Obligate Ligation-Gated Recombination (ObLiGaRe): Custom-designed nuclease-mediated targeted integration through nonhomologous end joining

Maresca, Marcello; Lin, Victor Guosheng; Guo, Ning; Yang, Yi

doi:10.1101/gr.145441.112

Cited by 283 publications

(265 citation statements)

References 47 publications

Supporting

Mentioning

260

Contrasting

Order By: Relevance

“…This technology has been successfully applied in several animal models including mouse, rat, zebrafish and Caenorhabditis elegans [4][5][6][7][8] . Gene targeting via non-homologous end-joining (NHEJ) has also been suggested to generate combined reporter and knockout alleles 9 . Combination of TALEN-based gene targeting with insertion of a few base pairs or transgenes using small ssDNA oligonucleotides or donor vectors with longer homologous arms was initially reported for zebrafish 10,11 and later extended to other animal models including mouse 12,13 .…”

mentioning

confidence: 99%

Efficient genome engineering by targeted homologous recombination in mouse embryos using transcription activator-like effector nucleases

et al. 2014

View full text Add to dashboard Cite

Generation of mouse models by introducing transgenes using homologous recombination is critical for understanding fundamental biology and pathology of human diseases. Here we investigate whether artificial transcription activator-like effector nucleases (TALENs)-powerful tools that induce DNA double-strand breaks at specific genomic locations-can be combined with a targeting vector to induce homologous recombination for the introduction of a transgene in embryonic stem cells and fertilized murine oocytes. We describe the generation of a conditional mouse model using TALENs, which introduce double-strand breaks at the genomic locus of the special AT-rich sequence-binding protein-1 in combination with a large 14.4 kb targeting template vector. We report successful germline transmission of this allele and demonstrate its recombination in primary cells in the presence of Cre-recombinase. These results suggest that TALEN-assisted induction of DNA double-strand breaks can facilitate homologous recombination of complex targeting constructs directly in oocytes.

show abstract

mentioning

confidence: 99%

Efficient genome engineering by targeted homologous recombination in mouse embryos using transcription activator-like effector nucleases

et al. 2014

View full text Add to dashboard Cite

show abstract

“…Thus, it should be possible to capture two different ssODNs simultaneously at different positions on the same chromosome (e.g., for the introduction of two loxP sites flanking a set of exons) depending on the efficiency of this process. Furthermore, combination of the segmental deletions with the application of ssODNs (Chen et al 2011;Bedell et al 2012) or the introduction of nuclease-stimulated ligation of an exogenous donor DNA (Cristea et al 2013;Maresca et al 2013) could facilitate the targeted replacement of a desired genomic segment with a specific sequence variant for a more complete deconvolution of function.…”

Section: Chromosomal Deletions and Inversions In Zebrafishmentioning

confidence: 99%

Targeted chromosomal deletions and inversions in zebrafish

et al. 2013

View full text Add to dashboard Cite

Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) provide powerful platforms for genome editing in plants and animals. Typically, a single nuclease is sufficient to disrupt the function of protein-coding genes through the introduction of microdeletions or insertions that cause frameshifts within an early coding exon. However, interrogating the function of cis-regulatory modules or noncoding RNAs in many instances requires the excision of this element from the genome. In human cell lines and invertebrates, two nucleases targeting the same chromosome can promote the deletion of intervening genomic segments with modest efficiencies. We have examined the feasibility of using this approach to delete chromosomal segments within the zebrafish genome, which would facilitate the functional study of large noncoding sequences in a vertebrate model of development. Herein, we demonstrate that segmental deletions within the zebrafish genome can be generated at multiple loci and are efficiently transmitted through the germline. Using two nucleases, we have successfully generated deletions of up to 69 kb at rates sufficient for germline transmission (1%–15%) and have excised an entire lincRNA gene and enhancer element. Larger deletions (5.5 Mb) can be generated in somatic cells, but at lower frequency (0.7%). Segmental inversions have also been generated, but the efficiency of these events is lower than the corresponding deletions. The ability to efficiently delete genomic segments in a vertebrate developmental system will facilitate the study of functional noncoding elements on an organismic level.

show abstract

“…Maresca et al added the ZFN site located in the genome into a drugresistant gene cassette vector, and cointroduced the ZFN and the targeting vector into human cultured cells to isolate the targeted clones with high efficacy through NHEJ activity (Fig. 1d) [66]. They named this method ObLiGaRe (obligate ligation-gated recombination), based on the Latin verb obligate ("to join to") [66].…”

Section: Nhej-mediated Knock-in Using Crisprobligare or Hitimentioning

confidence: 99%

“…1d) [66]. They named this method ObLiGaRe (obligate ligation-gated recombination), based on the Latin verb obligate ("to join to") [66]. In the ObLiGaRe method, highly effective transgene knock-in occurs in most human cultured cells, but the orientation of the transgene is not controlled and precise adjustment of junction sequences between a target locus and the transgene is not possible.…”

Section: Nhej-mediated Knock-in Using Crisprobligare or Hitimentioning

confidence: 99%

See 1 more Smart Citation

Updated summary of genome editing technology in human cultured cells linked to human genetics studies

2017

View full text Add to dashboard Cite

Current deep-sequencing technology provides a mass of nucleotide variations associated with human genetic disorders to accelerate the identification of causative mutations. To understand the etiology of genetic disorders, reverse genetics in human cultured cells is a useful approach for modeling a disease in vitro. However, gene targeting in human cultured cells is difficult because of their low activity of homologous recombination. Engineered endonucleases enable enhancement of the local activation of DNA repair pathways at the human genome target site to rewrite the desired sequence, thereby efficiently generating disease-modeling cultured cell clones. These edited cells can be used to explore the molecular functions of a causative gene product to uncover the etiological mechanisms. The correction of mutations in patient cells using genome editing technology could contribute to the development of unique gene therapies. This technology can also be applied to screening causative mutations. Rare genetic disorders and non-exonic mutation-caused diseases remain frontier in the field of human genetics as it is difficult to validate whether the extracted nucleotide variants are mutation or polymorphism. When isogenic human cultured cells with a candidate variant reproduce the pathogenic phenotypes, it is confirmed that the variant is a causative mutation.

show abstract

Obligate Ligation-Gated Recombination (ObLiGaRe): Custom-designed nuclease-mediated targeted integration through nonhomologous end joining

Cited by 283 publications

References 47 publications

Efficient genome engineering by targeted homologous recombination in mouse embryos using transcription activator-like effector nucleases

Efficient genome engineering by targeted homologous recombination in mouse embryos using transcription activator-like effector nucleases

Targeted chromosomal deletions and inversions in zebrafish

Updated summary of genome editing technology in human cultured cells linked to human genetics studies

Contact Info

Product

Resources

About