Highly efficient gene targeting in Penicillium chrysogenum using the bi-partite approach in Δlig4 or Δku70 mutants

Boer, Paulo de; Bastiaans, Jeroen; Touw, Hesselien; Kerkman, Richard; Bronkhof, Jurian; Berg, Marco van den; Offringa, Remko

doi:10.1016/j.fgb.2010.07.008

Cited by 30 publications

(18 citation statements)

References 42 publications

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“…In addition, suppression of random integration of the GT vector due to Lig4 deficiency might have a positive effect on GT. In fact, it has been reported that suppression of Ku70/80 or Lig4 function can increase the frequency of GT in several organisms, such as fungi (de Boer et al, 2010;Ushimaru et al, 2010;Nakazawa et al, 2011), bacteria (Zhang et al, 2012), vertebrates (Bertolini et al, 2009;Fattah et al, 2008;Iiizumi et al, 2008), and plants (Tanaka et al, 2010;Jia et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

Biallelic Gene Targeting in Rice

2015

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ORCID IDs: 0000-0002-9199-181X (M.E.); 0000-0003-0066-6446 (S.T.).Sequence-specific nucleases (SSNs) have been used successfully in homology-directed repair (HDR)-mediated gene targeting (GT) in many organisms. However, break-induced GT in plants remains challenging due to inefficient delivery of HDR templates and SSNs into plant nuclei. In many plants, including rice, Agrobacterium-mediated transformation is the most practical means of transformation because this biotic transformation system can deliver longer and more intact DNA payloads with less incorporation of fragmented DNA compared with physical transformation systems such as polyethylene glycol, electroporation, or biolistics. Following infection with Agrobacterium, transfer of transfer DNA (T-DNA) to the nucleus and its integration into the plant genome occur consecutively during cocultivation, thus timing the induction of DNA doublestrand breaks (DSBs) on the target gene to coincide with the delivery of the HDR template is crucial. To synchronize DSB induction and delivery of the HDR template, we transformed a Cas9 expression construct and GT vector harboring the HDR template with guide RNAs (gRNAs) targeting the rice acetolactate synthase (ALS) gene either separately or sequentially into rice calli. When gRNAs targeting ALS were transcribed transiently from double-stranded T-DNA containing the HDR template, DSBs were induced in the ALS locus by the assembled Cas9/gRNA complex and homologous recombination was stimulated. Contrary to our expectations, there was no great difference in GT efficiency between Cas9-expressing and nonexpressing cells. However, when gRNA targeting DNA ligase 4 was transformed with Cas9 prior to the GT experiment, GT efficiency increased dramatically and more than one line exhibiting biallelic GT at the ALS locus was obtained.

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Section: Discussionmentioning

confidence: 99%

Biallelic Gene Targeting in Rice

2015

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“…Our data indicate that suppression of the NHEJ pathway is expected to cause an increase in the occurrence of sequence-specific integration by HR and inhibition of random integration by NHEJ, resulting in a synergistic effect that will improve GT experiments. In fact, it has been reported that deletion of the Ku or Lig4 gene can increase the frequency of GT in several organisms, such as fungi (de Boer et al ., 2010; Ushimaru et al ., 2010; Nakazawa et al ., 2011), bacteria (Zhang et al ., 2012) , birds (Iiizumi et al ., 2008), mammals (Bertolini et al ., 2009; Fattah et al ., 2008; Iiizumi et al ., 2008) and plants (Tanaka et al ., 2010; Jia et al ., 2012). We are progressing towards the establishment of a high-efficiency GT procedure using KD rice plants targeting NHEJ-related genes.…”

Section: Discussionmentioning

confidence: 99%

Suppression of Ku70/80 or Lig4 leads to decreased stable transformation and enhanced homologous recombination in rice

et al. 2012

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SummaryEvidence for the involvement of the nonhomologous end joining (NHEJ) pathway in Agrobacterium-mediated transferred DNA (T-DNA) integration into the genome of the model plant Arabidopsis remains inconclusive.Having established a rapid and highly efficient Agrobacterium-mediated transformation system in rice (Oryza sativa) using scutellum-derived calli, we examined here the involvement of the NHEJ pathway in Agrobacterium-mediated stable transformation in rice. Rice calli from OsKu70,OsKu80 and OsLig4 knockdown (KD) plants were infected with Agrobacterium harboring a sensitive emerald luciferase (LUC) reporter construct to evaluate stable expression and a green fluorescent protein (GFP) construct to monitor transient expression of T-DNA.Transient expression was not suppressed, but stable expression was reduced significantly, in KD plants. Furthermore, KD-Ku70 and KD-Lig4 calli exhibited an increase in the frequency of homologous recombination (HR) compared with control calli. In addition, suppression of OsKu70,OsKu80 and OsLig4 induced the expression of HR-related genes on treatment with DNA-damaging agents.Our findings suggest strongly that NHEJ is involved in Agrobacterium-mediated stable transformation in rice, and that there is a competitive and complementary relationship between the NHEJ and HR pathways for DNA double-strand break repair in rice.

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“…Since GT was significantly increased in NHEJ mutants in yeast and fungi [3][4][5][6][7][8][9], our NHEJ mutants of Arabidopsis thaliana were used to study their GT frequency. The plants were transformed using a repair T-DNA with an incomplete mutated PPO gene with homology to the plant genome ( Figure 6(a)) through Agrobacterium floral dip transformation.…”

Section: Gene Targetingmentioning

confidence: 99%

“…Genes involved in homologous recombination, like Rad51 and Rad52, are required for the targeted integration of T-DNA in yeast [3]. Transient or stable inactivation of the NHEJ pathways was shown to be a very efficient method to improve targeted integration in yeast and fungi [4][5][6][7][8][9]. Estimates of GT frequencies in several different plant species vary from 10 −4 to 10 −6 [10][11][12][13][14][15][16][17].…”

Section: Introductionmentioning

confidence: 99%

Agrobacterium tumefaciensT-DNA Integration and Gene Targeting inArabidopsis thalianaNon-Homologous End-Joining Mutants

et al. 2012

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In order to study the role of AtKu70 and AtKu80 inAgrobacterium-mediated transformation and gene targeting, plant lines with a T-DNA insertion inAtKu80orAtKu70genes were functionally characterized. Such plant lines lacked both subunits, indicating that heterodimer formation between AtKu70 and AtKu80 is needed for the stability of the proteins. Homozygous mutants were phenotypically indistinguishable from wild-type plants and were fertile. However, they were hypersensitive to the genotoxic agent bleomycin, resulting in more DSBs as quantified in comet assays. They had lower end-joining efficiency, suggesting that NHEJ is a critical pathway for DSB repair in plants. BothAtkumutants and a previously isolatedAtmre11mutant were impaired inAgrobacteriumT-DNA integration via floral dip transformation, indicating that AtKu70, AtKu80, and AtMre11 play an important role in T-DNA integration inArabidopsis. The frequency of gene targeting was not significantly increased in theAtku80andAtku70mutants, but it was increased at least 10-fold in theAtmre11mutant compared with the wild type.

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Highly efficient gene targeting in Penicillium chrysogenum using the bi-partite approach in Δlig4 or Δku70 mutants

Cited by 30 publications

References 42 publications

Biallelic Gene Targeting in Rice

Biallelic Gene Targeting in Rice

Suppression of Ku70/80 or Lig4 leads to decreased stable transformation and enhanced homologous recombination in rice

Agrobacterium tumefaciensT-DNA Integration and Gene Targeting inArabidopsis thalianaNon-Homologous End-Joining Mutants

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