rapid In Situ Imaging Based on Cas9 Code Key System.

Autor: Hu R; The Center for Clinical Molecular Medical Detection, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400042, China.; Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China., Yang W; Laboratory Medicine Center, Department of Clinical Laboratory, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou, 310014, China., Li J; The Center for Clinical Molecular Medical Detection, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400042, China., Jiang L; The Center for Clinical Molecular Medical Detection, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400042, China., Li M; Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China., Zhang M; Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China., Kang Y; The Center for Clinical Molecular Medical Detection, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400042, China., Cheng X; The Center for Clinical Molecular Medical Detection, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400042, China., Zhu S; The Center for Clinical Molecular Medical Detection, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400042, China., Zhao L; Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China., He W; Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China., Guo M; Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China., Ding S; Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China., Wu H; Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China., Cheng W; The Center for Clinical Molecular Medical Detection, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400042, China.; Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China.
Jazyk: angličtina
Zdroj: Small methods [Small Methods] 2024 Dec; Vol. 8 (12), pp. e2400195. Date of Electronic Publication: 2024 May 03.
DOI: 10.1002/smtd.202400195
Abstrakt: Existing RNA in situ imaging strategies mostly utilize parallel repetitive nucleic acid self-assembly to achieve multiple analysis, with limitations of complicated systems and cumbersome steps. Here, a Cas9 code key system with key probe (KP) encoder and CRISPR/Cas9 signal exporter is developed. This system triggers T-protospacer adjacent motif (T-PAM structural transitions of multiple KP encoders to form coding products with uniform single-guide RNA (sgRNA) target sequences as tandem nodes. Only single sgRNA/Cas9 complex is required to cleave multiple coding products, enabling efficient "many-to-one" tandem signaling, and non-collateral cleavage activity-dependent automatic signaling output through active introduction of mismatched bases. Compared with conventional parallel multiple signaling analysis model, the proposed system greatly simplifies reaction process and enhances detection efficiency. Further, a rapid multiple RNA in situ imaging system is developed by combining the Cas9 code key system with a T-strand displacement amplification (T-SDA) signal amplifier. The constructed system is applied to tumor cells and clinicopathology slices, generating clear multi-mRNA imaging profiles in less than an hour with just one step. Therefore, this work provides reliable technical support for clinical tumor typing and molecular mechanism investigation.
(© 2024 Wiley‐VCH GmbH.)
Databáze: MEDLINE
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