THESIS
2020
xii, 56 pages : color illustrations ; 30 cm
Abstract
CRISPR-based gene editing technology has shown a great promise for biological and
biomedical basic and translational research. The off-target cleavage could be a major barrier
as one of the genetic therapeutics since it may generate undesired cleavage in a genome and
how protospacer adjacent motif helps the search and cleavage process still remains unknown.
The detailed mechanism of the cleavage activity of Cas9 is necessary to find out the steps of
which the off-target cleavage happens and how protospacer adjacent motif functions.
Moreover, the rationale of how currently available Cas9 proteins with high fidelity works is
essential to understand the function of the mutated residues.
Based on the confirmed experimental and computational evidence of allosteric behavior of
cas9 p...[
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CRISPR-based gene editing technology has shown a great promise for biological and
biomedical basic and translational research. The off-target cleavage could be a major barrier
as one of the genetic therapeutics since it may generate undesired cleavage in a genome and
how protospacer adjacent motif helps the search and cleavage process still remains unknown.
The detailed mechanism of the cleavage activity of Cas9 is necessary to find out the steps of
which the off-target cleavage happens and how protospacer adjacent motif functions.
Moreover, the rationale of how currently available Cas9 proteins with high fidelity works is
essential to understand the function of the mutated residues.
Based on the confirmed experimental and computational evidence of allosteric behavior of
cas9 protein, in this study we try to employ some statistical methods to study co-variance of
cas9 protein residues. Statistical coupling analysis and direct coupling analysis were used to
map the most important residues which play a role in allosteric transmission and activity of
cas9. In addition, based on the mechanisms and important residues, an overall kinetic
mechanism will be explained in detail with specific steps involved in a Cas9 activity to explain
how protospacer adjacent motif diversity comes from and what it influences.
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