Towards defining off-target cleavage sites for zinc finger nucleases in vitro

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Publication Type dissertation
School or College School of Medicine
Department Biochemistry
Author Mukherjee, Kusumika
Title Towards defining off-target cleavage sites for zinc finger nucleases in vitro
Date 2013-05
Description Zinc finger nucleases (ZFNs) are chimeric, modular proteins used for targeted mutagenesis in many model organisms and human cell lines. ZFNs bind to the designed target through a DNA binding domain and make a double-strand break (DSB) at the site. This DSB can then be repaired by the cell's repair machinery. Previous studies have revealed ZFN-induced toxicity in many cases which most likely occurs due to nonspecific cleavage by the ZFNs at secondary targets. The nonspecific DSBs can lead to unwanted secondary mutations. However, if cleavage at secondary sites is extensive, the DNA repair system is overwhelmed, leading to death of the organism or cells. The work in this dissertation describes the attempts made to develop an unbiased and comprehensive method to define these secondary targets for a pair of ZFNs in vitro in the Drosophila melanogaster genome. The strategy involves capturing all ends made by a ZFN in the Drosophila genome and then subjecting them to deep sequencing in order to define these sequences and the frequencies at which the target is cut. Towards this end, a protocol was developed to capture all ZFN-induced ends in the Drosophila genome in vitro using the rosy (ry) ZFNs. The stepwise development of the protocol is detailed here. The method was then applied to a pair of ZFNs targeting yellow (y) which are known to be toxic in Drosophila. The ends generated at the designed ZFN target were successfully captured. However, capture of other ZFN-induced ends failed. The probable causes of the failure of this strategy were also investigated and are discussed. Additional work involved the nematode Caenorhabditis elegans. ZFNs have been used successfully in nematode somatic tissue. However, attempts to generate targeted offspring using ZFNs have been futile due to widespread silencing of the transgenes in the germline. This dissertation also describes attempts to overcome this germline silencing and express ZFNs in the nematode germline.
Type Text
Publisher University of Utah
Subject In vitro; Noncanonical; Off-target; T4 DNA ligase; Template-independent; Zinc finger nucleases
Dissertation Institution University of Utah
Dissertation Name Doctor of Philosophy
Language eng
Rights Management Copyright © Kusumika Mukherjee 2013
Format Medium application/pdf
Format Extent 2,296,745 bytes
Identifier etd3/id/3444
ARK ark:/87278/s6574m9g
Setname ir_etd
Date Created 2015-04-27
Date Modified 2021-05-06
ID 197000
Reference URL