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A pair of ZFNs, each with three zinc fingers binding to target DNA, are shown introducing a double-strand break, at the FokI domain, depicted in yellow. Subsequently, the double strand break is shown as being repaired through either homology-directed repair or non-homologous end joining. [3]
The dimerization of two ZFNs is required to produce the necessary double-strand break within the CCR5 gene because the interaction between the FokI enzyme and DNA is weak. [11] This break is repaired by the natural repair mechanisms of the cell, specifically non-homologous end joining. [11]
Zinc fingers were first identified in a study of transcription in the African clawed frog, Xenopus laevis in the laboratory of Aaron Klug.A study of the transcription of a particular RNA sequence revealed that the binding strength of a small transcription factor (transcription factor IIIA; TFIIIA) was due to the presence of zinc-coordinating finger-like structures. [6]
A double-strand break repair model refers to the various models of pathways that cells undertake to repair double strand-breaks (DSB). DSB repair is an important cellular process, as the accumulation of unrepaired DSB could lead to chromosomal rearrangements, tumorigenesis or even cell death. [ 1 ]
Double-strand break repair models that act via homologous recombination. Two primary models for how homologous recombination repairs double-strand breaks in DNA are the double-strand break repair (DSBR) pathway (sometimes called the double Holliday junction model) and the synthesis-dependent strand annealing (SDSA) pathway. [43]
Through targeted deletions, the custom ZFN disables the C-C chemokine receptor 5 gene, which encodes a co-receptor that is used by the HIV virus to enter the cell. [60] As a result of the high degree of sequence homology between C-C chemokine receptors this ZFN also cleaves CCR2 , leading to off-target ~15kb deletions and genomic rearrangements.
The CompoZr Zinc finger nuclease (ZFN) platform is a technology developed by Sigma-Aldrich that allows researchers to target and manipulate the genome of living cells thereby creating cell lines or entire organisms with permanent and heritable gene deletions, insertions, or modifications. The technology was released in September 2008. [1]
In addition, it has been used to engineer stably modified human embryonic stem cell and induced pluripotent stem cell (IPSCs) clones and human erythroid cell lines, [11] [28] to generate knockout C. elegans, [12] knockout rats, [13] knockout mice, [29] and knockout zebrafish. [14] [30] Moreover, the method can be used to generate knockin organisms.