Article

Ku70, an essential gene, modulates the frequency of rAAV-mediated gene targeting in human somatic cells

Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Medical School, Minneapolis, MN 55455, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 07/2008; 105(25):8703-8. DOI: 10.1073/pnas.0712060105
Source: PubMed

ABSTRACT

Gene targeting has two important applications. One is the inactivation of genes ("knockouts"), and the second is the correction of a mutated allele back to wild-type ("gene therapy"). Central to these processes is the efficient introduction of the targeting DNA into the cells of interest. In humans, this targeting is often accomplished through the use of recombinant adeno-associated virus (rAAV). rAAV is presumed to use a pathway of DNA double-strand break (DSB) repair termed homologous recombination (HR) to mediate correct targeting; however, the specifics of this mechanism remain unknown. In this work, we attempted to generate Ku70-null human somatic cells by using a rAAV-based gene knockout strategy. Ku70 is the heterodimeric partner of Ku86, and together they constitute an end-binding activity that is required for a pathway [nonhomologous end joining (NHEJ)] of DSB repair that is believed to compete with HR. Our data demonstrated that Ku70 is an essential gene in human somatic cells. More importantly, however, in Ku70(+/-) cells, the frequency of gene targeting was 5- to 10-fold higher than in wild-type cells. RNA interference and short-hairpinned RNA strategies to deplete Ku70 phenocopied these results in wild-type cells and greatly accentuated them in Ku70(+/-) cell lines. Thus, Ku70 protein levels significantly influenced the frequency of rAAV-mediated gene targeting in human somatic cells. Our data suggest that gene-targeting frequencies can be significantly improved in human cells by impairing the NHEJ pathway, and we propose that Ku70 depletion can be used to facilitate both knockout and gene therapy approaches.

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    • "In addition, suppression of random integration of the GT vector due to Lig4 deficiency might have a positive effect on GT. In fact, it has been reported that suppression of Ku70/80 or Lig4 function can increase the frequency of GT in several organisms , such as fungi (de Boer et al., 2010;Ushimaru et al., 2010;Nakazawa et al., 2011), bacteria (Zhang et al., 2012), vertebrates (Bertolini et al., 2009;Fattah et al., 2008;Iiizumi et al., 2008), and plants (Tanaka et al., 2010;Jia et al., 2012). Thus, although Lig4 deficiency appears effective in enhancing GT, NHEJ is an important DNA repair pathway and somaclonal mutations might be increased in a Lig4 knockout background. "
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    ABSTRACT: Sequence-specific nucleases (SSNs) have been used successfully in homology-directed repair (HDR)-mediated gene targeting (GT) in many organisms. However, break-induced GT in plants remains challenging due to inefficient delivery of HDR templates and SSNs into plant nuclei. In many plants, including rice, Agrobacterium-mediated transformation is the most practical means of transformation because this biotic transformation system can deliver longer and more intact DNA payloads with less incorporation of fragmented DNA compared with physical transformation systems such as PEG, electroporation or biolistics. Following infection with Agrobacterium, transfer of T-DNA to the nucleus and its integration into the plant genome occur consecutively during co-cultivation, thus timing the induction of DNA double-strand breaks (DSBs) on the target gene to coincide with the delivery of the HDR template is crucial. To synchronize DSB induction and delivery of the HDR template, we transformed a Cas9 expression construct and GT vector harboring the HDR template with gRNAs targeting the rice acetolactate synthase (ALS) gene either separately or sequentially into rice calli. When gRNAs targeting ALS were transcribed transiently from double-stranded T-DNA containing the HDR template, DSBs were induced in the ALS locus by the assembled Cas9/gRNA complex and homologous recombination was stimulated. Contrary to our expectations, there was no great difference in GT efficiency between Cas9-expressing and non-expressing cells. However, when gRNA targeting DNA ligase 4 (Lig4) was transformed with Cas9 prior to the GT experiment, GT efficiency increased dramatically and more than one line exhibiting bi-allelic GT at the ALS locus was obtained.
    Preview · Article · Dec 2015 · Plant physiology
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    • "Non-homologous end-joining (NHEJ), which repairs DNA double-strand breaks (DSBs) in a Ku-dependent manner [4], is responsible for nearly all random integration events in lower eukaryotes, such as Neurospora crassa, and thus NHEJ deficiency dramatically enhances gene targeting [5], [6]. Unfortunately, however, this is not the case for human somatic cells, as apparently NHEJ is not the sole mechanism of random integration [7], [8] (Figure 1). Although earlier studies have suggested a substantial role of NHEJ in random integration [9], [10], we have previously observed robust random integration events in cells lacking DNA ligase IV (LIG4, a critical NHEJ factor [11]). "
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    ABSTRACT: Random integration of targeting vectors into the genome is the primary obstacle in human somatic cell gene targeting. Non-homologous end-joining (NHEJ), a major pathway for repairing DNA double-strand breaks, is thought to be responsible for most random integration events; however, absence of DNA ligase IV (LIG4), the critical NHEJ ligase, does not significantly reduce random integration frequency of targeting vector in human cells, indicating robust integration events occurring via a LIG4-independent mechanism. To gain insights into the mechanism and robustness of LIG4-independent random integration, we employed various types of targeting vectors to examine their integration frequencies in LIG4-proficient and deficient human cell lines. We find that the integration frequency of targeting vector correlates well with the length of homology arms and with the amount of repetitive DNA sequences, especially SINEs, present in the arms. This correlation was prominent in LIG4-deficient cells, but was also seen in LIG4-proficient cells, thus providing evidence that LIG4-independent random integration occurs frequently even when NHEJ is functionally normal. Our results collectively suggest that random integration frequency of conventional targeting vectors is substantially influenced by homology arms, which typically harbor repetitive DNA sequences that serve to facilitate LIG4-independent random integration in human cells, regardless of the presence or absence of functional NHEJ.
    Full-text · Article · Sep 2014 · PLoS ONE
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    • "We suspect that this is because of the preferred choice of nonhomologous end joining (NHEJ) pathway during repairing of double strand breaks (DSBs). It was reported that disruption of ku70 could increase the frequency of HR mediated knock-in in various organisms from fungus, plant to human cells21222324. Ku70, an evolutionarily conserved protein from bacteria to human, is part of the ku heterodimer, which binds to DNA DSB and is required for NHEJ pathway. "
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    ABSTRACT: CRISPR/Cas9, a bacterial adaptive immune system derived genome-editing technique, has become to be one of the most compelling topics in biotechnology. Bombyx mori is an economically important insect and a model organism for studying lepidopteran and arthropod biology. Here we reported highly efficient and multiplex genome editing in B. mori cell line and heritable site-directed mutagenesis of Bmku70, which is required for NHEJ pathway and also related to antigen diversity, telomere length maintenance and subtelomeric gene silencing, using CRISPR/Cas9 system. We established a simple and practicable method and obtained several Bmku70 knockout B. mori lines, and showed that the frequency of HR was increased in embryos of the Bmku70 knockout B. mori. The mutant lines obtained in this study could be a candidate genetic resource for efficient knock-in and fundamental research of DNA repair in B. mori. We also provided a strategy and procedure to perform heritable genome editing of target genes with no significant phenotype effect.
    Full-text · Article · Mar 2014 · Scientific Reports
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