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Title: | STRUCTURE AND FUNCTION OF Lb2CAS12A AND ITS RNA-DNA COMPLEXS UNCOVER THE CRRNA CAPTURING AND DNA CLEAVAGE MECHANISMS AND ITS APPLICATIONS | Authors: | LI JIANWEI | ORCID iD: | orcid.org/0000-0001-9181-4760 | Keywords: | CRISPR-Cas, cryo-EM, apo Cas12a, Structure rearrangement, SARS-CoV2 | Issue Date: | 5-Jan-2021 | 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. | 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. | URI: | https://scholarbank.nus.edu.sg/handle/10635/199972 |
Appears in Collections: | Ph.D Theses (Open) |
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