scholarly journals Efficient in planta gene targeting in tomato using geminiviral replicons and the CRISPR/Cas9 system

2018 ◽  
Vol 95 (1) ◽  
pp. 5-16 ◽  
Author(s):  
Tal Dahan-Meir ◽  
Shdema Filler-Hayut ◽  
Cathy Melamed-Bessudo ◽  
Samuel Bocobza ◽  
Henryk Czosnek ◽  
...  
Keyword(s):  
Gene Targeting ◽  
In Planta

scholarly journals Homology-directed repair of a defective glabrous gene in Arabidopsis with Cas9-based gene targeting

2018 ◽  
Author(s):  
Florian Hahn ◽  
Marion Eisenhut ◽  
Otho Mantegazza ◽  
Andreas P.M. Weber
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Strand Break ◽  
Double Strand Break ◽  
In Planta ◽  
End Joining ◽  
Trichome Formation ◽  
Break Site ◽  
Repair Template ◽  
Non Homologous End Joining

ABSTRACTThe CRISPR/Cas9 system has emerged as a powerful tool for targeted genome editing in plants and beyond. Double-strand breaks induced by the Cas9 enzyme are repaired by the cell’s own repair machinery either by the non-homologous end joining pathway or by homologous recombination. While the first repair mechanism results in random mutations at the double-strand break site, homologous recombination uses the genetic information from a highly homologous repair template as blueprint for repair of the break. By offering an artificial repair template, this pathway can be exploited to introduce specific changes at a site of choice in the genome. However, frequencies of double-strand break repair by homologous recombination are very low. In this study, we compared two methods that have been reported to enhance frequencies of homologous recombination in plants. The first method boosts the repair template availability through the formation of viral replicons, the second method makes use of an in planta gene targeting approach. Additionally, we comparatively applied a nickase instead of a nuclease for target strand priming. To allow easy, visual detection of homologous recombination events, we aimed at restoring trichome formation in a glabrous Arabidopsis mutant by repairing a defective glabrous1 gene. Using this efficient visual marker, we were able to regenerate plants repaired by homologous recombination at frequencies of 0.12% using the in planta gene targeting approach, while both approaches using viral replicons did not yield any trichome-bearing plants.

scholarly journals In planta gene targeting can be enhanced by the use of CRISPR/Cas12a

2019 ◽  
Vol 100 (5) ◽  
pp. 1083-1094 ◽  
Author(s):  
Felix Wolter ◽  
Holger Puchta
Keyword(s):  
Gene Targeting ◽  
In Planta

scholarly journals In-planta Gene Targeting in Barley using Cas9, with and without Geminiviral Replicons

2021 ◽  
Author(s):  
Tom Lawrenson ◽  
Alison Hinchliffe ◽  
Martha Clarke ◽  
Yvie Morgan ◽  
Wendy Harwood
Keyword(s):  
Genome Editing ◽  
Gene Targeting ◽  
Target Gene ◽  
In Planta ◽  
Crop Species ◽  
Increase Copy Number ◽  
Independent Gene ◽  
Copy Numbers ◽  
Wide Range ◽  
Repair Template

AbstractAdvances in the use of RNA-guided Cas9-based genome editing in plants have been rapid over the last few years. A desirable application of genome editing is gene targeting (GT), as it allows a wide range of precise modifications, however this remains inefficient especially in key crop species. Here we describe successful, heritable gene targeting in barley using an in-planta strategy but fail to achieve the same using a wheat dwarf virus replicon to increase copy number of the repair template. Without the replicon, we were able to delete 150bp of the coding sequence of our target gene whilst simultaneously fusing in-frame mCherry in its place. Starting from 14 original transgenic plants, two plants appeared to have the required gene targeting event. From one of these T0 plants, three independent gene targeting events were identified, two of which were heritable. When the replicon was included, 39 T0 plants were produced and shown to have high copy numbers of the repair template. However, none of the 17 lines screened in T1 gave rise to significant or heritable gene targeting events despite screening twice the number of plants in T1 compared to the non-replicon strategy. Investigation indicated that high copy numbers of repair template created by the replicon approach cause false positive PCR results which are indistinguishable at the sequence level to true GT events in junction PCR screens widely used in GT studies. In the successful non-replicon approach, heritable gene targeting events were obtained in T1 and subsequently the T-DNA was found to be linked to the targeted locus. Thus, physical proximity of target and donor sites may be a factor in successful gene targeting.

CRISPR/Cas-Mediated In Planta Gene Targeting

2017 ◽  
pp. 3-11 ◽  
Author(s):  
Simon Schiml ◽  
Friedrich Fauser ◽  
Holger Puchta
Keyword(s):  
Gene Targeting ◽  
In Planta

scholarly journals In-planta Gene Targeting in Barley Using Cas9 With and Without Geminiviral Replicons

2021 ◽  
Vol 3 ◽  
Author(s):  
Tom Lawrenson ◽  
Alison Hinchliffe ◽  
Martha Clarke ◽  
Yvie Morgan ◽  
Wendy Harwood
Keyword(s):  
Genome Editing ◽  
Gene Targeting ◽  
Target Gene ◽  
Wheat Dwarf Virus ◽  
In Planta ◽  
Crop Species ◽  
Independent Gene ◽  
Copy Numbers ◽  
Wide Range ◽  
Repair Template

Advances in the use of RNA-guided Cas9-based genome editing in plants have been rapid over the last few years. A desirable application of genome editing is gene targeting (GT), as it allows a wide range of precise modifications; however, this remains inefficient especially in key crop species. Here, we describe successful, heritable gene targeting in barley at the target site of Cas9 using an in-planta strategy but fail to achieve the same using a wheat dwarf virus replicon to increase the copy number of the repair template. Without the replicon, we were able to delete 150 bp of the coding sequence of our target gene whilst simultaneously fusing in-frame mCherry in its place. Starting from 14 original transgenic plants, two plants appeared to have the required gene targeting event. From one of these T0 plants, three independent gene targeting events were identified, two of which were heritable. When the replicon was included, 39 T0 plants were produced and shown to have high copy numbers of the repair template. However, none of the 17 lines screened in T1 gave rise to significant or heritable gene targeting events despite screening twice the number of plants in T1 compared with the non-replicon strategy. Investigation indicated that high copy numbers of repair template created by the replicon approach cause false-positive PCR results which are indistinguishable at the sequence level to true GT events in junction PCR screens widely used in GT studies. In the successful non-replicon approach, heritable gene targeting events were obtained in T1, and subsequently, the T-DNA was found to be linked to the targeted locus. Thus, physical proximity of target and donor sites may be a factor in successful gene targeting.

scholarly journals Using CRISPR/tt Lb Cas12a for in planta Gene Targeting in A. thaliana

2020 ◽  
Vol 5 (3) ◽  
Author(s):  
Laura Merker ◽  
Patrick Schindele ◽  
Holger Puchta
Keyword(s):  
Gene Targeting ◽  
In Planta

scholarly journals Enhancing in planta gene targeting efficiencies in Arabidopsis using temperature‐tolerant CRISPR/ Lb Cas12a

2020 ◽  
Vol 18 (12) ◽  
pp. 2382-2384 ◽  
Author(s):  
Laura Merker ◽  
Patrick Schindele ◽  
Teng‐Kuei Huang ◽  
Felix Wolter ◽  
Holger Puchta
Keyword(s):  
Gene Targeting ◽  
In Planta

scholarly journals In planta gene targeting

2012 ◽  
Vol 109 (19) ◽  
pp. 7535-7540 ◽  
Author(s):  
F. Fauser ◽  
N. Roth ◽  
M. Pacher ◽  
G. Ilg ◽  
R. Sanchez-Fernandez ◽  
...  
Keyword(s):  
Gene Targeting ◽  
In Planta

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.

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