CRISPR/Cas9 – mediated genome editing of bread wheat to modulate heading time

This study was conducted during two crop seasons to investigate the effect of nitrogen fertilizer quantity and application form on grain yield and quality in Eskisehir Transitional Zone Agricultural Research Institute by using seven bread wheat cultivars in rainfed and eight in irrigated conditions. In the study, urea as a source of nitrogen was applied in heading time (Zadoks 59) in granular and foliar form. In all treatments grain yield, some technological quality characteristics were determined of the genotypes. In both conditions while sedimentation value, protein content, aggregation energy (AGGEN) and maximum torque (BEM) values increased, peak maximum time (PMT) decreased with foliar urea applications. it was determined that there were statistically significant differences between the varieties in terms of SDS sedimentation values in both rainfed and irrigated conditions. In this study, In irrigated and rainfed conditions the values obtained from foliar urea applications compared to granular ones with relation higher bread quality in the genotypes which contain Glu-A1 locus 2* subunit; 9% for grain yield, 6.5% for protein content, 17.0% for sedimentation value and 3.8% increase of BEM, Glu-D1 locus; 5+10 subunit 8% for grain yield, 5.6% for protein content, sedimentation 16.0%, 6.4% for BEM increase, for Glu-B1 locus; 7+9 subunit 7.0% for grain yield, 6.0% for protein content, 19.6% for sedimentation value, and 7.2% for BEM value increase. This study was observed that the foliar application during the heading time provided the improvement of bread wheat grain yield and quality characteristics.

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Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes

Nature Communications ◽

10.1038/ncomms14261 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 313

Author(s):

Zhen Liang ◽

Kunling Chen ◽

Tingdong Li ◽

Yi Zhang ◽

Yanpeng Wang ◽

...

Keyword(s):

Genome Editing ◽

Bread Wheat ◽

Deep Sequencing ◽

Crop Plants ◽

Good Prospect ◽

Crop Breeding ◽

Transgene Integration ◽

Ribonucleoprotein Complexes ◽

Wheat Embryos ◽

Foreign Dna

Abstract Substantial efforts are being made to optimize the CRISPR/Cas9 system for precision crop breeding. The avoidance of transgene integration and reduction of off-target mutations are the most important targets for optimization. Here, we describe an efficient genome editing method for bread wheat using CRISPR/Cas9 ribonucleoproteins (RNPs). Starting from RNP preparation, the whole protocol takes only seven to nine weeks, with four to five independent mutants produced from 100 immature wheat embryos. Deep sequencing reveals that the chance of off-target mutations in wheat cells is much lower in RNP mediated genome editing than in editing with CRISPR/Cas9 DNA. Consistent with this finding, no off-target mutations are detected in the mutant plants. Because no foreign DNA is used in CRISPR/Cas9 RNP mediated genome editing, the mutants obtained are completely transgene free. This method may be widely applicable for producing genome edited crop plants and has a good prospect of being commercialized.

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An optimised CRISPR/Cas9 protocol to create targeted mutations in homoeologous genes and an efficient genotyping protocol to identify edited events in wheat

Plant Methods ◽

10.1186/s13007-019-0500-2 ◽

2019 ◽

Vol 15 (1) ◽

Cited By ~ 9

Author(s):

Xiucheng Cui ◽

Margaret Balcerzak ◽

Johann Schernthaner ◽

Vivijan Babic ◽

Raju Datla ◽

...

Keyword(s):

Genome Editing ◽

Bread Wheat ◽

Plant Species ◽

Transgenic Wheat ◽

Comparative Assessment ◽

Guide Rna ◽

Large Deletions ◽

Identification And Characterization ◽

Number Of Plant Species

Abstract Background Targeted genome editing using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has been applied in a large number of plant species. Using a gene-specific single guide RNA (sgRNA) and the CRISPR/Cas9 system, small editing events such as deletions of few bases can be obtained. However larger deletions are required for some applications. In addition, identification and characterization of edited events can be challenging in plants with complex genomes, such as wheat. Results In this study, we used the CRISPR/Cas9 system and developed a protocol that yielded high number of large deletions employing a pair of co-expressed sgRNA to target the same gene. The protocol was validated by targeting three genes, TaABCC6, TaNFXL1 and TansLTP9.4 in a wheat protoplast assay. Deletions of sequences located between the two sgRNA in each gene were the most frequent editing events observed for two of the three genes. A comparative assessment of editing frequencies between a codon-optimized Cas9 for expression in algae, crCas9, and a plant codon-optimized Cas9, pcoCas9, showed more consistent results with the vector expressing pcoCas9. Editing of TaNFXL1 by co-expression of sgRNA pair was investigated in transgenic wheat plants. Given the ploidy of bread wheat, a rapid, robust and inexpensive genotyping protocol was also adapted for hexaploid genomes and shown to be a useful tool to identify homoeolog-specific editing events in wheat. Conclusions Co-expressed pairs of sgRNA targeting single genes in conjunction with the CRISPR/Cas9 system produced large deletions in wheat. In addition, a genotyping protocol to identify editing events in homoeologs of TaNFXL1 was successfully adapted.

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Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA

Nature Communications ◽

10.1038/ncomms12617 ◽

2016 ◽

Vol 7 (1) ◽

Cited By ~ 306

Author(s):

Yi Zhang ◽

Zhen Liang ◽

Yuan Zong ◽

Yanpeng Wang ◽

Jinxing Liu ◽

...

Keyword(s):

Durum Wheat ◽

Genome Editing ◽

Bread Wheat ◽

Transient Expression ◽

Plant Species ◽

Genome Engineering ◽

Plant Genome ◽

Engineering Research ◽

Plant Genomes ◽

Highly Efficient

Abstract Editing plant genomes is technically challenging in hard-to-transform plants and usually involves transgenic intermediates, which causes regulatory concerns. Here we report two simple and efficient genome-editing methods in which plants are regenerated from callus cells transiently expressing CRISPR/Cas9 introduced as DNA or RNA. This transient expression-based genome-editing system is highly efficient and specific for producing transgene-free and homozygous wheat mutants in the T0 generation. We demonstrate our protocol to edit genes in hexaploid bread wheat and tetraploid durum wheat, and show that we are able to generate mutants with no detectable transgenes. Our methods may be applicable to other plant species, thus offering the potential to accelerate basic and applied plant genome-engineering research.

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