An update on targeted gene repair in mammalian cells: methods and mechanisms

Jensen, Nanna Møller; Dalsgaard, Trine Kastrup; Jakobsen, Maria; Nielsen, Roni; Sørensen, Charlotte Brandt; Bolund, Lars; Jensen, Thomas G.

doi:10.1186/1423-0127-18-10

Cited by 38 publications

(26 citation statements)

References 126 publications

(264 reference statements)

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“…In the absence of a targeted mega-nuclease like Cas9, the ODN donors may use spontaneously occurring genomic lesions in order to perform PGE. Indeed, it was proposed that ODNs could be incorporated into the gaps generated by nucleotide excision repair (Faruqi et al 2000;Kuan and Glazer 2004) or lagging strand synthesis during DNA replication (Ferrara and Kmiec 2004;Wu et al 2005;Huen et al 2006), although the specific mechanisms are still disputed (Suzuki 2008;Jensen et al 2011). Interestingly, both of these models involve the physical incorporation of the ODNs into the genome via an ssDI-like mechanism; a prediction that was later supported by experiments carried out by Radecke et al (2006b).…”

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confidence: 61%

Mechanisms of precise genome editing using oligonucleotide donors

et al. 2017

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The use of programmable meganucleases is transforming genome editing and functional genomics. CRISPR/Cas9 was developed such that targeted genomic lesions could be introduced in vivo with unprecedented ease. In the presence of homology donors, these lesions facilitate high-efficiency precise genome editing (PGE) via homology-directed repair (HDR) pathways. However, the identity and hierarchy of the HDR (sub)pathways leading to the formation of PGE products remain elusive. Here, we established a green to blue fluorescent protein conversion system to systematically characterize oligodeoxynucleotide (ODN)-mediated PGE using Cas9 and its nickase variants in human cells. We demonstrate that, unlike double-stranded DNA (dsDNA) donors with central heterologies, ODNs generated short conversion tracts with Gaussianlike distributions. Interestingly, single-nick-induced PGE using ODN donors produced conversion tracts biased either mostly uni-or bidirectional depending on the relative strandedness of the ODNs and the nick. Moreover, the ODNs were physically incorporated into the genome only in the bidirectional, but not in the unidirectional, conversion pathway. In the presence of double-stranded genomic lesions, the unidirectional conversion pathway was preferentially utilized even though the knock-in mutation could theoretically have been converted by both pathways. Collectively, our results suggest that ODN-mediated PGE utilizes synthesis-dependent strand annealing and single-stranded DNA incorporation pathways. Both of these pathways generate short conversion tracts with Gaussian-like distributions. Although synthesis-dependent strand annealing is preferentially utilized, our work unequivocally establishes the existence of a single-stranded DNA incorporation pathway in human cells. This work extends the paradigms of HDR-mediated gene conversion and establishes guidelines for PGE in human cells.

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confidence: 61%

Mechanisms of precise genome editing using oligonucleotide donors

et al. 2017

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“…Therefore, gene disruption and ultimately gene knockout may be identifi ed from the resulting mutation repertoire. In contrast, HDR is a template-dependent process [ 6 ] and thus can be harnessed for user-defi ned gene editing when a well-designed DNA donor is co-delivered together with engineered endonucleases.…”

Section: Introductionmentioning

confidence: 97%

Gene Editing Using ssODNs with Engineered Endonucleases

Chen

Pruett-Miller

Davis

2014

Methods in Molecular Biology

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Gene editing using engineered endonucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nucleases, requires the creation of a targeted, chromosomal DNA double-stranded break (DSB). In mammalian cells, these DSBs are typically repaired by one of the two major DNA repair pathways: nonhomologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ is an error-prone repair process that can result in a wide range of end-joining events that leads to somewhat random mutations at the site of DSB. HDR is a precise repair pathway that can utilize either an endogenous or exogenous piece of homologous DNA as a template or "donor" for repair. Traditional gene editing via HDR has relied on the co-delivery of a targeted, engineered endonuclease and a circular plasmid donor construct. More recently, it has been shown that single-stranded oligodeoxynucleotides (ssODNs) can also serve as DNA donors and thus obviate the more laborious and time-consuming plasmid vector construction process. Here we describe the use of ssODNs for making defined genome modifications in combination with engineered endonucleases.

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“…Despite the multitude of advances recently made to improve delivery methods and minimize unwanted side-effects, many gene therapy technologies are still moving cautiously toward clinical applications (Jensen et al 2011 ) . The fi nding by Jasin and colleagues that double-strand breaks (DSBs) introduced at speci fi c genomic locations can signi fi cantly stimulate gene repair by homologous recombination with a corrective DNA template (Rouet et al 1994 ) has motivated efforts to utilize this particular strategy for gene correction.…”

Section: Introductionmentioning

confidence: 99%

Engineered Zinc Finger Nucleases for Targeted Genome Editing

Ramirez

Joung

2012

Site-Directed Insertion of Transgenes

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Zinc fi nger nucleases (ZFNs) are arti fi cial proteins consisting of engineered zinc fi nger proteins fused to the FokI endonuclease domain. These nucleases bind to speci fi c DNA recognition sites and introduce double-strand breaks (DSBs). Repair of these DSBs by normal cellular processes can be exploited to either disrupt genes or signi fi cantly increase the frequency of homologous recombination with a user-de fi ned repair template. Several platforms have been developed that enable engineering of zinc fi nger proteins that bind with high af fi nity and speci fi city to 9-18 bp target DNA sequences. ZFNs have already been used in basic research, most prominently with the development of methods to ef fi ciently modify various model organisms and cell lines.

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An update on targeted gene repair in mammalian cells: methods and mechanisms

Cited by 38 publications

References 126 publications

Mechanisms of precise genome editing using oligonucleotide donors

Mechanisms of precise genome editing using oligonucleotide donors

Gene Editing Using ssODNs with Engineered Endonucleases

Engineered Zinc Finger Nucleases for Targeted Genome Editing

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