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: 9.81). 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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    • "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.
    PLoS ONE 09/2014; 9(9):e108236. DOI:10.1371/journal.pone.0108236 · 3.23 Impact Factor
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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.
    Scientific Reports 03/2014; 4:4489. DOI:10.1038/srep04489 · 5.58 Impact Factor
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    • "Indeed, mutations of Ku and DNA ligase IV have different outcomes in mouse and chicken [18], [20]. It is important to note, however, that KU70/KU80 are most likely essential genes in human somatic cells [51]–[53]. In fact, despite efforts to isolate Ku70-null Nalm-6 cells, we have been unable to disrupt the second KU70 allele in KU70+/− mutant (our unpublished observations). "
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    ABSTRACT: Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are two major pathways for repairing DNA double-strand breaks (DSBs); however, their respective roles in human somatic cells remain to be elucidated. Here we show using a series of human gene-knockout cell lines that NHEJ repairs nearly all of the topoisomerase II- and low-dose radiation-induced DNA damage, while it negatively affects survival of cells harbouring replication-associated DSBs. Intriguingly, we find that loss of DNA ligase IV, a critical NHEJ ligase, and Artemis, an NHEJ factor with endonuclease activity, independently contribute to increased resistance to replication-associated DSBs. We also show that loss of Artemis alleviates hypersensitivity of DNA ligase IV-null cells to low-dose radiation- and topoisomerase II-induced DSBs. Finally, we demonstrate that Artemis-null human cells display increased gene-targeting efficiencies, particularly in the absence of DNA ligase IV. Collectively, these data suggest that DNA ligase IV and Artemis act cooperatively to promote NHEJ, thereby suppressing HR. Our results point to the possibility that HR can only operate on accidental DSBs when NHEJ is missing or abortive, and Artemis may be involved in pathway switching from incomplete NHEJ to HR.
    PLoS ONE 08/2013; 8(8):e72253. DOI:10.1371/journal.pone.0072253 · 3.23 Impact Factor
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