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  <doc>
    <id>1832</id>
    <completedYear/>
    <publishedYear>2023</publishedYear>
    <thesisYearAccepted/>
    <language>eng</language>
    <pageFirst/>
    <pageLast/>
    <pageNumber>52</pageNumber>
    <edition/>
    <issue/>
    <volume/>
    <type>bachelorthesis</type>
    <publisherName/>
    <publisherPlace/>
    <creatingCorporation/>
    <contributingCorporation/>
    <belongsToBibliography>0</belongsToBibliography>
    <completedDate>2023-11-19</completedDate>
    <publishedDate>--</publishedDate>
    <thesisDateAccepted>--</thesisDateAccepted>
    <title language="eng">Identification of Cas9-nickase Fusions that Promote HDR-mediated Genome Editing</title>
    <abstract language="eng">At the heart of this project was the exploration and improvement of the CRISPR/Cas9 gene editing platform with the goal of enhancing efficiency and ensuring safety. The research carried out was primarily concerned with testing and developing a gene editing system that bypasses the need for double strand breaks (DSBs). Given that DSBs are the most damaging events for a cell and pose a major safety risk in genome editing, eliminating them was a key focus of the project. The project aimed to increase the frequency of homology directed repair (HDR) while simultaneously decreasing non homologous end joining (NHEJ), thereby improving the accuracy of genome editing.&#13;
&#13;
The methodology involved the use of a Cas9 protein fused to factors involved in DNA repair. Based on previous evidence that this strategy improves the HDR mediated repair of a Cas9 induced DSB, intention here was to apply the same strategy to Cas9 nickases for a safer alternative to standard genome editing procedures.&#13;
&#13;
A Cas9 nuclease can be transformed into a Cas9 nickase (nCas9) that cuts only one strand of DNA by mutating one of the two nuclease domains of the Cas9 either via a D10A point mutation for RuvC or an H840A for HNH. Preliminary experiments had shown that using nickases could reduce the occurrence of DSBs at both target and non target sites. However, because genome editing efficiency of nickases is limited, the project focused on amplifying nickase induced HDR frequency by fusing the D10A nCas9 with DNA repair factors that guide the repair of single strand breaks via HDR.&#13;
&#13;
Experiments evaluated the ability of each Cas9 nickase to direct the resolution of single strand breaks via HDR. An eGFP or mCherry cassette under its own promoter was employed as a reporter gene to measure the success of the transfection. The experiments were performed in three genomic loci, namely AAVS1, CLYBL, and H2AX. The degree of eGFP or mCherry expression after 14 days, as quantified by flow cytometry, was used to estimate the knock in success.&#13;
&#13;
The results obtained support the hypothesis that fusing D10A nCas9 with selected DNA repair factors leads to an increase in the knock in efficiencies as compared to the unmodified nCas9. The choice of the promoter used to express the nuclease, either CMV or CAG, did not seem to influence the knock in efficiencies. Instead, the type of repair template used did influence the knock in efficiencies with nicked plasmid templates contributing to greater HDR levels than non nicked ones. Knock in efficiencies of Cas9 nickases varied depending on which locus they were tested in.&#13;
&#13;
More research remains to be done to test alternative Cas9 nickase fusions. However, this approach represents a promising direction for the creation of a more efficient and safer gene editing instrument for clinical use.</abstract>
    <identifier type="urn">urn:nbn:de:hbz:1383-opus4-18322</identifier>
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    <licence>Bestimmungen des deutschen Urheberrechts</licence>
    <author>Julia Erkkilä</author>
    <collection role="institutes" number="">Fakultät Life Sciences</collection>
    <thesisPublisher>Hochschule Rhein-Waal</thesisPublisher>
    <thesisGrantor>Hochschule Rhein-Waal</thesisGrantor>
    <file>https://opus4.kobv.de/opus4-rhein-waal/files/1832/Thesis_Julia_Erkkilae.pdf</file>
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