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Kinetic Analysis of AceCas9

Title: Kinetic Analysis of AceCas9.

Inaccessible until Aug 10, 2020 due to copyright restrictions.

Name(s): Duboy, Emily C, author
Type of Resource: text
Genre: Text
Bachelor Thesis
Date Issued: 2018-08-03
Physical Form: computer
online resource
Extent: 1 online resource
Language(s): English
Abstract/Description: The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (CRISPR-Cas) systems utilize Cas proteins and CRISPR RNA (crRNA) to cleave target DNA. The crRNA are responsible for guiding the Cas enzyme to the target DNA sequences whereas the Cas enzyme cleaves the target. Cas9 is one of the most intensively studied CRISPR-Cas systems owing to its potentials for genome editing and clinical applications. To be cleaved by Cas9, the target substrate must have an about 20-nucleotide long protospacer region which is complementary to the guide region of the crRNA, and a protospacer adjacent motif (PAM) that is recognized and bound by the PAM-interacting domain (PID) of Cas9. Opposed to the most commonly characterized Streptococcus pyogenes Cas9 protein, which optimally utilizes a 20-nt long sgRNA, AceCas9, from organism Acidothermus cellulolycticus, reaches most optimal cleavage activity utilizing a 24-nt long sgRNA, at 50°C. Further various assays reveal that the PID within AceCas9 is possibly involved in substrate specificity and off-target cleavage. Based off in vivo survival assays, we hypothesized that, in addition to PAM recognition and binding, the PID is also involved in AceCas9 substrate specificity. After kinetic studies, varying cleavage rates of AceCas9 PID variants supports that the PID is somewhat responsible for off-target activity, but to what extent is still not understood. The continued characterization of various types of Cas9s will lead to a broadened selection of Cas9s while also improving the proteins application within biomedical research with a greater understanding of Cas9 specificity.
Identifier: FSU_libsubv1_scholarship_submission_1533314598_f4f3c168 (IID)
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Owner Institution: FSU

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Duboy, E. C. (2018). Kinetic Analysis of AceCas9. Retrieved from