scholarly journals Efficient Gene Targeting in Maize using Inducible CRISPR-Cas9 and Marker-Free Donor Template

2020 ◽  
Author(s):  
Pierluigi Barone ◽  
Emily Wu ◽  
Brian Lenderts ◽  
Ajith Anand ◽  
William Gordon-Kamm ◽  
...  
Keyword(s):  
Gene Targeting ◽  
Selectable Marker ◽  
Marker Gene ◽  
In Planta ◽  
Genomic Deletions ◽  
Efficient Gene ◽  
Marker Free ◽  
Genomic Gene ◽  
Donor Locus ◽  
Donor Template

AbstractCRISPR-Cas9 is a powerful tool for generating targeted mutations and genomic deletions. However, precise gene insertion or sequence replacement remains a major hurdle before application of CRISPR-Cas9 technology is fully realized in plant breeding. Here we report high frequency, selectable marker-free intra-genomic gene targeting (GT) in maize. Heat shock-inducible Cas9 was used for generating targeted double-strand breaks (DSBs) and simultaneous mobilization of the donor template from pre-integrated T-DNA. The construct was designed such that release of the donor template and subsequent DNA repair activated expression of the selectable marker gene within the donor locus. This approach generated up to 4.7% targeted insertion of the donor sequence into the target locus in T0 plants, with up to 86% detected donor template release and 99% mutation rate were observed at the donor loci and the genomic target site, respectively. Unlike previous in planta or intra-genomic homologous recombination reports, that required multiple generations and extensive screening, our method provides non-chimeric, heritable GT in the T0 generation.

scholarly journals Efficient, selectable marker free gene targeting in soybean using novel Ochrobactrum haywardense-mediated delivery

2021 ◽  
Author(s):  
Sandeep Kumar ◽  
Zhan-Bin Lui ◽  
Nathalie Sanyour-Doyel ◽  
Brian Landerts ◽  
Andrew Worden ◽  
...  
Keyword(s):  
High Resolution ◽  
Gene Targeting ◽  
Plasmid Dna ◽  
Selectable Marker ◽  
Low Frequency ◽  
Transformation Method ◽  
Dna Templates ◽  
Marker Free ◽  
Free Gene ◽  
Donor Template

We report robust selectable marker-free gene targeting (GT) system in soybean, one of the most economically important crops. A novel efficient Ochrobactrum haywardense-mediated embryonic axis transformation method was used for the delivery of CRISPR-Cas9 components and donor template to regenerate T0 plants in 6-8 weeks after transformation. This approach generated up to 3.4% targeted insertion of the donor sequence into the target locus in T0 plants, with ~ 90% mutation rate observed at the genomic target site. The GT was demonstrated in two genomic sites using two different donor DNA templates without a need of a selectable marker within the template. High-resolution Southern by Sequencing (SbS) analysis identified T1 plants with precise targeted insertion and without unintended plasmid DNA. Unlike previous low-frequency GT reports in soybean that involved particle bombardment-mediated delivery and extensive selection, the method described here is fast, efficient, reproducible, does not require selectable marker within the donor DNA, and generates non-chimeric plants with heritable GT.

scholarly journals Efficient Gene Targeting in Maize Using Inducible CRISPR-Cas9 and Marker-free Donor Template

2020 ◽  
Vol 13 (8) ◽  
pp. 1219-1227 ◽  
Author(s):  
Pierluigi Barone ◽  
Emily Wu ◽  
Brian Lenderts ◽  
Ajith Anand ◽  
William Gordon-Kamm ◽  
...  
Keyword(s):  
Gene Targeting ◽  
Efficient Gene ◽  
Marker Free ◽  
Donor Template

scholarly journals Inheritance of NmDef02 synthetic defensin gene and hpt selectable marker in four successive generations of transgenic rice (Oryza sativa, cv. J-104)

2021 ◽  
Vol 8 (9) ◽  
pp. 34-41
Author(s):  
Maylin Pérez-Bernal ◽  
Magali Delgado ◽  
Daymí Abreu ◽  
Onel Valdivia ◽  
Raúl Armas
Keyword(s):  
Transgenic Rice ◽  
Selectable Marker ◽  
Marker Gene ◽  
Rice Cultivar ◽  
Future Research ◽  
Maturation Stage ◽  
Defensin Gene ◽  
Regenerated Plants ◽  
Marker Free ◽  
Successive Generations

The evaluation of the inheritance and stability of the transgenes is essential for the application of transgenic plants in agriculture. In this work, we studied the inheritance of two transgenes in T1, T2, T3 and T4 rice progenies. Transgenic rice plants (cv. J-104) was obtained by biolistic method using a synthetic defensin gene (NmDef02) and hpt as selectable marker gene, co-transformed in a 4:1 proportion, respectively. Regenerated plants were acclimated under natural conditions. The study started from the primary transformants that fulfilled the agronomic characters reported by the experts for the J-104 rice cultivar in the maturation stage. The integration and relative expression of NmDef02 in T1 plants was verified by Southern blot and qRT-PCR, respectively. The inheritance of transgenes over four generations was analyzed by PCR. The following transgene combinations were identified: NmDef02(+)hpt(+), NmDef02(+)hpt(-) and NmDef02(-)hpt(-). The most advantageous combination was NmDef02(+)hpt(-), which corresponded to the marker-free plants harboring the gene of interest. The inheritance of NmDef02 was confirmed in T1 and T2 progenies, but in some T3 and T4 lines the loss of this gene was verified. This inheritance analysis provided information concerning the transgenic population, but the mechanisms that destabilize the inheritance of the gene of interest will be the goal of future research.

Excision of a selectable marker in transgenic lily (Sorbonne) using the Cre/loxP DNA excision system

2013 ◽  
Vol 93 (5) ◽  
pp. 903-912
Author(s):  
Sh. Li ◽  
Y.-P. Du ◽  
Zh.-Y. Wu ◽  
C.-L. Huang ◽  
X.-H. Zhang ◽  
...  
Keyword(s):  
Southern Blotting ◽  
Molecular Detection ◽  
Selectable Marker ◽  
Marker Gene ◽  
Inducible Promoter ◽  
Transformation Rate ◽  
Site Specific ◽  
Marker Free ◽  
Drought And Salt Stresses ◽  
Excision Rate

Li, S. H., Du, Y.-P., Wu, Z. H.-Y., Huang, C.-L., Zhang, X.-H., Wang, Z. H.-X. and Jia, G.-X. 2013. Excision of a selectable marker in transgenic lily (Sorbonne) using the Cre/loxP DNA excision system. Can. J. Plant Sci. 93: 903–912. To generate transgenic lily plants with no selectable marker and improved tolerance to abiotic stress, two vectors were co-transformed into the Lilium oriental hybrid Sorbonne by particle bombardment. The pKSB vector included the Cre/loxp-mediated site-specific cDNA excision system under control of the inducible promoter rd29A, and the pBPC-P5CS-F129A vector carried the P5CS gene, which we hypothesized would improve resistance to drought and salt stresses in transgenic lily plantlets. The presence of the two genes was simultaneously detected by PCR and Southern blotting in two resistant plantlets. The co-transformation rate was 0.16%. Subsequently, inducer expression was tested under varying conditions to optimize the deletion of marker gene. Results from molecular detection assays revealed that maintaining bases of bulblet scales at 4°C for 12 h resulted in an increase in the excision rate, reaching 60%. Expression of P5CS improved resistance to salt stress in transgenic lily plantlets. These results demonstrated the feasibility of using the Cre/loxP-based marker elimination system to generate marker-free transgenic plantlets with improved stress tolerance.

Generation of selectable marker-free transgenic rice using double right-border (DRB) binary vectors

2001 ◽  
Vol 28 (3) ◽  
pp. 241 ◽  
Author(s):  
Hui-Juan Lu ◽  
Xue-Rong Zhou ◽  
Zhu-Xun Gong ◽  
Narayana M. Upadhyaya
Keyword(s):  
Selectable Marker ◽  
Binary Vector ◽  
Marker Gene ◽  
Marker Genes ◽  
Binary Vectors ◽  
Selectable Marker Genes ◽  
Right Border ◽  
Marker Free ◽  
Dna Vectors ◽  
Transgenic Progeny

Currently employed transformation systems require selectable marker genes encoding antibiotic or herbicide resistance, along with the gene of interest (GOI), to select transformed cells from among a large population of untransformed cells. The continued presence of these selectable markers, especially in food crops such as rice (Oryza sativa L.), is of increasing public concern. Techniques based on DNA recombination and Agrobacterium-mediated co-transformation with two binary vectors in a single or two different Agrobacterium strains, or with super-binary vectors carrying two sets of T-DNA border sequences (twin T-DNA vectors), have been employed by researchers to produce selectable marker-free (SMF) transgenic progeny. We have developed a double right-border (DRB) binary vector carrying two copies of T-DNA right-border (RB) sequences flanking a selectable marker gene, followed by a GOI and one copy of the left border sequence. Two types of T-DNA inserts, one initiated from the first RB containing both the selectable gene and the GOI, and the other from the second RB containing only the GOI, were expected to be produced and integrated into the genome. In the subsequent generation, these inserts could segregate away from each other, allowing the selection of the progeny with only the GOI. We tested this vector using two selectable marker genes and successfully obtained progeny plants in which the second selectable marker gene segregated away from the first. Using the DRB binary vector system, we recovered SMF transgenic lines containing a rice ragged stunt virus (RRSV)-derived synthetic resistance gene in the rice cultivars Jarrah and Xiu Shui. Approximately 36–64% of the primary transformants of these cultivars yielded SMF progeny. Among SMF Jarrah transgenic progeny <50% of plants contained the RRSV transgene. Thus, we have developed an efficient vector for producing SMF plants that allows straightforward cloning of any GOIs in comparison with the published ‘twin T-DNA’ vectors.

In planta removal of nptII selectable marker gene from transgenic tobacco plants using CRISPR/Cas9 system

Plant Gene ◽  
2021 ◽  
Vol 26 ◽  
pp. 100288
Author(s):  
Arsalan Rezaei ◽  
Mohammad Farsi ◽  
Saeid Malekzadeh-Shafaroudi ◽  
Alireza Seifi
Keyword(s):  
Transgenic Tobacco ◽  
Selectable Marker ◽  
Marker Gene ◽  
Transgenic Tobacco Plants ◽  
Selectable Marker Gene ◽  
In Planta ◽  
Tobacco Plants

scholarly journals Production of selectable marker gene-free Cavendish banana (Musa spp.) using a steroid-inducible recombinase platform

2019 ◽  
Vol 29 (1) ◽  
pp. 81-93 ◽  
Author(s):  
Jennifer Kleidon ◽  
Anthony Brinin ◽  
Jean-Yves Paul ◽  
Robert Harding ◽  
James Dale ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Selectable Marker ◽  
Marker Gene ◽  
Single Copy ◽  
Selectable Marker Gene ◽  
New Genes ◽  
Cavendish Banana ◽  
Marker Free ◽  
Green Fluorescent ◽  
Regulatory Acceptance

Abstract Genetic improvement of commercially accepted banana cultivars is strongly reliant on the ability to introduce genes that encode important agro-traits such as disease resistance. In most cases this can only be achieved using a transgenic approach. Public and regulatory acceptance of these events would greatly increase with “clean” single copy integration events free of the selectable marker gene and extraneous vector backbone. This would also allow for the successive addition of new genes and traits as they become available. In this study, we used the pMarker Free 1 (pMF1) vector containing the green fluorescent protein (gfp) reporter gene to assess the effectiveness of steroid-inducible recombination and positive/negative dual selection to regenerate transgenic Cavendish banana plants that were potentially free of the selectable marker gene. By examining the interaction of two different Agrobacterium strains with two different cultivars of Cavendish banana, namely Williams and Grand Naine, we describe a transformation and regeneration strategy that successfully produced marker-free, single transgene copy, gfp-expressing events. The system will provide a useful means of serially improving banana into the future.

scholarly journals Construction of Marker-Free Genetically Modified Maize Using a Heat-Inducible Auto-Excision Vector

Genes ◽  
2019 ◽  
Vol 10 (5) ◽  
pp. 374 ◽  
Author(s):  
Dengxiang Du ◽  
Ruchang Jin ◽  
Jinjie Guo ◽  
Fangdong Zhang
Keyword(s):  
Transgenic Plants ◽  
Selectable Marker ◽  
Marker Gene ◽  
Transformation Process ◽  
Transgenic Maize ◽  
Vector System ◽  
Marker Genes ◽  
Site Specific ◽  
Selectable Marker Genes ◽  
Marker Free

Gene modification is a promising tool for plant breeding, and gradual application from the laboratory to the field. Selectable marker genes (SMG) are required in the transformation process to simplify the identification of transgenic plants; however, it is more desirable to obtain transgenic plants without selection markers. Transgene integration mediated by site-specific recombination (SSR) systems into the dedicated genomic sites has been demonstrated in a few different plant species. Here, we present an auto-elimination vector system that uses a heat-inducible Cre to eliminate the selectable marker from transgenic maize, without the need for repeated transformation or sexual crossing. The vector combines an inducible site-specific recombinase (hsp70::Cre) that allows for the precise elimination of the selectable marker gene egfp upon heating. This marker gene is used for the initial positive selection of transgenic tissue. The egfp also functions as a visual marker to demonstrate the effectiveness of the heat-inducible Cre. A second marker gene for anthocyanin pigmentation (Rsc) is located outside of the region eliminated by Cre and is used for the identification of transgenic offspring in future generations. Using the heat-inducible auto-excision vector, marker-free transgenic maize plants were obtained in a precisely controlled genetic modification process. Genetic and molecular analyses indicated that the inducible auto-excision system was tightly controlled, with highly efficient DNA excision, and provided a highly reliable method to generate marker-free transgenic maize.

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