Browsing by Subject "Synthesis dependent strand annealing"

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    The Mechanism of Precise Genome Engineering in Human Cells
    (2015-09) Kan, Yinan
    Genome engineering is the intentional alteration of the genetic information in living cells or organisms. Since Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated 9 (CRISPR/Cas9) was repurposed for genome engineering, the “CRISPR Craze” is quickly bridging the genotype and phenotype worlds and transforming the biological, biomedical and biotechnological research. Interestingly, CRISRP/Cas9 does not perform precise genome engineering (PGE) by itself, but it only induces a targeted genomic lesion and invites the HDR pathways to introduce the desired modifications. Although PGE has a wide application in genome modification and gene therapy, the identity, property and hierarchy of the HDR pathways leading to the formation of PGE products remain obscure. In my doctorial dissertation, I demonstrated that double-strand DNA (dsDNA) donors with a sizable central heterology preferentially utilize the double-strand break repair (DSBR) pathway in the absence and presence of chromosomal double-strand breaks (DSBs). This pathway generates long, bidirectional conversion tracts with linear distribution. In contrast, single-strand oligonucleotide (ODN) donors utilize the synthesis-dependent strand annealing (SDSA) and single-strand DNA incorporation (ssDI) pathways, respectively, depending on the strandedness of the genomic lesions and ODN donors. These pathways produce short, unidirectional and bidirectional conversion tracts with Gaussian distributions. The SDSA pathway is preferentially utilized in the presence of compound genomic lesions such as DSBs and paired nicks. In summary, this work systematically determined the identity, property and hierarchy of the HDR pathways underlying PGE with definitive molecular evidence, and provided practical guidelines for the improvement of PGE.