Targeted Chromosomal Gene Modification in Human Cells by Single-Stranded Oligodeoxynucleotides in the Presence of a DNA Double-Strand Break

Radecke, Frank; Peter, Ingrid; Radecke, Sarah; Gellhaus, Katharina; Schwarz, Klaus; Cathomen, Toni

doi:10.1016/j.ymthe.2006.06.008

Cited by 46 publications

(40 citation statements)

References 39 publications

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“…3A), even though the lesion can likely initiate effective ssDI as well. This result nicely explains the previous paradox that the physical incorporation of ODN donors was detected in the absence (Radecke et al 2006b), but not presence (Radecke et al 2006a), of targeted chromosomal DSBs; without a targeted lesion, the ODNs were likely physically incorporated via the ssDI pathways (Faruqi et al 2000;Ferrara and Kmiec 2004;Kuan and Glazer 2004;Wu et al 2005;Huen et al 2006) using naturally occurring gaps, whereas with a DSB most of the PGE products are generated via the SDSA pathway as a result of DNA synthesis using ODNs only as templates (Figs. 2E, 3A).…”

Section: Odns Stimulate Efficient Pge In the Presence Of Genomic Lesionssupporting

confidence: 83%

“…The bidirectional conversion tracts generated by ODN donors that were the same as the strand with the nick were consistent with an ssDI model, in which the ODN is physically assimilated into a single-strand gap and displaces the flanking sequences on both sides ( Fig. 3B; Radecke et al 2006a;Storici et al 2006;Davis and Maizels 2014). Although various forms of the ssDI model have been proposed (Faruqi et al 2000;Ferrara and Kmiec 2004;Kuan and Glazer 2004;Wu et al 2005;Huen et al 2006), the efficiency of this pathway was so low in the absence of targeted genomic lesions that it was generally not regarded as a naturally occurring HDR pathway (also note that ssDI is different from the more familiar process of single-strand annealing [SSA]) (San Filippo et al 2008;Jasin and Rothstein 2013).…”

Section: Odns Stimulate Efficient Pge In the Presence Of Genomic Lesionssupporting

confidence: 75%

“…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). Confusingly, however, the same group of authors also demonstrated that ODNs were not physically incorporated into the genome during double-strand break (DSB)-induced PGE, suggesting the existence of an alternative mechanism(s) (Radecke et al 2006a). Finally, in the presence of mega-nucleaseinduced targeted single-strand nicks, it was proposed by Davis and Maizels that certain forms of the SDSA and the ssDI pathways could be utilized depending on the strandedness of the donor ODNs Maizels 2011, 2014), a hypothesis that was supported by recent work from the Corn laboratory (Richardson et al 2016).…”

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confidence: 99%

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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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Section: Odns Stimulate Efficient Pge In the Presence Of Genomic Lesionssupporting

confidence: 83%

Section: Odns Stimulate Efficient Pge In the Presence Of Genomic Lesionssupporting

confidence: 75%

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confidence: 99%

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Mechanisms of precise genome editing using oligonucleotide donors

et al. 2017

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“…ssODNs have been shown to be able to seamlessly repair and modify targeted single DSB sites via HDR. 46 These are convenient repair templates that allow for small, targeted gene modifications to be made without the need for constructing large The sgRNA target sites were designed around an oncogenic c.35G>T mutation in the KRAS gene, with the cleavage sites depicted as arrowheads. Two primer sets were used to amplify cleavage regions that were 5ʹ and 3ʹ of the point mutation.…”

Section: Micro-deletions Can Be Repaired Precisely By Hdrmentioning

confidence: 99%

Multiplexed CRISPR/Cas9 genome editing increases the efficacy of homologous-dependent repair of donor sequences in mammalian cells

Fok¹,

Penny²,

Mhlanga³

et al. 2015

S. Afr. J. Sci.

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Multiplexed CRISPR/Cas9 genome editing increases the efficacy of homologous-dependent repair of donor sequences in mammalian cellsEfficient and robust genome editing tools and strategies allow for specific and exact genetic changes to be captured in model systems, thereby accelerating both forward and reverse genetics studies. The development of CRISPR/Cas9 as a facile designer nuclease toolset has allowed for defined genetic modifications to be efficiently made through homology-directed repair of targeted DNA double-stranded breaks (DSBs) using exogenous repair templates. However, traditional single DSB strategies are still relatively inefficient as the short gene conversion tracts of mammalian cell systems limit the extent of achievable gene alteration from the DSB site. In order to improve on the inefficiency, we devised a dual cut strategy, which relies on reconstituting entire deleted gene fragments to precisely modify extensive gene regions of interest. Using the CRISPR/Cas9 system, we were able to introduce targeted deletions and repair of the endogenous KRAS gene locus in cell culture. The use of two simultaneous DSBs can be employed for efficient application of homology-directed repair with a large dsDNA donor sequence, thereby improving the efficacy of deriving cells with a desired gene editing outcome. In conclusion, a multiplexed CRISPR/Cas9 editing strategy represents an efficient tool for the editing of complex, heterologous sequence tracts. IntroductionPrecise genome editing represents a powerful new paradigm for both forward and reverse genetics studies in model systems. Sequence-specific nucleases have been used to expand our ability for precision engineering of the genome, and can be programmed to introduce targeted chromosomal double-stranded breaks (DSBs) to trigger endogenous DNA repair pathways.1-3 The error-prone non-homologous end joining (NHEJ) repair pathway involves the re-ligation of the broken DNA ends, and in the process introduces small mutagenic insertions and deletions (indels). The homology-dependent repair (HDR) pathway seamlessly repairs the damaged site by utilising a homologous template, a process which can be exploited for targeted gene modifications.4-6 These tools have enabled functional studies based on the systematic knockout and knockin of genes 7 , the modelling of human diseases in cell-or animal-based systems [8][9][10] , and the generation of isogenic cell lines for stable transgene expression 11,12 .The clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated (Cas) protein system constitute an adaptive immune system found in prokaryotes, which functions to silence foreign DNA by RNA-guided nucleolytic digestion. 13-15 CRISPR/Cas was adapted to target DNA by using short chimeric single-guide RNA (sgRNA) and a Cas9 nuclease reconstituted to function in cultured mammalian cells. [16][17][18][19] The Cas9 nuclease can be directed to a specific cognate DNA target via a ~20-nucleotide 'guide' sequence within the sgRNA. The ease at whi...

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“…Sequence conversion in mammalian cells by SSO donors at targeted DSBs has been described (57,58). The DSB was introduced by the well known homing endonuclease I-SceI in stably integrated marker genes.…”

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confidence: 99%

Sequence Conversion by Single Strand Oligonucleotide Donors via Non-homologous End Joining in Mammalian Cells

Liu

Majumdar

Liu

et al. 2010

Journal of Biological Chemistry

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Double strand breaks (DSBs) can be repaired by homology independent nonhomologous end joining (NHEJ) pathways involving proteins such as Ku70/80, DNAPKcs, Xrcc4/Ligase 4, and the Mre11/Rad50/Nbs1 (MRN) complex. DSBs can also be repaired by homology-dependent pathways (HDR), in which the MRN and CtIP nucleases produce single strand ends that engage homologous sequences either by strand invasion or strand annealing. The entry of ends into HDR pathways underlies protocols for genomic manipulation that combine site-specific DSBs with appropriate informational donors. Most strategies utilize long duplex donors that participate by strand invasion. Work in yeast indicates that single strand oligonucleotide (SSO) donors are also active, over considerable distance, via a single strand annealing pathway. We examined the activity of SSO donors in mammalian cells at DSBs induced either by a restriction nuclease or by a targeted interstrand cross-link. SSO donors were effective immediately adjacent to the break, but activity declined sharply beyond ϳ100 nucleotides. Overexpression of the resection nuclease CtIP increased the frequency of SSO-mediated sequence modulation distal to the break site, but had no effect on the activity of an SSO donor adjacent to the break. Genetic and in vivo competition experiments showed that sequence conversion by SSOs in the immediate vicinity of the break was not by strand invasion or strand annealing pathways. Instead these donors competed for ends that would have otherwise entered NHEJ pathways. Non-homologous end joining (NHEJ)3 repair is un-templated, as there is no requirement, or even apparent use, for an informational reference sequence acting in trans. NHEJ might be regarded as a collection of operations (end processing by nucleases, limited single strand exposure of the ends, fill in by polymerases, ligation), without requirement for a specific order of events (1). In mammalian cells the best characterized version of NHEJ, often termed "classical" (C-NHEJ) (2, 3), utilizes several factors, including Ku70/80, Artemis, XLF, DNAPKcs, Xrcc4/DNA ligase 4, pol, pol, and XLF/Cernunnos (4 -8).Although precise joining of restriction enzyme cleaved ends can occur, the pathway is frequently mutagenic, resulting in the introduction of short deletions at the break site (9 -11).Deletions at the DSB are sometimes bounded by short homology elements located on either side of the break. These have been described as the product of a microhomology mediated end joining (MMEJ) pathway (5, 12). Deletions without microhomologies are also recovered and recent work suggests that the MRN complex plays an important role in both versions of deletional NHEJ (2, 13-15). A backup pathway involving poly(ADP-ribose) polymerase 1 and ligase III has been described as well (16,17). It should be noted that whether end joining in the absence of one of the canonical factors is evidence of different discrete pathways, or simply a variation on a fundamental scheme, is a matter for discussion (7). Nonetheless, NHEJ is the dom...

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Targeted Chromosomal Gene Modification in Human Cells by Single-Stranded Oligodeoxynucleotides in the Presence of a DNA Double-Strand Break

Cited by 46 publications

References 39 publications

Mechanisms of precise genome editing using oligonucleotide donors

Mechanisms of precise genome editing using oligonucleotide donors

Multiplexed CRISPR/Cas9 genome editing increases the efficacy of homologous-dependent repair of donor sequences in mammalian cells

Sequence Conversion by Single Strand Oligonucleotide Donors via Non-homologous End Joining in Mammalian Cells

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