Enhanced targeted integration mediated by translocated I-SceI during the Agrobacterium mediated transformation of yeast

Rolloos, Martijn; Hooykaas, Paul J. J.; Zaal, Bert J. van der

doi:10.1038/srep08345

Cited by 20 publications

(18 citation statements)

References 40 publications

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“…The effect of TrwC as a DNA chaperone in combination with a site-specific nuclease would promote integration of the incoming DNA molecule by homologous recombination. In support of this approach, it has been reported that concomitant translocation of I-SceI homing site-specific endonuclease together with VirD2 relaxase-T-DNA complexes through the A. tumefaciens T4SS enhanced T-DNA site-specific integration into the yeast chromosome when the I-SceI target site was present (33).…”

Section: Discussionmentioning

confidence: 94%

The Conjugative Relaxase TrwC Promotes Integration of Foreign DNA in the Human Genome

González-Prieto

Rinaldi

Dehio

et al. 2017

Appl Environ Microbiol

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Bacterial conjugation is a mechanism of horizontal DNA transfer. The relaxase TrwC of the conjugative plasmid R388 cleaves one strand of the transferred DNA at the gene, covalently attaches to it, and leads the single-stranded DNA (ssDNA) into the recipient cell. In addition, TrwC catalyzes site-specific integration of the transferred DNA into its target sequence present in the genome of the recipient bacterium. Here, we report the analysis of the efficiency and specificity of the integrase activity of TrwC in human cells, using the type IV secretion system of the human pathogen to introduce relaxase-DNA complexes. Compared to Mob relaxase from plasmid pBGR1, we found that TrwC mediated a 10-fold increase in the rate of plasmid DNA transfer to human cells and a 100-fold increase in the rate of chromosomal integration of the transferred DNA. We used linear amplification-mediated PCR and plasmid rescue to characterize the integration pattern in the human genome. DNA sequence analysis revealed mostly reconstituted sequences, indicating that TrwC is active and recircularizes transferred DNA in human cells. One TrwC-mediated site-specific integration event was detected, proving that TrwC is capable of mediating site-specific integration in the human genome, albeit with very low efficiency compared to the rate of random integration. Our results suggest that TrwC may stabilize the plasmid DNA molecules in the nucleus of the human cell, probably by recircularization of the transferred DNA strand. This stabilization would increase the opportunities for integration of the DNA by the host machinery. Different biotechnological applications, including gene therapy strategies, require permanent modification of target cells. Long-term expression is achieved either by extrachromosomal persistence or by integration of the introduced DNA. Here, we studied the utility of conjugative relaxase TrwC, a bacterial protein with site-specific integrase activity in bacteria, as an integrase in human cells. Although it is not efficient as a site-specific integrase, we found that TrwC is active in human cells and promotes random integration of the transferred DNA in the human genome, probably acting as a DNA chaperone until it is integrated by host mechanisms. TrwC-DNA complexes can be delivered to human cells through a type IV secretion system involved in pathogenesis. Thus, TrwC could be used to transfer the DNA of interest into the appropriate cell and promote its integration. If used in combination with a site-specific nuclease, it could lead to site-specific integration of the incoming DNA by homologous recombination.

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Section: Discussionmentioning

confidence: 94%

The Conjugative Relaxase TrwC Promotes Integration of Foreign DNA in the Human Genome

González-Prieto

Rinaldi

Dehio

et al. 2017

Appl Environ Microbiol

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“…S4). However, we have successfully cloned the gene, hyg, in the four transformants mentioned above, demonstrating that the knockout cassette was randomly inserted into the chromosome of these strains by off-target mutagenesis [ 36 ].…”

Section: Resultsmentioning

confidence: 99%

Constitutive hyperproduction of sorbicillinoids in Trichoderma reesei ZC121

Lin

Sun

et al. 2018

Biotechnol Biofuels

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BackgroundIn addition to its outstanding cellulase production ability, Trichoderma reesei produces a wide variety of valuable secondary metabolites, the production of which has not received much attention to date. Among them, sorbicillinoids, a large group of hexaketide secondary metabolites derived from polyketides, are drawing a growing interest from researchers because they exhibit a variety of important biological functions, including anticancer, antioxidant, antiviral, and antimicrobial properties. The development of fungi strains with constitutive, hyperproduction of sorbicillinoids is thus desired for future industry application but is not well-studied. Moreover, although T. reesei has been demonstrated to produce sorbicillinoids with the corresponding gene cluster and biosynthesis pathway proposed, the underlying molecular mechanism governing sorbicillinoid biosynthesis remains unknown.ResultsRecombinant T. reesei ZC121 was constructed from strain RUT-C30 by the insertion of the gene 12121-knockout cassette at the telomere of T. reesei chromosome IV in consideration of the off-target mutagenesis encountered during the unsuccessful deletion of gene 121121. Strain ZC121, when grown on cellulose, showed a sharp reduction of cellulase production, but yet a remarkable enhancement of sorbicillinoids production as compared to strain RUT-C30. The hyperproduction of sorbicillinoids is a constitutive process, independent of culture conditions such as carbon source, light, pH, and temperature. To the best of our knowledge, strain ZC121 displays record sorbicillinoid production levels when grown on both glucose and cellulose. Sorbicillinol and bisvertinolone are the two major sorbicillinoid compounds produced. ZC121 displayed a different morphology and markedly reduced sporulation compared to RUT-C30 but had a similar growth rate and biomass. Transcriptome analysis showed that most genes involved in cellulase production were downregulated significantly in ZC121 grown on cellulose, whereas remarkably all genes in the sorbicillinoid gene cluster were upregulated on both cellulose and glucose.ConclusionA constitutive sorbicillinoid-hyperproduction strain T. reesei ZC121 was obtained by off-target mutagenesis, displaying an overwhelming shift from cellulase production to sorbicillinoid production on cellulose, leading to a record for sorbicillinoid production. For the first time, T. reesei degraded cellulose to produce platform chemical compounds other than protein in high yield. We propose that the off-target mutagenesis occurring at the telomere region might cause chromosome remodeling and subsequently alter the cell structure and the global gene expression pattern of strain ZC121, as shown by phenotype profiling and comparative transcriptome analysis of ZC121. Overall, T. reesei ZC121 holds great promise for the industrial production of sorbicillinoids and serves as a good model to explore the regulation mechanism of sorbicillinoids’ biosynthesis.Electronic supplementary materialThe online version of this article (10....

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“…VirD2 precisely integrate T-DNA in the genome of yeast and mammalian cells [50][51][52] ; similarly, fusion of VirD2 with the homing endonuclease I-SceI increased the rate of targeted integration of T-DNA in yeast cells 39 . Therefore, we expect that our chimeric Cas9-VirD2 system will be equally adoptable across other eukaryotic species.…”

Section: Discussionmentioning

confidence: 98%

“…Our data show that the covalent binding of the repair-template to the bimodular protein (Cas9-VirD2) improved the HDR rate (up to 5.5-fold) in plant cells compared to the use of Cas9 alone. Previously an alternative approach of targeted insertion of the repair-template into the host genome via I-SceI-VirD2 fusion translocation from Agrobacterium was applied to engineer the yeast genome 39 . Similarly, Cas9-VirD2-sgRNA-T-DNA complex translocation would be worth trying in plants.…”

Section: Discussionmentioning

confidence: 99%

“…We preferred VirD2 over three other strategies used in mammalian cultured cells [24][25][26] because VirD2 requires only a 25-nt sequence for binding, and after covalently binding to the RB sequence, VirD2 only adds three extra nucleotides to the end of the repair-template 38 . Additionally, VirD2 naturally evolved with plants to engineer plant genomes and VirD2 bound T-DNAs with homologous ends or VirD2-meganuclease fusions bound to T-DNAs can enhance gene targeting via HDR 39 .…”

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confidence: 99%

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Fusion of the Cas9 endonuclease and the VirD2 relaxase facilitates homology-directed repair for precise genome engineering in rice

et al. 2020

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Precise genome editing by systems such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) requires high-efficiency homologydirected repair (HDR). Different technologies have been developed to improve HDR but with limited success. Here, we generated a fusion between the Cas9 endonuclease and the Agrobacterium VirD2 relaxase (Cas9-VirD2). This chimeric protein combines the functions of Cas9, which produces targeted and specific DNA double-strand breaks (DSBs), and the VirD2 relaxase, which brings the repair template in close proximity to the DSBs, to facilitate HDR. We successfully employed our Cas9-VirD2 system for precise ACETOLACTATE SYN-THASE (OsALS) allele modification to generate herbicide-resistant rice (Oryza sativa) plants, CAROTENOID CLEAVAGE DIOXYGENASE-7 (OsCCD7) to engineer plant architecture, and generate in-frame fusions with the HA epitope at HISTONE DEACETYLASE (OsHDT) locus. The Cas9-VirD2 system expands our ability to improve agriculturally important traits in crops and opens new possibilities for precision genome engineering across diverse eukaryotic species.

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Enhanced targeted integration mediated by translocated I-SceI during the Agrobacterium mediated transformation of yeast

Cited by 20 publications

References 40 publications

The Conjugative Relaxase TrwC Promotes Integration of Foreign DNA in the Human Genome

The Conjugative Relaxase TrwC Promotes Integration of Foreign DNA in the Human Genome

Constitutive hyperproduction of sorbicillinoids in Trichoderma reesei ZC121

Fusion of the Cas9 endonuclease and the VirD2 relaxase facilitates homology-directed repair for precise genome engineering in rice

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