WO2023242872A1

WO2023242872A1 - Recombinant expression cassettes for modification of glucosinolate content in plants

Info

Publication number: WO2023242872A1
Application number: PCT/IN2023/050566
Authority: WO
Inventors: Juhi KUMARI; Avni MANN; Roshan Kumar; Naveen C. BISHT
Original assignee: National Institute Of Plant Genome Research
Priority date: 2022-06-17
Filing date: 2023-06-15
Publication date: 2023-12-21

Abstract

The present disclosure discloses recombinant expression cassette comprising guide RNAs and Cas9 protein, and the said expression cassette is effective in modifying the glucosinolate content in a plant. Also provided is a process for modifying the tissue-specific glucosinolate accumulation in a plant and a process for producing a transgene-free edited plant using the recombinant expression cassette.

Description

RECOMBINANT EXPRESSION CASSETTES FOR MODIFICATION OF GLUCOSINOLATE CONTENT IN PLANTS

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

An official copy of the Sequence Listing a file named "PD046803IN-SC_SEQ LISTING.xml" created on June 14, 2023, having a size of 112 kb filed electronically concurrently with the Specification is part of the Specification.

FIELD OF INVENTION

[001] The present disclosure broadly relates to the field of plant biotechnology. In particular, the present disclosure provides with recombinant expression cassettes comprising guide RNA and Cas9 protein for modifying the glucosinolate content in plants belonging to Brassica spp. The disclosure further provides with a method of preparing a transgene-free edited plant having low glucosinolate content in the seeds and high glucosinolate content in leaves and pods.

BACKGROUND OF INVENTION

[002] Rapeseed and mustard are the second-largest cultivated oilseed Brassica crops after soybean in the world oilseed production (FAO, 2020). The presence of a high amount of glucosinolates and erucic acid in the oilseed Brassica crops causes health problems and reduces the meal palatability for animal feed (Cartea, M.E. and Velasco, P. (2008) Glucosinolates in Brassica foods: bioavailability in food and significance for human health. Phytochem. Rev. 7, 213-229; Ishida, M., Hara, M., Fukino, N., Kakizaki, T., & Morimitsu, Y. (2014). Glucosinolate metabolism, functionality and breeding for the improvement of Brassicaceae vegetables. Breeding Science, 64(1), 48-59. https://doi.org/10.1270/jsbbs.64.48; Bell, L., Oloyede, O. O., Lignou, S., Wagstaff, C., & Methven, L. (2018). Taste and Flavor Perceptions of Glucosinolates, Isothiocyanates, and Related Compounds. Molecular Nutrition & Food Research, 62(18), 1700990. https://doi.org/10.1002/ mnfr.201700990). The improvement of oilseed quality through the development of ‘Canola’ quality lines, having low levels of seed glucosinolates (SGC <30 pmol g ¹ dry weight) and erucic acid (<2% of the free fatty acid pool) in the seeds is a major breeding objective in the oilseed Brassica crops.

[003] Glucosinolates, specifically their hydrolysis products are the key defense arsenals of the Brassicaceae family members against invading pests and pathogens (Halkier, B. A., & Gershenzon, J. (2006). Biology and biochemistry of glucosinolates. Annual Review of Plant Biology, 57(1), 303-333. https://doi.org/10.1146/annurev. arplant.57.032905.105228; Clay, N. K., Adio, A. M., Carine, C., Jander, G., & Ausubel, F. M. (2009). Glucosinolate metabolites required for an Arabidopsis innate immune response. Science, 323, 95-101; Hopkins, R. J., van Dam, N. M., & van Loon, J. J. A. (2009). Role of glucosinolates in insect plant relationships and multitrophic interactions. Annual Review of Entomology, 54, 57-83). In recent years, the ‘Canola’ varieties are reportedly suffering from a range of existing and newly emerging pests and diseases, which are overcoming the plant’s defense due to compromised glucosinolates biosynthesis in the vegetative tissues (Sekulic G and Rempel CB (2016). Evaluating the role of seed treatments in canola/oilseed rape production: integrated pest management, pollinator health, and biodiversity. Plants 5:32. doi: 10.3390/plants5030032; Milovac Z, Zoric M, Franeta F, Terzi c S, Petrovic Obradovic O, and Maij anovic Jeromela A (2017) Analysis of oilseed rape stem weevil chemical control using a damage rating scale. Pest Manag Sci. 73, 1962-1971. doi: 10.1002/ps.4568; Zheng X, Koopmann B, Ulber B and von Tiedemann A (2020) A Global Survey on Diseases and Pests in Oilseed Rape - Current Challenges and Innovative Strategies of Control. Front. Agronomy. 2: 590908. doi: 10.3389/fagro.2020.590908). As a consequence, disease control in oilseed rape cultivation largely relies on harmful agrochemicals including pesticides and fungicides. The increasing cost and restrictions on agrochemicals use worldwide, particularly in Europe, and their declining efficacy has threatened the profitability of rapeseed-mustard production (Insecticide Resistance Action Group, 2019. Insecticide Resistance status in UK oilseed rape crop).

[004] For the development of plants with low glucosinolate content, genetic manipulation using either TILLING- mutagenesis or RNAi-based constitutive suppression of GTR1 and/or GTR2 homologs has been tested in B. juncea, however, only 60-74% reduction of the SGC compared to the wild-type seeds could be achieved without establishing the ‘Canola’ quality mustard lines (Nour-Eldin HH, Madsen SR, Engelen S, Jprgcnscn ME, Olsen CE, Andersen JS, Seynnaeve D, Verhoye T, Fulawka R, Denolf P, et al. (2017) Reduction of antinutritional glucosinolates in Brassica oilseeds by mutation of genes encoding transporters. Nat Biotechnol 35: 377-382. 2017; Nambiar DM, Kumari J, Augustine R, Kumar P, Bajpai PK, Bisht NC (2021) GTR1 and GTR2 transporters differentially regulate tissue-specific glucosinolate contents and defence responses in the oilseed crop Brassica juncea. Plant Cell & Environment 44, 2729-2743). In the related oilseed crop, B. napus, the editing of BnaGTR2 homologs provided a reduced accumulation of glucosinolates in the sink (seeds) and, surprisingly, also in the source tissues (leaf and siliques), which could impact the plant defense negatively ((He Y, Yang Z, Tang M, Yang QY, Zhang Y, Liu S. (2022) Enhancing canola breeding by editing a glucosinolate transporter gene lacking natural variation. Plant Physiol, (in press) doi: 10.1093/plphys/kiac021; Tan Z, Xie Z, Dai L, Zhang Y, Zhao H, Tang S, Wan L, Yao X, Guo L, Hong D (2022) Genome- and transcriptome- wide association studies reveal the genetic basis and the breeding history of seed glucosinolate content in Brassica napus. Plant Biotechnol J 20: 211-225).

[005] W02012004013A2 provides methods to alter the glucosinolate content in plants, in particular in specific plant parts, by modifying glucosinolate transporter protein (GTR) activity in plants or parts thereof.

[006] The aforesaid drawback in the prior art demands the development of superior low SGC genotypes in rapeseed-mustard while retaining a high amount of foliar glucosinolates, necessary for uncompromised plant defense.

[007] US10988772B2 relates to Pennycress (Thlaspi arvense) seed, seed lots, seed meal, and compositions with reduced glucosinolate content as well as plants that yield such seed, seed lots, seed meal, and compositions.

SUMMARY OF THE INVENTION

[008] In an aspect of the present disclosure, there is provided a recombinant expression cassette comprising: (a) a guide polynucleotide (gRNA) complementary to a target sequence, operably linked to a promoter, wherein the target sequence has a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8. SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, and wherein the guide polynucleotide has contiguous nucleotides complementary to the target sequence in the range of 15-20 nucleotides; (b) a scaffold region intervening gRNAl, gRNA2, and gRNA3 having nucleotide sequence as set forth in SEQ ID NO: 15; and (c) a polynucleotide encoding Cas9 protein having a nucleotide sequence selected from sequences as set forth in SEQ ID NO: 14, SEQ ID NO: 20 or SEQ ID NO: 21, operably linked with a promoter, wherein the recombinant expression cassette is effective in modifying the glucosinolate content in a plant.

[009] In another aspect of the present disclosure, there is provided a recombinant expression cassette comprising: (a) a guide polynucleotide (gRNA) construct comprising gRNAl, gRNA2, and gRNA3, each operably linked to a promoter, wherein gRNAl is complementary to a target sequence having a nucleotide sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, gRNA2 is complementary to a target sequence having a nucleotide sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and gRNA3 is complementary to a target sequence having a nucleotide sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and wherein the gRNAl, gRNA2 and gRNA3 each has contiguous nucleotides complementary to the target sequence in the range of 15-20 nucleotides; (b) a scaffold region intervening gRNAl, gRNA2, and gRNA3 having nucleotide sequence as set forth in SEQ ID NO: 15; and (c) a polynucleotide encoding Cas9 protein having a nucleotide sequence selected from sequences as set forth in SEQ ID NO:14, SEQ ID NO: 20 or SEQ ID NO: 21, operably linked with a promoter, wherein the recombinant expression cassette is effective in modifying the glucosinolate content in a plant.

[0010] In another aspect of the present disclosure, there is provided a process for producing a transgene-free plant with modified glucosinolate content, said process comprising: (a) transforming a plant cell with the recombinant vector comprising the recombinant expression cassette as described hereinabove or the host cell comprising the recombinant vector comprising the recombinant expression cassette as described hereinabove, to obtain stably transformed plant cells, (b) selecting a transgene-free edited plant cell from the stably transformed plant cells, (c) growing the transgene - free edited plant cell for producing a transgene-free edited plant with modified glucosinolate content, wherein the transgene-free plant has reduced glucosinolate content in seeds and increased glucosinolate content in leaves and pod walls compared to a control non-edited plant.

[0011] In yet another aspect of the present disclosure, there is provided a transgene- free edited plant having reduced glucosinolate content in seeds and increased glucosinolate content in leaves and pod walls produced by the process as described hereinabove.

[0012] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

[0013] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0014] Figure 1 depicts development of CRISPR/Cas9 construct targeting BjuGTRl and BjuGTR2 homologs, seed GSL content (SGC) and mutation screening of BjuGTR- edited lines, (a) Sequence alignment showing the BjuGTR targets of gRNAl (blue), gRNA2 (green) and gRNA3 (violet). The PAM site is marked red. (b) T-DNA map of BjuGTRl ::GTR2(GEd) construct used for targeting ten BjuGTRs. The pZP200 binary vector contains bar gene as the plant selection marker and CsVMV driven SpCas9. (c) Bar graph showing the mean SGC (in pmoles g ¹ DW) of 37 independent TO events along with wild-type (cv. Varuna) and vector control (VC) plants. Three independent measurements for each line were made using HPLC. (d) Analysis of the CRISPR/Cas9-induced mutations in ten BjuGTR homologs of representative low (<30 pmoles g ¹ DW) and high (>70 pmoles g ¹ DW) SGC events. The edited (orange) and un-edited (green) BjuGTR homologs of each TO event are depicted, in accordance with an embodiment of the present disclosure.

[0015] Figure 2 depicts glucosinolate content, seed weight and genotype of the transgene-free B/z/G77 -cditcd B. juncea lines. Bar graph showing the mean GSL content in (a) seeds (SGC) in the T3 generation, (b) flag leaf (LGC), GSL content (in pmoles g ¹ DW) of 5-12 T2 plants (represented as dots) of the 23 transgene-free edited lines was estimated using HPLC. Different letters on top indicate significant differences across chemotypes using Tukey’s post hoc test at P <0.05. (c) Summary of the mean GSL content (in pmoles g ¹ DW) in the mature seeds (SGC), leaf (LGC), pod walls (PWGC), developing green seeds (gSGC) and roots (RGC) of lines belonging to two GSL chemotypes viz., Ct-I (13 lines) and Ct-II (10 lines), (d) Analysis of the genetic zygosity of BjuGTR mutations in the representative low SGC lines belonging to two chemotypes (Ct-I and Ct-II). The true BHo (mutl/mutl') and BHt (mul l/mul2) null mutations are marked orange; and unedited (wt/wt) alleles are in green. The BjuGTR mutated allele showing a shorter deletion, in the multiple of 3 bp, is represented as asterisk, and also marked in green. The altered GSL phenotype in source (leaf, LGC) and sink (mature seeds, SGC) organs in representative lines belonging to two GSL chemotypes is also provided, in accordance with an embodiment of the present disclosure.

[0016] Figure 3 demonstrates the vector map of pZP200:lox-debar::ptCsVMV- SpCas9-pA vector plus insert sequence (PtAtU6-26-gRNAl-scaffold::PtAtU6-26- gRNA2-scaffold::PtAtU6-26-gRNA3-scaffold), in accordance with an embodiment of the present disclosure.

[0017] Figure 4 demonstrates the vector map of pZP200:lox-debar::ptCaMV35S- synJ-BcoCas9-HFl-pA vector plus insert sequence (PtAtU6-26-gRNAl- scaffold::PtAtU6-26-gRNA2-scaffold::PtAtU6-26-gRNA3 scaffold), in accordance with an embodiment of the present disclosure.

[0018] Figure 5 demonstrates the summary of the mutation frequency and SGC in the lines generated using BcoCas9:BjuGTRl::GTR2(GEd) construct, (a) A total of 40 TO lines were tested for the editing of representative BjuGTR homologs and the mutation frequency is provided along with, (b) The mean SGC in T1 seeds of a few representative lines showing the effect of BjuGTR-editing generated using BcoCas9 construct. The SGC of wild-type (WT) and vector control (VC) plants are also provided, in accordance with an embodiment of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

[0019] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications.

The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.

[0020] The Table 1 below provides the list of primers used in the study: Table 1:

Definitions

[0021] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0022] The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0023] The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.

[0024] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps. [0025] The term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.

[0026] For the purposes of the present disclosure, the term “recombinant” refers to sequences of nucleotide or amino acid which are genetically engineered using human intervention and molecular biology tools.

[0027] The term “gRNA” or “guide polynucleotide” is a short synthetic RNA composed of a scaffold sequence necessary for Cas-binding and a user-defined ~20 nucleotide spacer that defines the genomic target to be modified. [0028] The term “Cas9 protein” refers to RNA-guided enzyme that cleaves foreign nucleic acids bearing sequence complementary to the RNA loaded into the enzyme.

[0029] The term “modifying” refers to increase or decrease in the levels of certain enzyme or protein. In the present disclosure, the term “modifying” relates to increase or decrease in the glucosinolate content in plants.

[0030] The term “guide polynucleotide (gRNA) construct” refers to a polynucleotide sequence comprising the gRNAl, gRNA 2 and gRNA3.

[0031] The term “host cell” refers to a cell which is capable of being transformed with a recombinant DNA, construct or vector.

[0032] The term “transgene-free edited plant” refers to a plant that has been produced by transforming a recombinant DNA, construct or vector and in the T1 generation the mutations in BjuGTR homologs theoretically segregate independently of the T-DNA locus that encodes the recombinant DNA.

[0033] The term “stably transformed plant” or “stably transformed plant cell” is one where the foreign DNA is fully integrated into the host genome and expressed in later generations of the plant.

[0034] The term “selecting” or “selection” refers to a process of picking/ choosing/ preserving/ propagating transgenic plants with desirable characteristics and eliminating those with less or no desirable characteristics.

[0035] The term “control non-edited plant” refers to a wild type or parent plant which has not been transformed with a recombinant DNA.

[0036] Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub -ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.

[0038] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein.

[0039] Glucosinolate biosynthesis and transport processes have been well- characterized in the model plant Arabidopsis thaliana and to some extent in the oilseed Brassica crops (Halkier, B. A., & Gershenzon, J. (2006). Biology and biochemistry of glucosinolates. Annual Review of Plant Biology, 57(1), 303-333. https://doi.org/10.1146/annurev. arplant.57.032905.105228; Spnderby, I. E., Geu- Flores, F., and Halkier, B. A. (2010) Biosynthesis of glucosinolates-gene discovery and beyond. Trends in Plant Science, 15(5), 283-290. https://doi.Org/10.1016 j.tpIants.20I0.02.005; Augustine, R., and Bisht, N. C. (2017) Regulation of glucosinolate metabolism: From model plant Arabidopsis thaliana to Brassica crops. In K. Ramawat, & J.-M. M Ari I Ion (Eds.), Reference series in phytochemistry: Glucosinolates (pp. 163-199). Germany: Springer Press). Of seminal discovery, in glucosinolate research are the two glucosinolate transporters (GTR1 and GTR2), which dictate the source-sink dynamics of the glucosinolates accumulation (Nour-Eldin HH, Andersen TG, Burow M, Madsen SR, Jprgensen ME, Olsen CE, Dreyer I, Hedrich R, Geiger D, Halkier BA (2012) NRT/PTR transporters are essential for translocation of glucosinolate defence compounds to seeds. Nature 488: 531-534). An initial study in Arabidopsis demonstrates that gtrlgtr2 double mutant accumulates SGC below detection limits, however, neither the gtrl nor gtr2 single mutant could provide the reduction of SGC to the expected level (Nour-Eldin HH, Andersen TG, Burow M, Madsen SR, Jprgcnscn ME, Olsen CE, Dreyer I, Hedrich R, Geiger D, Halkier BA (2012) NRT/PTR transporters are essential for translocation of glucosinolate defence compounds to seeds. Nature 488: 531-534). A major breeding objective in the oilseed Brassica crops- B. napus (rapeseed) and B. juncea (mustard) has been to bring the glucosinolate content in the seed meal from 150 to less than 30 pmoles g ¹ dry weight (DW) of the defatted seed meal to make it more palatable and nutritious to livestock and poultry. The genetic manipulation of glucosinolate transporters has been attempted in oilseed mustard, however to date, the development of Canola quality lines having SGC <30 pmoles g ¹ DW has not been achieved (Nour-Eldin HH, Madsen SR, Engelen S, Jprgcnscn ME, Olsen CE, Andersen JS, Seynnaeve D, Verhoye T, Fulawka R, Denolf P, et al. (2017) Reduction of antinutritional glucosinolates in Brassica oilseeds by mutation of genes encoding transporters. Nat Biotechnol 35: 377-382; Nambiar DM, Kumari J, Augustine R, Kumar P, Bajpai PK, Bisht NC (2021) GTR1 and GTR2 transporters differentially regulate tissue-specific glucosinolate contents and defence responses in the oilseed crop Brassica juncea. Plant Cell & Environment 44, 2729-2743).

[0040] Such lines developed by manipulating the biosynthesis pathway genes are low glucosinolate both in the source and sink tissues rendering them vulnerable to generalist pests and pathogens. Thus, the globally cultivated Canola quality rapeseed-mustard varieties are suffering from a range of existing and newly emerging pests and pathogens, which are overcoming plant’s defense due to compromised glucosinolates biosynthesis.

[0041] Ideal lines of crops should have normal glucosinolate levels in the source and low in the sink (seeds) with normal growth and development of the plants. Molecular analysis suggests that functional mutations in multiple BjuGTR homologs are required for the generation of an ideal mustard genotype with low SGC while having a concomitant over- accumulation of glucosinolates in source organs (leaves and pod walls), important for enhancing both nutrition and plant defense traits.

[0042] In view of the above, the present disclosure describes the development of such ideal lines in mustard by editing multiple genes encoding for glucosinolate transporters. In particular, the present disclosure describes the deployment of the CRISPR/Cas9 strategy for editing multiple BjuGTRl and BjuGTR2 homologs in B. juncea to generate an ‘ideal glucosinolate chemotype’ with low SGC and high glucosinolates in the vegetative tissues, in the most efficient way possible.

[0043] The present disclosure provides with B. juncea lines having seed glucosinolates content (SGC) as low as 6.21 pmoles g ¹ DW, through CRISPR/Cas9- based editing of glucosinolate transporter (BjuGTR) gene family. The transgenic lines have been developed in the oilseed mustard by CRISPR/Cas9-based concomitant editing of most, but not all, of the GTR1 and GTR2 family genes.

[0044] In an embodiment of the present disclosure, there is provided a recombinant expression cassette comprising: (a) a guide polynucleotide (gRNA) complementary to a target sequence, operably linked to a promoter, wherein the target sequence has a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8. SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, and wherein the guide polynucleotide has contiguous nucleotides complementary to the target sequence in the range of 15-20 nucleotides; (b) a scaffold region intervening gRNAl, gRNA2, and gRNA3 having nucleotide sequence as set forth in SEQ ID NO: 15; and (c) a polynucleotide encoding Cas9 protein having a nucleotide sequence selected from sequences as set forth in SEQ ID NO: 14, SEQ ID NO: 20 or SEQ ID NO: 21, operably linked with a promoter, wherein the recombinant expression cassette is effective in modifying the glucosinolate content in a plant. In another embodiment, the gRNA is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. In yet another embodiment, the gRNA comprises SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

[0045] In an embodiment of the present disclosure, there is provided a recombinant expression cassette comprising: (a) a guide polynucleotide (gRNA) construct comprising gRNAl, gRNA2, and gRNA3, each operably linked to a promoter, wherein gRNAl is complementary to a target sequence having a nucleotide sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, gRNA 2 is complementary to a target sequence having a nucleotide sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and gRNA3 is complementary to a target sequence having a nucleotide sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and wherein the gRNAl, gRNA2 and gRNA3 each has contiguous nucleotides complementary to the target sequence in the range of 15-20 nucleotides; (b) a scaffold region intervening gRNAl, gRNA2, and gRNA3 having nucleotide sequence as set forth in SEQ ID NO: 15; and (c) a polynucleotide encoding Cas9 protein having a nucleotide sequence selected from sequences as set forth in SEQ ID NO: 14, SEQ ID NO: 20 or SEQ ID NO: 21, operably linked with a promoter, wherein the recombinant expression cassette is effective in modifying the glucosinolate content in a plant.

[0046] In an embodiment of the present disclosure, there is provided a recombinant expression cassette as described herein, wherein the promoter driving the expression of the gRNA is AtU6-26 promoter, or orthologs thereof from Brassica species. In yet another embodiment AtU6-26 promoter has a sequence as set forth in SEQ ID NO: 18.

[0047] In an embodiment of the present disclosure, there is provided a recombinant expression cassette as described herein, wherein the promoter driving the expression of Cas9 protein is selected from the group consisting of CsVMV promoter, CaMV 35S promoter, FMV promoter, MMV promoter, and RbcS promoter. In yet another embodiment, the promoter driving the expression of Cas9 protein is CsVMV promoter having sequence as set forth in SEQ ID NO: 19.

[0048] In an embodiment of the present disclosure, there is provided a recombinant expression cassette as described herein, wherein the expression cassette further comprises 5’ untranslated region (UTR) upstream to the polynucleotide encoding the Cas9 protein, and wherein the 5’ UTR is synthetic sequence (synJ). In another embodiment, the 5’ UTR enhances the expression of the Cas9 protein.

[0049] In an embodiment of the present disclosure, there is provided a recombinant vector comprising the recombinant expression cassette as described hereinabove. In another embodiment of the present disclosure, the vector is lab modified binary vector pZP200:lox::bar containing bar gene as a selectable marker.

[0050] In an embodiment of the present disclosure, there is provided a host cell comprising the recombinant vector comprising the recombinant expression cassette as described hereinabove, wherein the host cell is E. coli or Agrobacterium tumefaciens. In another embodiment of the present disclosure, the host cell is E. coli. In yet another embodiment of the present disclosure, the host cell is Agrobacterium tumefaciens. [0051] In an embodiment of the present disclosure, there is provided a process for modifying the glucosinolate content in a plant, comprising targeting the expression of sequences selected from the group consisting of SEQ ID NO: 4 to 13 in said plant by the recombinant expression cassette as described hereinabove.

[0052] In an embodiment of the present disclosure, there is provided a process for producing a transgene-free edited plant with modified glucosinolate content, said process comprising: (a) transforming a plant cell with the recombinant vector comprising the recombinant expression cassette as described hereinabove or the host cell comprising the recombinant vector comprising the recombinant expression cassette as described hereinabove, to obtain stably transformed plant cells, (b) selecting a transgene-free edited plant cell from the stably transformed plant cells, (c) growing the transgene-free edited plant cell for producing a transgene-free edited plant with modified glucosinolate content, wherein the transgene-free edited plant has reduced glucosinolate content in seeds and increased glucosinolate content in leaves and pods compared to a control non-edited plant.

[0053] In an embodiment of the present disclosure, there is provided a process for producing a transgene-free edited plant with modified glucosinolate content wherein the plant is selected from a group consisting of Brassica nigra, B. rapa, B. oleracea, B. juncea, B. napus, B. carinata, Camelina sativa, Capsella rubella, Sinapis alba, and Arabidopsis thaliana.

[0054] In an embodiment of the present disclosure, there is provided a transgene- free edited plant having reduced glucosinolate content in seeds and increased glucosinolate content in leaves and pod walls produced by the process as described hereinabove.

[0055] In an embodiment of the present disclosure, there is provided a transgene- free edited plant having reduced glucosinolate content in seeds and increased glucosinolate content in leaves and pod walls produced by the process as described hereinabove, wherein the transgene-free edited plant has glucosinolate content in seeds in a range of 6-30.00 pmoles g ¹ dry weight and glucosinolate content in leaves in the range of 75.84 to 105.32 pmoles g^-1 dry weight, and pods in the range of 25.83 to 64.69 pmoles g ¹ dry weight. In one of the embodiments, the transgene-free edited plant has glucosinolate content in seeds in a range of 6-30.00 pmoles g ¹ dry weight. In yet another embodiment, the transgene-free plant has glucosinolate content in seeds in a range of 15.12-29.03 pmoles g^-1 dry weight

[0056] In an embodiment of the present disclosure, there is provided a recombinant expression cassette as described herein, wherein the sequence as set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8. SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13 represent the homologues of GTR1 and/or GTR2. GTR1 has a sequence as set forth in SEQ ID NO: 16 and GTR2 has a sequence as set forth in SEQ ID NO: 17 and the GTRs belong to Arabidopsis spp.

[0057] In an embodiment of the present disclosure, there is provided a recombinant expression cassette as described herein, wherein the guide polynucleotide has contiguous nucleotides complementary to the target sequence in the range of 15-20. In one of the embodiments of the present disclosure, the guide polynucleotide has contiguous nucleotides complementary to the target sequence in the range of 18-20. In one of the embodiments of the present disclosure, the guide polynucleotide has 20 contiguous nucleotides complementary to the target sequence.

[0058] Although the subject matter has been described in considerable detail with reference to certain examples and implementations thereof, other implementations are possible.

EXAMPLES

[0059] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may apply.

Example 1:

Designing gRNAs and generation of CRISPR/Cas9-based editing construct/s

[0060] Full-length genomic sequences of B. juncea GTR1 BjuGTRl) and GTR2 (BjuGTRT) homologs were used as queries to identify the 20 nucleotide (nt) gRNAs with PAM (NGG) using B. juncea as the target genome in CRISPR-P v2.0 (http://crispr.hzau.edu.cn/CRISPR2/). Further to accommodate the requirement of transcription initiation (G) by AtU6-26 promoter and PAM sequence specificity (NGG as represented by SEQ ID NO: 23) for the three gRNAs targeting all the expressed BjuGTR homologs (as shown in Figure 1A and represented by SEQ ID NO:4 to SEQ ID NO: 13) were selected based on maximum on-score and minimum off-target associated with each of the predicted gRNA.

[0061] To generate sgRNA (single guide RNA) scaffold, the 20 nucleotides seed sequence of gRNA was introduced between AtU6-26 promoter and the scaffold, in a two-step PCR reaction using customized PCR primers. In the first step, two PCR reactions were performed to amplify the ‘promoter-gRNA’ and ‘gRNA-scaffold’ fragments, independently. In the second step, the two fragments thus obtained were linked through another round of overlapping PCR to generate the complete sgRNA fragment i.e. AtU6-26 promoter:gRNA:scaffold.

[0062] The S. pyogenes wild-type Cas9 gene (SpCas9 as represented by SEQ ID NO: 14), driven by the constitutive Cassava Vein Mosaic Virus (CsVMV) promoter, was cloned into the lab modified binary vector pZP200:lox::bar containing bar gene as a selectable marker within the ‘lox’ tandem repeats for marker excision, to develop the pZP200debar:SpCas9 binary vector. All the three sgRNA fragments were cloned within appropriate restriction sites of the pZP200debar:SpCas9 vector to develop the GTREGTR2 -editing construct (Figure lb).

Example 2:

Genetic transformation of B. juncea and development of G edited lines

[0063] A high glucosinolate B. juncea cultivar (Varuna) was used for the genetic transformation experiments. The 5-days-old hypocotyl explants were used to genetically transform B. juncea using Agro/zacterznm-mediated genetic transformation, as per the publicly available and established protocol (Augustine, R., Mukhopadhyay, A., & Bisht, N. C. (2013) Targeted silencing of BjMYB28 transcription factor gene directs development of low glucosinolate lines in oilseed Brassica juncea. Plant Biotechnology Journal, 11, 855-866. https://doi.org/ 10.1111/pbi.12078). The TO transformed plants were grown and maintained under contained net-house field conditions of NIPGR from November to April, as per the guidelines laid by the Department of Biotechnology, Government of India. TO transformants were confirmed through Basta spray (200 mg I’¹). The confirmed TO events were maintained by self-pollination to obtain the T1 and T2 seeds. Both open- pollinated and self-pollinated seeds were harvested separately upon maturity.

Example 3:

Isolation of genomic DNA and mutation screening of BjuGTR homologs in edited B. juncea lines

[0064] Genomic DNA of TO events, Cas9-free T1 progeny, and the wild-type plants was extracted using the cetyltrimethylammonium bromide (CTAB) method. The flanking genomic sequence around the CRISPR target sites (~200bp) for each of the BjuGTR homologs was amplified using homolog- specific primers and TaKaRa ExTaq polymerase in a standard PCR amplification reaction known in the art. The PCR products were gel-eluted and sequenced using the Sanger sequencing method. Further to identify the overall editing efficiency, type of mutations, and the mutation frequency, the chromatogram file of edited lines for each BjuGTR homolog was compared with the wild-type plant in the ICE (Inference of CRISPR edits) analysis tool of Synthego.

[0065] The percentage of transformed plants showing editing of the target gene was used to calculate the editing frequency. Further, the mutation efficiency for each gene i.e., percent of sequences mutated in a pool of cells, was calculated using ICE tool (Synthego). The edited sequences were aligned using different modules of the DNASTAR software (Lasergene, USA). Example 4:

Identification of transgene-free BjuGTR-edited lines

[0066] Approximately 40-50 T1 seeds from each independent event were grown in the contained net-house and herbicide Basta (active ingredient phosphinothricin) was painted on the young leaves of 3-4 weeks old plants. T1 progeny from each transgenic event were segregated into Basta resistant (Cas9-containing) and sensitive (Cas9-free) phenotypes. The Basta-sensitive transgene-free edited lines were further confirmed through PCR using bar gene-specific primers. Both Basta resistant and Basta sensitive T1 plants were propagated by selfing to obtain homozygous mutations for BjuGTR homologs in the subsequent T2 generation.

Example 5:

Glucosinolate estimation in BjuGTR-edited lines

The total and component glucosinolates from different tissue types namely seeds, leaves, green pods, and developing seeds were determined using the established HPLC -based protocol (Augustine, R., Mukhopadhyay, A., & Bisht, N. C. (2013) Targeted silencing of BjMYB28 transcription factor gene directs development of low glucosinolate lines in oilseed Brassica juncea. Plant Biotechnology Journal, 11, 855- 866. https://doi.org/ 10.1111/pbi.12078). Briefly, glucosinolates were extracted from 10-20 mg of the lyophilized tissue in 1 ml of 70% methanol containing the internal standard (50 pM sinalbin), extract is passed through a customized Sephadex-A25 column, and treated with Sulphatase overnight. The desulpho-glucosinolates were eluted in 1 ml water and 10 pl run in a Shimadzu CLASS-VP V 6.14 HPLC machine. The program was set at solvent B (acetonitrile) gradient of 1-19% with respect to solvent A (water) through a 25 min cycle using the 250 mm HPLC column. The flow rate was maintained at 1 ml min ¹ and detection was made at 229 nm. Glucosinolate concentration was determined by identifying the substrate peak of known glucosinolates and referencing it with the internal standard peak (sinalbin) and applying the relative response factors. The final values were expressed as pmoles g~ ¹ DW. The total glucosinolate content and profiles were estimated from independent BjuGTR-edited lines and control plants, each in 3-4 replications. Data was checked for normal distribution and homogeneity of variance through Shapiro-Wilk and Levene’s test respectively in SPSS. A two-way mixed-design repeated measure ANOVA was performed on natural log transformed data and Tukey’ s post-hoc range tests were applied. Box plots were plotted using GraphPad Prism 6

Results:

[0067] B. juncea is an allotetraploid crop (AABB genome) resulted from interspecific hybridization between the mesopolyploid crop species B. rapa (AA) and B. nigra (BB) (Panjabi P, Jagannath A, Bisht NC, Padmaja L, Sharma S, Gupta V, Pradhan AK, Pental D (2008) Comparative mapping of Brassica juncea and Arabidopsis thaliana using Intron Polymorphism (IP) markers: homeologous relationships, diversification and evolution of the A, B and C Brassica genomes. BMC Genomics 9: 113; Paritosh K, Yadava SK, Singh P, Bhayana L, Mukhopadhyay A, Gupta V, Bisht NC, Zhang J, Kudrna DA, Copetti D, Wing RA, Reddy Lachagari VB, Pradhan AK, Pental D (2021) A chromosome-scale assembly of allotetraploid Brassica juncea (AABB) elucidates comparative architecture of the A and B genomes. Plant Biotechnol J. 19: 602-614. doi: 10.1111/pbi.13492). As a consequence, the oilseed B. juncea genome contains up to six homologs each of the BjuGTRl and BjuGTR2 genes (Nambiar DM, Kumari J, Augustine R, Kumar P, Bajpai PK, Bisht NC (2021) GTR1 and GTR2 transporters differentially regulate tissue-specific glucosinolate contents and defence responses in the oilseed crop Brassica juncea. Plant Cell & Environment 44, 2729-2743; Nour-Eldin HH, Madsen SR, Engelen S, Jprgensen ME, Olsen CE, Andersen JS, Seynnaeve D, Verhoye T, Fulawka R, Denolf P, et al. (2017) Reduction of antinutritional glucosinolates in Brassica oilseeds by mutation of genes encoding transporters. Nat Biotechnol 35: 377-382). For this study, the most conserved region from the second exon was selected and three guide RNAs (gRNAs) were designed that all together can target the 10 well-expressed BjuGTRl and BjuGTR2 homologs (Figure la). The two least expressed BjuGTRl homologs viz., BjuGTRl -A3 and BjuGTRl -B3 (Nambiar DM, Kumari J, Augustine R, Kumar P, Bajpai PK, Bisht NC (2021) GTR1 and GTR2 transporters differentially regulate tissue-specific glucosinolate contents and defence responses in the oilseed crop Brassica juncea. Plant Cell & Environment 44, 2729- 2743) displayed sequence divergence with three gRNAs selected and were therefore not considered for editing.

[0068] To examine the potential of the GTRl:GTR2(GEd) construct as prepared in Example 1 (Figure lb) for achieving the low SGC, the B. juncea cultivar Varuna as maintained in Example 2, which accumulates a high amount of SGC (134.44+5.01 pmoles g ¹ dry weight (DW)) was transformed with the construct. A total of 37 primary transformants (TO) were generated and SGC content in the T1 seeds ranged from 6.21-145.88 pmoles g ¹ DW (Figure 1c). A significant reduction of SGC at par with the Canola quality standard (<30 pmoles g ¹ DW) was observed in 30 out of 37 tested events. The major anti-nutritive glucosinolates of B. juncea seeds (sinigrin, gluconapin) were found to be reduced significantly in the low SGC events. The low SGC was subsequently tested in the T2 seeds of 8-10 T1 segregating progeny in each of the 17 representative events and was found to be inherited stably.

[0069] Further the editing of all the 10 target BjuGTR homologs among low (<30 pmoles g^-1 DW; 17 events) and high (>70 pmoles g^-1 DW; 5 events) SGC events (TO) generated using the GTRl::GTR2(GEd) construct was checked (Figure Id). The use of three gRNAs in a single construct provided >86% mutation efficiency in the four BjuGTRl and six BjuGTR2 targets. More than 90% of mutations in BjuGTR , were found to be of single nucleotide insertion (+1N). Further, a high frequency of biallelic homozygous (BHo; mutl / mutT) and biallelic heterozygous (BHt; mutl / mut2) mutations were observed in most of the target BjuGTR homologs. A close comparison suggested that the lines with low SGC exhibit functional mutations in BjuGTRl and BjuGTR2 homologs, showing either frame-shift or missense mutations. However, in the events containing high SGC most of the BjuGTR2 homologs in particular exhibited functional alleles, either due to monoallelic mutations (Mo; wt/ mutl) or the presence of shorter deletions in the multiple of 3 bp (marked as mutl * in Figure Id).

[0070] Overall, the data suggested that editing of a few BjuGTRl and at least four BjuGTR2 homologs, namely BjuGTR2-Al, BjuGTR2-A2, BjuGTR2-A3 and BjuGTR2-B2 is needed for achieving the low SGC in oilseed mustard. [0071] The use of CRISPR/Cas9 mutagenesis provided with an opportunity to identify the transgene-free (Cas9-free) edited plants in the T1 generation since the mutations in BjuGTR homologs segregated independently of the T-DNA locus that encodes the Cas9 nuclease and sgRNA. A total of 764 T1 progeny were screened for Basta segregation analysis, from which a total of 23 independent transgene-free (Basta sensitive) edited lines with low SGC were obtained and propagated to the subsequent T2 generation (Example 3). Mutation screening of a few representative transgene-free T1 progeny further confirmed the inheritance of CRISPR/Cas9- induced mutations in BjuGTRs, which correlates well with the reduced SGC observed in T2 seeds (Figure Id).

[0072] Glucosinolate is a maternally influenced trait, wherein leaf and siliques act as the source tissues for glucosinolates that accumulate in seeds (Chen S, Petersen BL, Olsen CE, Schulz A, Halkier BA (2001) Long-Distance Phloem Transport of Glucosinolates in Arabidopsis, Plant Physiology, 127:194-201, https://doi.org/10.1104/pp.127.L194; Jprgcnscn ME, Nour-Eldin HH, Halkier BA (2015) Transport of defense compounds from source to sink: lessons learned from glucosinolates Trends in Plant Science 20: 508-514 https://doi.Org/10.1016/j.tplants.2015.04.006; Nour-Eldin HH, Andersen TG, Burow M, Madsen SR, Jprgensen ME, Olsen CE, Dreyer I, Hedrich R, Geiger D, Halkier BA (2012) NRT/PTR transporters are essential for translocation of glucosinolate defence compounds to seeds. Nature 488: 531-534). Therefore, the potential of BjnGTR-editing in shaping the glucosinolates accumulation in different tissue types, namely flag leaf (post-bolting stage) and siliques of the 23 transgene-free low SGC B. juncea lines was assessed. The leaf glucosinolate content (LGC) in T2 progeny was found to be ranging from 75.84-105.32 pmoles g ¹ DW, which was broadly categorized into two distinct glucosinolate chemotypes, namely Ct-I and Ct-II (Figure 2b). A total of 13 out of 25 tested lines categorized under Ct-I accumulated significantly higher LGC (99.34 ±1.35 pmoles g ¹ DW) compared to the control plants (69.87 ±1.44 pmoles g ¹ DW). The Ct-II class comprises 10 lines that had marginally higher LGC (84.90 ±0.75 pmoles g ¹ DW) compared to the control plants (Figure 2c). [0073] Since silique is the economically important organ determining the seed yield, nutritional quality and stress tolerance to the encapsulated developing seeds (Bennett EJ, Roberts JA, Wagstaff C (2011) The role of the pod in seed development: strategies for manipulating yield. New Phytology 190: 838-853. doi: 10.1111/j.l469- 8137.2011.03714.x; Smith MR, Rao IM, Merchant A (2018) Source-Sink Relationships in Crop Plants and Their Influence on Yield Development and Nutritional Quality. Front Plant Sci. 9: 1889. doi: 10.3389/fpls.2018.01889), the glucosinolate content in the pod wall (PWGC) and the developing green seeds (gSGC) of the 23 transgene-free edited lines was also estimated. As also observed for the LGC, a high amount of PWGC was accumulated in the lines belonging to Ct- I (53.24 ±1.55 pmoles g ¹ DW), whereas Ct-II lines accumulated PWGC (37.04 ±1.11 pmoles g ¹ DW) marginally lower than the wild-type plants (39.93 ±1.47 pmoles g ¹ DW) (Figure 2c). All the tested lines accumulated negligible to low amounts of gSGC compared to the control plants (Figure 2c). This low gSGC accumulation in all possibilities could be attributed to the limiting glucosinolate transport from the pod wall to the developing green seeds in the BjuGTR-edited lines. Further, up on maturity, the seeds of all the lines showed a significant reduction of mean SGC ranging from 15.12 to 29.03 pmoles g ¹ DW (Figure 2a).

[0074] The generation of two distinct glucosinolate chemotypes (Ct-I and Ct-II) led to exploration the genetic basis of this phenomenon by analyzing the BjuGTRs mutations in the advanced T2 progeny (Figure 2d). It was observed that the plants belonging to Ct-I have loss-of-function mutations in at least four BjuGTR2 homologs (BjuGTR2-Al , BjuGTR2-Bl, BjuGTR2-A2 and BjuGTR2-B2) while at least one of the well-expressed BjuGTRl remained functional which providing a low SGC with concomitant over- accumulation of EGC and PWGC (Figure 2d). However, in Ct-II lines, the loss-of-function of all well-expressed BjuGTRl homologs along with four non-functional BjuGTR2 homologs leads to a minor reduction of EGC and PWGC. [0075] In Arabidopsis, GTR1 is primarily involved in the distribution within the leaf, potentially including import into the glucosinolate-rich S-cells located adjacent to the phloem, whereas GTR2 has a major role in apoplasmic phloem-loading of glucosinolates (Nour-Eldin HH, Andersen TG, Burow M, Madsen SR, Jprgcnscn ME, Olsen CE, Dreyer I, Hedrich R, Geiger D, Halkier BA (2012) NRT/PTR transporters are essential for translocation of glucosinolate defence compounds to seeds. Nature 488: 531-534). The present study thus demonstrates that for the generation of B. juncea lines with superior glucosinolate chemotype, a few well- expressed BjuGTRl are needed to be functional to maintain the optimal distribution of glucosinolates in leaves and pod wall.

[0076] For biotechnological applications, the CRISPR/Cas9-based manipulation of these transporters needs to be achieved in a very precise manner, without having any detrimental effects on the plants. To check the effect of B/7/G77 -cditing on various growth and agronomical parameters, the selected 23 transgene-free edited lines with low SGC were grown under the contained field condition in a net-house. Proper seed germination, growth phenotype, pollen viability, and seed set in all the lines belonging to Ct-I and Ct-II was observed. Furthermore, the 1000 seed weight and various seed quality parameters including oil content, protein content, and fatty acid compositions in the B/wG77 -cditcd lines were also found to be comparable to the wild-type Varuna - the national check cultivar of B. juncea.

[0077] Figure 5 depicts editing pattern of BjuGTR homologs in 40 independent TO events, generated using BcoCas9:BjuGTRl::GTR2(GEd) construct.

[0078] From the Figure 5, it can be seen that the construct BcoCas9:BjuGTRl::GTR2 (sequence of BcoCas9 (codon optimized Cas9) as represented in SEQ ID NO: 20) was able to reduce the content of seed glucosinolate in E42 and E54 lines.

Advantages of the present disclosure

[0079] The present disclosure discloses expression system comprising gRNA and Cas9 protein for modifying the glucosinolate content in plants. The said expression cassette allows development of ideal low seed glucosinolate chemotype of oilseed mustard by editing of multiple GTR1 and GTR2 genes. Accordingly, B.juncea lines having seed glucosinolates content (SGC) as low as 6.21 pmoles g ¹ DW has been developed in the present disclosure. The transgene-free B/wG77 -cditcd mustard lines with low SGC content is a significant improvement to those reported earlier in the rapeseed-mustard cultivars. [0080] Furthermore, the higher accumulation of glucosinolates in the leaf and pod wall of the several BjuGTR-edited lines will improve the plant’s ability to defend against a variety of pests and pathogens. These findings are of broader significance owing to the extreme importance of glucosinolates in plant defense (Clay, N. K., Adio, A. M., Carine, C., Jander, G., & Ausubel, F. M. (2009). Glucosinolate metabolites required for an Arabidopsis innate immune response. Science, 323, 95- 101. https://doi.org/10.1126/science.1164627; Hopkins, R. J., van Dam, N. M., & van Loon, J. J. A. (2009). Role of glucosinolates in insect plant relationships and multitrophic interactions. Annual Review of Entomology, 54, 57-83)., While the Canola quality seed meal will overcome the dependence on the expensive soybean imports in regions, this improved strategy will also allow a substantial reduction in the amount of insecticide used in the global rapeseed-mustard cultivation. The study also opens a new generation of opportunities for CRISPR/Cas9-mediated targeting of the complex traits towards fulfilling the breeding milestones of the Brassica crops.

Claims

I/We Claim:

1. A recombinant expression cassette comprising:

(a) a guide polynucleotide (gRNA) complementary to a target sequence, operably linked to a promoter, wherein the target sequence has a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8. SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, and wherein the guide polynucleotide has contiguous nucleotides complementary to the target sequence in the range of 15-20 nucleotides;

(b) a scaffold region intervening gRNAl, gRNA2, and gRNA3 having nucleotide sequence as set forth in SEQ ID NO: 15; and

(c) a polynucleotide encoding Cas9 protein having a nucleotide sequence selected from sequences as set forth in SEQ ID NO: 14, SEQ ID NO: 20 or SEQ ID NO: 21, operably linked with a promoter, wherein the recombinant expression cassette is effective in modifying the glucosinolate content in a plant.

2. The recombinant expression cassette as claimed in claim 1, wherein the gRNA is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

3. The recombinant expression cassette as claimed in claim 1, wherein the gRNA comprises SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

4. A recombinant expression cassette comprising:

(a) a guide polynucleotide (gRNA) construct comprising gRNAl, gRNA2, and gRNA3, each operably linked to a promoter, wherein gRNAl is complementary to a target sequence having a nucleotide sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, gRNA2 is complementary to a target sequence having a nucleotide sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, , and gRNA3 is complementary to a target sequence having a nucleotide sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and wherein the gRNAl, gRNA2 and gRNA3 each has contiguous nucleotides complementary to the target sequence in the range of 15-20 nucleotides;

(c) a polynucleotide encoding Cas9 protein having a nucleotide sequence selected from sequences as set forth in SEQ ID NO: 14, SEQ ID NO: 20 or SEQ ID NO: 21, operably linked with a promoter, wherein the recombinant expression cassette is effective in modifying the glucosinolate content in a plant. The recombinant expression cassette as claimed in claim 1, wherein the promoter driving the expression of the gRNA is AtU6-26 promoter, or orthologs thereof from Brassica species. The recombinant expression cassette as claimed in claim 1, wherein the promoter driving the expression of Cas9 protein is selected from the group consisting of CsVMV promoter, CaMV 35S promoter, FMV promoter, MMV promoter, and RbcS promoter. The recombinant expression cassette as claimed in claim 1 or 4, wherein the expression cassette further comprises 5’ untranslated region (UTR) upstream to the polynucleotide encoding the Cas9 protein, and wherein the 5’ UTR is a synthetic sequence (synJ). A recombinant vector comprising the recombinant expression cassette as claimed in any one of claims 1-7. A host cell comprising the recombinant vector as claimed in claim 8, wherein the host cell is E. coli or Agrobacterium tumefaciens. A process for modifying the glucosinolate content in a plant, comprising targeting the expression of sequences selected from the group consisting of SEQ ID NO: 4 to 13 in said plant by the recombinant expression cassette as claimed in claims 1-7. A process for producing a transgene- free edited plant with modified glucosinolate content, said process comprising: transforming a plant cell with the recombinant vector as claimed in claim 8 or the host cell as claimed in claim 9 to obtain stably transformed plant cells, selecting a transgene-free edited plant cell from the stably transformed plant cells, and growing the transgene-free edited plant cell for producing a transgene - free edited plant with modified glucosinolate content, wherein the transgene-free edited plant has reduced glucosinolate content in seeds and increased glucosinolate content in leaves and pods compared to a control non-edited plant. The process as claimed in claim 8, wherein the plant is selected from the group consisting of Brassica nigra, B. rapa, B. oleracea, B. juncea, B. napus, B. carinata, Camelina sativa, Capsella rubella, Sinapis alba, and Arabidopsis thaliana. A transgene-free edited plant having reduced glucosinolate content in seeds and increased glucosinolate content in leaves and pods produced by the process as claimed in claim 11. The transgene-free edited plant as claimed in claim 13, wherein the edited plant has glucosinolate content in seeds in a range of 6-30.00 pmoles g ¹ dry weight and glucosinolate content in leaves in the range of 75.84 to 105.32 pmoles g ¹ dry weight, and pods in the range of 25.83 to 64.69 pmoles g ¹ dry weight.