CRISPR/Cas9-Mediated Multiplex Genome Editing of the BnWRKY11 and BnWRKY70 Genes in Brassica napus L.

Targeted genome editing is a desirable means of basic science and crop improvement. The clustered, regularly interspaced, palindromic repeat (CRISPR)/Cas9 (CRISPR-associated 9) system is currently the simplest and most commonly used system in targeted genomic editing in plants. Single and multiplex genome editing in plants can be achieved under this system. In Arabidopsis, AtWRKY11 and AtWRKY70 genes were involved in JA- and SA-induced resistance to pathogens, in rapeseed (Brassica napus L.), BnWRKY11 and BnWRKY70 genes were found to be differently expressed after inoculated with the pathogenic fungus, Sclerotinia sclerotiorum (Lib.) de Bary. In this study, two Cas9/sgRNA constructs targeting two copies of BnWRKY11 and four copies of BnWRKY70 were designed to generate BnWRKY11 and BnWRKY70 mutants respectively. As a result, twenty-two BnWRKY11 and eight BnWRKY70 independent transformants (T0) were obtained, with the mutation ratios of 54.5% (12/22) and 50% (4/8) in BnWRKY11 and BnWRKY70 transformants respectively. Eight and two plants with two copies of mutated BnWRKY11 and BnWRKY70 were obtained respectively. In T1 generation of each plant examined, new mutations on target genes were detected with high efficiency. The vast majority of BnWRKY70 mutants showed editing in three copies of BnWRKY70 in examined T1 plants. BnWRKY70 mutants exhibited enhanced resistance to Sclerotinia, while BnWRKY11 mutants showed no significant difference in Sclerotinia resistance when compared to non-transgenic plants. In addition, plants that overexpressed BnWRKY70 showed increased sensitivity when compared to non-transgenic plants. Altogether, our results demonstrated that BnWRKY70 may function as a regulating factor to negatively control the Sclerotinia resistance and CRISPR/Cas9 system could be used to generate germplasm in B. napus with high resistance against Sclerotinia.

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Highly efficient generation of canker resistant sweet orange enabled by an improved CRISPR/Cas9 system

10.1101/2021.10.20.465103 ◽

2021 ◽

Author(s):

Xiaoen Huang ◽

Nian Wang

Keyword(s):

Genome Editing ◽

Gene Editing ◽

Target Genes ◽

High Efficiency ◽

Sweet Orange ◽

Susceptibility Gene ◽

Transformation Rate ◽

Biallelic Mutation ◽

Important Species ◽

Citrus Production

Sweet orange (Citrus sinensis) is the most economically important species for the citrus industry. However, it is susceptible to many diseases including citrus bacterial canker caused by Xanthomonas citri subsp. citri (Xcc) that triggers devastating effects on citrus production. Conventional breeding has not met the challenge to improve disease resistance of sweet orange due to the long juvenility and other limitations. CRISPR-mediated genome editing has shown promising potentials for genetic improvements of plants. Generation of biallelic/homozygous mutants remains difficult for sweet orange due to low transformation rate, existence of heterozygous alleles for target genes and low biallelic editing efficacy using the CRISPR technology. Here, we report improvements in the CRISPR/Cas9 system for citrus gene editing. Based on the improvements we made previously (dicot codon optimized Cas9, tRNA for multiplexing, a modified sgRNA scaffold with high efficiency, CsU6 to drive sgRNA expression), we further improved our CRISPR/Cas9 system by choosing superior promoters (CmYLCV or CsUbi promoter) to drive Cas9 and optimizing culture temperature. This system was able to generate a biallelic mutation rate of up to 89% for Carrizo citrange and 79% for Hamlin sweet orange. Consequently, this system was used to generate canker resistant Hamlin sweet orange by mutating the effector binding element (EBE) of canker susceptibility gene CsLOB1, which is required for causing canker symptoms by Xcc. Six biallelic Hamlin sweet orange mutant lines in the EBE were generated. The biallelic mutants are resistant to Xcc. Biallelic mutation of the EBE region abolishes the induction of CsLOB1 by Xcc. This study represents a significant improvement in sweet orange gene editing efficacy and generating disease resistant varieties via CRISPR-mediated genome editing. This improvement in citrus genome editing makes genetic studies and manipulations of sweet orange more feasible.

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Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system

10.1101/734442 ◽

2019 ◽

Cited By ~ 2

Author(s):

Remi L. Gratacap ◽

Tim Regan ◽

Carola E. Dehler ◽

Samuel A.M. Martin ◽

Pierre Boudinot ◽

...

Keyword(s):

Disease Resistance ◽

Cell Line ◽

Genome Editing ◽

Target Genes ◽

High Efficiency ◽

Genetic Resistance ◽

Model Organisms ◽

Fish Cell ◽

Genome Wide ◽

Lentivirus Transduction

1AbstractGenome editing is transforming bioscience research, but its application to non-model organisms, such as farmed animal species, requires optimisation. Salmonids are the most important aquaculture species by value, and improving genetic resistance to infectious disease is a major goal. However, use of genome editing to evaluate putative disease resistance genes in cell lines, and the use of genome-wide CRISPR screens is currently limited by a lack of available tools and techniques. In the current study, an optimised protocol using lentivirus transduction for efficient integration of constructs into the genome of a Chinook salmon (Oncorhynchus tshwaytcha) cell line (CHSE-214) was developed. As proof-of-principle, two target genes were edited with high efficiency in an EGFP-Cas9 stable CHSE cell line; specifically, the exogenous, integrated EGFP and the endogenous RIG-I locus. Finally, the effective use of antibiotic selection to enrich the successfully edited targeted population was demonstrated. The optimised lentiviral-mediated CRISPR method reported here increases possibilities for efficient genome editing in salmonid cells, in particular for future applications of genome-wide CRISPR screens for disease resistance.

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Precision Genome Engineering Through Cytidine Base Editing in Rapeseed (Brassica napus. L)

Frontiers in Genome Editing ◽

10.3389/fgeed.2020.605768 ◽

2020 ◽

Vol 2 ◽

Author(s):

Limin Hu ◽

Olalekan Amoo ◽

Qianqian Liu ◽

Shengli Cai ◽

Miaoshan Zhu ◽

...

Keyword(s):

Genome Engineering ◽

Molecular Breeding ◽

Target Genes ◽

Agronomic Traits ◽

Crop Improvement ◽

Brassica Napus L ◽

Editing Efficiency ◽

Base Editing ◽

Expected Gain ◽

Genome Wide

Rapeseed is one of the world's most important sources of oilseed crops. Single nucleotide substitution is the basis of most genetic variation underpinning important agronomic traits. Therefore, genome-wide and target-specific base editing will greatly facilitate precision plant molecular breeding. In this study, four CBE systems (BnPBE, BnA3A-PBE, BnA3A1-PBE, and BnPBGE14) were modified to achieve cytidine base editing at five target genes in rapeseed. The results indicated that genome editing is achievable in three CBEs systems, among which BnA3A1-PBE had the highest base-editing efficiency (average 29.8% and up to 50.5%) compared to all previous CBEs reported in rapeseed. The editing efficiency of BnA3A1-PBE is ~8.0% and fourfold higher, than those of BnA3A-PBE (averaging 27.6%) and BnPBE (averaging 6.5%), respectively. Moreover, BnA3A1-PBE and BnA3A-PBE could significantly increase the proportion of both the homozygous and biallelic genotypes, and also broaden the editing window compared to BnPBE. The cytidine substitution which occurred at the target sites of both BnaA06.RGA and BnaALS were stably inherited and conferred expected gain-of-function phenotype in the T1 generation (i.e., dwarf phenotype or herbicide resistance for weed control, respectively). Moreover, new alleles or epialleles with expected phenotype were also produced, which served as an important resource for crop improvement. Thus, the improved CBE system in the present study, BnA3A1-PBE, represents a powerful base editor for both gene function studies and molecular breeding in rapeseed.

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Genome Editing in Cereals: Approaches, Applications and Challenges

International Journal of Molecular Sciences ◽

10.3390/ijms21114040 ◽

2020 ◽

Vol 21 (11) ◽

pp. 4040 ◽

Cited By ~ 4

Author(s):

Waquar A. Ansari ◽

Sonali U. Chandanshive ◽

Vacha Bhatt ◽

Altafhusain B. Nadaf ◽

Sanskriti Vats ◽

...

Keyword(s):

Genome Editing ◽

Target Genes ◽

Crop Improvement ◽

Whole Genome Sequence ◽

Cereal Crops ◽

Sequence Information ◽

World Population ◽

Base Editing ◽

Crop Varieties ◽

Current Scenario

Over the past decades, numerous efforts were made towards the improvement of cereal crops mostly employing traditional or molecular breeding approaches. The current scenario made it possible to efficiently explore molecular understanding by targeting different genes to achieve desirable plants. To provide guaranteed food security for the rising world population particularly under vulnerable climatic condition, development of high yielding stress tolerant crops is needed. In this regard, technologies upgradation in the field of genome editing looks promising. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 is a rapidly growing genome editing technique being effectively applied in different organisms, that includes both model and crop plants. In recent times CRISPR/Cas9 is being considered as a technology which revolutionized fundamental as well as applied research in plant breeding. Genome editing using CRISPR/Cas9 system has been successfully demonstrated in many cereal crops including rice, wheat, maize, and barley. Availability of whole genome sequence information for number of crops along with the advancement in genome-editing techniques provides several possibilities to achieve desirable traits. In this review, the options available for crop improvement by implementing CRISPR/Cas9 based genome-editing techniques with special emphasis on cereal crops have been summarized. Recent advances providing opportunities to simultaneously edit many target genes were also discussed. The review also addressed recent advancements enabling precise base editing and gene expression modifications. In addition, the article also highlighted limitations such as transformation efficiency, specific promoters and most importantly the ethical and regulatory issues related to commercial release of novel crop varieties developed through genome editing.

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CRISPR/Cas9-mediated genome editing reveals differences in the contribution of INDEHISCENT homologues to pod shatter resistance in Brassica napus L.

Theoretical and Applied Genetics ◽

10.1007/s00122-019-03341-0 ◽

2019 ◽

Vol 132 (7) ◽

pp. 2111-2123 ◽

Cited By ~ 11

Author(s):

Yungu Zhai ◽

Shengli Cai ◽

Limin Hu ◽

Yang Yang ◽

Olalekan Amoo ◽

...

Keyword(s):

Brassica Napus ◽

Genome Editing ◽

Brassica Napus L ◽

Pod Shatter

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Response of Rapes (Brassica napus L.) to Nano- Iron Fertilization under semi-arid Region conditions

Bionatura ◽

10.21931/rb/2019.04.01.3 ◽

2019 ◽

Vol 4 (1) ◽

Author(s):

Lamiaa A. Mutlag ◽

Nagam A. Meshaimsh ◽

Hasan H. Mahdi ◽

Raghad S. Mouhamad ◽

Hasan H. Khamat ◽

...

Keyword(s):

Brassica Napus ◽

Agricultural Research ◽

Block Design ◽

Nutrient Content ◽

Brassica Napus L ◽

Arid Conditions ◽

Significant Difference ◽

Randomized Complete Block Design ◽

Semi Arid Region ◽

Semi Arid

The field experiment was conducted in the season of 2017-2018 at the Agricultural Research Department-AL-Azafrinih/Baghdad-Iraq. This study aimed to determine the effect of adding the Foliar Fe fertilization (Nano-Fe) and ground fertilization of (N, P) in the growth and production yield of rapes (Brassica napus L.). The experiment included three levels of foliar fertilization (Nano-Fe) that is (0, 5, 10 Kg. ha-1) and one level of ground fertilization (N 100 Kg. ha-1, P 250 Kg. ha-1). The experiment designed according to Randomized Complete Block Design (R.C.B.D) with three replications each parameter. The studied traits were nutrients content (macro-micro) in seeds and soil, the percentage of protein, oil, and carbohydrate. All data were analyzed and used statistically. Results showed a significant difference between Nano-Fe fertilization and nutrient content (N, Mn, K, Ca, Mg, S, and Cu), pH in soil at the level of 10 kg. ha-1, compared to no-fertilization. Moreover, the significant difference between Nano-Fe fertilization, yield (1425.6 kg. ha-1), the percentage of oil (52.16%), protein content in seeds (46.5%), carbohydrate (26.8%), Nutrient content N and P in seeds at the level of 10 kg. ha-1, compared to no-fertilization and the nutrient content (Ca, Mg, Fe) in seeds at levelof5 kg. ha-1, compared to no-fertilization. Current results suggested that Nano-Fe fertilization at the level of 10 kg. ha-1 can be adopted as the best level for rapes (Brassica napus L.) cultivation under semi-arid conditions.

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Evaluation of two self-incompatibility alleles in three summer rape (Brassica napus L.) cultivars by UV fluorescence microscopy, seed set and outcrossing rates

Canadian Journal of Plant Science ◽

10.4141/p99-070 ◽

2000 ◽

Vol 80 (2) ◽

pp. 255-260

Author(s):

L. J. Lewis ◽

D. L. Woods ◽

H. W. Klein-Gebbinck

Keyword(s):

Brassica Napus ◽

Seed Set ◽

Brassica Napus L ◽

Significant Difference ◽

S Alleles ◽

Outcrossing Rates ◽

Incompatibility Alleles ◽

Sporophytic Incompatibility ◽

S Allele ◽

Yellow Petal

S-alleles W1 and T2 and an incompletely dominant white petal character were introgressed into the self-compatible (SC) summer rape (Brassica napus L. ssp. oleifera {Metzg.}) cultivars Global, Topas and Westar. The derived self-incompatible (SI) lines were evaluated for strength of incompatibility by ultraviolet fluorescence of pollen tubes, and by seed set. Pollen tube and seed set analyses showed the W1 and T2 alleles were strongly, moderately and weakly expressed in Topas, Global and Westar, respectively. Seed set data showed a significant difference between SI lines, but not between S-alleles, or between homozygous or heterozygous lines from the same SI cultivar. SI cultivar yellow petal (wild type) lines were field pollinated with SC white petal lines. Seed collected from the SI cultivars were evaluated for proportion of outcrossed progeny by recording the frequency of yellow petal and cream petal plants, which were the result of self- and cross-pollination, respectively. The proportion of outcrossed progeny (i.e., outcrossing rates) ranged from 23% to 79%. Topas SI lines had significantly higher outcrossing rates than Global SI lines, which corresponded to SI line seed set data. Environment, S-allele selection and genotype significantly affected outcrossing rates. Key words: Brassica napus, sporophytic incompatibility, S-allele, outcrossing rate

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Temporal genetic patterns of root growth in Brassica napus L. revealed by a low-cost, high-efficiency hydroponic system

Theoretical and Applied Genetics ◽

10.1007/s00122-019-03356-7 ◽

2019 ◽

Vol 132 (8) ◽

pp. 2309-2323

Author(s):

Jie Wang ◽

Lieqiong Kuang ◽

Xinfa Wang ◽

Guihua Liu ◽

Xiaoling Dun ◽

...

Keyword(s):

Brassica Napus ◽

Root Growth ◽

High Efficiency ◽

Low Cost ◽

Brassica Napus L ◽

Hydroponic System ◽

Genetic Patterns

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Transcriptome analysis reveals gene responses to herbicide, tribenuron methyl, in Brassica napus L. during seed germination

BMC Genomics ◽

10.1186/s12864-021-07614-1 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Liuyan Wang ◽

Ruili Wang ◽

Wei Lei ◽

Jiayi Wu ◽

Chenyang Li ◽

...

Keyword(s):

Brassica Napus ◽

Seed Germination ◽

Transcriptome Analysis ◽

Crop Production ◽

Target Genes ◽

Acetolactate Synthase ◽

Plant Tolerance ◽

Brassica Napus L ◽

Antioxidant Genes ◽

Tribenuron Methyl

Abstract Background Tribenuron methyl (TBM) is an herbicide that inhibits sulfonylurea acetolactate synthase (ALS) and is one of the most widely used broad-leaved herbicides for crop production. However, soil residues or drifting of the herbicide spray might affect the germination and growth of rapeseed, Brassica napus, so it is imperative to understand the response mechanism of rape to TBM during germination. The aim of this study was to use transcriptome analysis to reveal the gene responses in herbicide-tolerant rapeseed to TBM stress during seed germination. Results 2414, 2286, and 1068 differentially expressed genes (DEGs) were identified in TBM-treated resistant vs sensitive lines, treated vs. control sensitive lines, treated vs. control resistant lines, respectively. GO analysis showed that most DEGs were annotated to the oxidation-reduction pathways and catalytic activity. KEGG enrichment was mainly involved in plant-pathogen interactions, α-linolenic acid metabolism, glucosinolate biosynthesis, and phenylpropanoid biosynthesis. Based on GO and KEGG enrichment, a total of 137 target genes were identified, including genes involved in biotransferase activity, response to antioxidant stress and lipid metabolism. Biotransferase genes, CYP450, ABC and GST, detoxify herbicide molecules through physical or biochemical processes. Antioxidant genes, RBOH, WRKY, CDPK, MAPK, CAT, and POD regulate plant tolerance by transmitting ROS signals and triggering antioxidant enzyme expression. Lipid-related genes and hormone-related genes were also found, such as LOX3, ADH1, JAZ6, BIN2 and ERF, and they also played an important role in herbicide resistance. Conclusions This study provides insights for selecting TBM-tolerant rapeseed germplasm and exploring the molecular mechanism of TBM tolerance during germination.

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Sulphur Nutrition and its Effect on Yield and Oil Content of Oilseed Rape (Brassica Napus L.)

Acta Universitatis Agriculturae et Silviculturae Mendelianae Brunensis ◽

10.11118/actaun201765020555 ◽

2017 ◽

Vol 65 (2) ◽

pp. 555-562 ◽

Cited By ~ 3

Author(s):

Mária Varényiová ◽

Ladislav Ducsay ◽

Pavel Ryant

Keyword(s):

Brassica Napus ◽

Oilseed Rape ◽

Oil Content ◽

Oil Production ◽

Brassica Napus L ◽

Agricultural Cooperative ◽

Sulphur Nutrition ◽

Significant Difference ◽

Scale Experiment ◽

Effect On Yield

The aim of the experiment was to study the importance of sulphur in oilseed rape (Brassica napus L.) nutrition as well as the effect of rising doses of sulphur in combination with nitrogen on yield, oiliness, oil production, nutrients content in seed and nutrients uptake by rapeseed. The plot–scale experiment was established in years 2013/14 and 2014/15 within the agricultural cooperative in Mojmírovce. There were four fertilization treatments on 600 m2 experimental plots in three replications in this experiment. The first treatment was unfertilized control. Other three treatments were fertilized by the same nitrogen dose of 160 kg.ha−1 and by increasing doses of sulphur. The second treatment was fertilized by a dose of 15 kg.ha−1 S, the third by a dose of 40 kg.ha−1 and a dose of 65 kg.ha−1 S was applied at the fourth treatment. The highest average yield 3.96 t.ha-1 was found when a dose of 40 kg.ha−1 S was applied. The application of sulphur in a dose of 65 kg.ha−1 was accompanied by a yield decrease by 11.4 % as compared to the treatment where a sulphur dose of 40 kg.ha−1 was used. An average oil content of 45.1, 45.5, and 44.0 % was found in treatments in which the doses of sulphur of 15, 40 and 65 kg.ha−1 were applied. No significant difference among the treatments fertilized by sulphur was found. The average oil production reached 1809, 1802 and 1595 kg.ha−1 in cases of treatments fertilized by sulphur doses of 15, 40 and 65 kg.ha−1.

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