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https://scholarbank.nus.edu.sg/handle/10635/199972
DC Field | Value | |
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dc.title | STRUCTURE AND FUNCTION OF Lb2CAS12A AND ITS RNA-DNA COMPLEXS UNCOVER THE CRRNA CAPTURING AND DNA CLEAVAGE MECHANISMS AND ITS APPLICATIONS | |
dc.contributor.author | LI JIANWEI | |
dc.date.accessioned | 2021-08-31T18:00:32Z | |
dc.date.available | 2021-08-31T18:00:32Z | |
dc.date.issued | 2021-01-05 | |
dc.identifier.citation | LI JIANWEI (2021-01-05). STRUCTURE AND FUNCTION OF Lb2CAS12A AND ITS RNA-DNA COMPLEXS UNCOVER THE CRRNA CAPTURING AND DNA CLEAVAGE MECHANISMS AND ITS APPLICATIONS. ScholarBank@NUS Repository. | |
dc.identifier.uri | https://scholarbank.nus.edu.sg/handle/10635/199972 | |
dc.description.abstract | The CRISPR-Cas protein, Cas12a, processes precursor crispr (cr) RNA and cleaves invading genetic material. We report the crystal structures of apo Lachnospiraceae bacterium MA2020 (Lb2)Cas12a and the Lb2Cas12a-crRNA complex, and cryo-EM structures of Lb2Cas12a-crRNA-DNA heteroduplex in different conformations along with functional studies. Apo Lb2Cas12a is in the unique elongated open conformation and its single-stranded (ss) DNA cleavage activity is triggered by Mn2+ accompanied by a “Lid” movement, while the crRNA, crRNA-DNA duplex bound forms are in the closed/intermediate conformations. The Lb2Cas12a recognition lobe displays the unique mobility of 102 Å movement and 103 rotation aiding to position the RNA-DNA duplex near the catalytic site. Further, we present an in vitro single crRNA array strategy to detect multiple-genes, reducing the probability of detection escape, and demonstrated with SARS-CoV-2, MERS, and DENV. Our findings provide insight for crRNA capture, crRNA-independent ssDNA cleavage, open/close conformation of Cas12a for activity and a clinically relevant method for nucleic acid detection. | |
dc.language.iso | en | |
dc.subject | CRISPR-Cas, cryo-EM, apo Cas12a, Structure rearrangement, SARS-CoV2 | |
dc.type | Thesis | |
dc.contributor.department | BIOLOGICAL SCIENCES | |
dc.contributor.supervisor | Jayaraman Sivaraman | |
dc.description.degree | Ph.D | |
dc.description.degreeconferred | DOCTOR OF PHILOSOPHY (FOS) | |
dc.identifier.orcid | 0000-0001-9181-4760 | |
Appears in Collections: | Ph.D Theses (Open) |
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Li Jianwei thesis.pdf | 10.95 MB | Adobe PDF | OPEN | None | View/Download |
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