Claims:

CLAIMS

l. A method for isolating a nucleic acid sequence encoding glutamate decarboxylase gene that exhibits enhanced tolerance to environmental stresses; comprising, incorporating into a plant's genome a DNA construct comprising a promoter operably linked to a nucleotide sequence that encodes a functional glutamate decarboxylase (GAD) enzyme and method of editing the GAD gene using CRISPR/Cas.

2. The method according to claim 1, wherein the nucleotide sequence that encodes a functional glutamate decarboxylase enzyme comprises a nucleotide sequence set forth in SEQ IDNo.l.

3. The method according to claim 1, wherein the promoter is selected from the group consisting of a constitutive promoter, an inducible promoter, a tissue specific promoter and a cell type specific promoter operably linked to the nucleotide sequence set forth in SEQ ID No. l.

4. The method according to claim 2, wherein the glutamate decarboxylase enzyme comprises an amino acid sequence set forth in SEQ ID NO. 2.

5. The method according to claim 4, wherein the amino acid sequence as set forth in SEQ ID No.2 is effective to catalyze a reaction of glutamic acid to gamma-amino-butyric acid (GABA).

6. The method according to claim I, wherein the target plant is selected from the group consisting of monocots, dicots, cereals, forage crops, legumes, pulses, vegetables, fruits, oil seeds, fiber crops, flowers, horticultural, medicinal and aromatic plants.

7. The method according to claim l-6 wherein a cell tissue or organ from a host plant is transformed with the DNA construct mediated by using particle gun, biolistic or Agrobacterium or protoplast delivery.

8. The method according to claims I - 8, wherein the plant exhibits significantly enhanced tolerance to environmental stress selected from the group consisting of salt stress, drought, mechanical shock, heat, cold, salt, flooding, wounding, anoxia, pathogens, ultraviolet-B. nutritional deprivation, and combinations thereof.

9. The method according to claims I - 8, wherein, the product of Glutamate decarboxylase gene expression, GABA, is useful in the development of medicines or food materials.

Dated this 23 day of July, 2020

Avesthagen Limited

Signature:¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬¬__________________________

Name of the Signatory: Dr. Villoo Morawala Patell

To
The Controller of Patents,
The Patent Office at CHENNAI

, Description:FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003

COMPLETE SPECIFICATION
(See Section 10, rule 13)

Title of Invention

"CRISPR/Cas Genome editing of Glutamate Decarboxylase to improve environmental stress tolerance in the field conditions"

Applicant Name:

Avesthagen Limited
THE dry lab, Yolee Grande
2nd Floor, 14, Pottery Road,
Richards Town,
Bangalore, Karnataka
India - 560005
Email: villoo@avesthagen.com
M No. +91-9886037291

Name and Address of the Applicant’s Patent Agent/Patent Counsel:
Mr. HARIKRISHNA S HOLLA, Holla Associates-Advocates and IPR Consultants,
#193, ‘Kashi Bhavan’, 6th Cross, Gandhi Nagar
Bangalore 560 009, Karnataka State, India
Tel: +91-80-2228 0778; +91-80-2228 0778; Cell: +91-98440 36805
E-mail: hariholla@gmail.com

The following specification particularly describes and ascertains the invention and the manner in which it is to be performed.

FIELD OF THE INVENTION

The present invention relates to the isolation of plant nucleic acids and DNA encoding Glutamate decarboxylase (GAD). The transcripts and protein of GAD emerges in the plants subjected to environmental stress thereby conferring valuable pharma and agro properties. In addition, present invention also refers to genome editing of GAD gene coding sequence, 5' UTR and 3' UTR using sequences specific nucleases to develop organisms with stress tolerance, pharma, biomass and nutritional properties.

BACKGROUND OF THE INVENTION

Salinity stress negatively impacts agricultural yield throughout the world. The plant response to salinity consists of numerous processes that must function in coordination to alleviate both cellular hyperosmolarity and ion disequilibrium. In the present invention methods and materials for making plants having an enhanced ability to withstand environmental stress and having desirable morphological and/or agronomic characteristics or the like, are provided through plant genetic engineering. More particularly, the invention relates to genetic transformation of plants with genes that enhance a plant's ability to synthesis glutamate decarboxylase enzyme, which catalyzes the conversion of glutamic acid to GABA thereby enhancing the plant's ability to withstand stress or imparts other desirable characteristics.

As a background to the invention, the enzyme GAD (glutamic acid decarboxylase) has been shown to catalyze the formation of gamma amino butyric acid (GABA) from glutamate (Glu), and several plant GAD genes have been cloned. The rapid accumulation of GABA in plant cells after exposure to stress has been well documented. The production of GABA by a-decarboxylation of glutamate facilitated by the enzyme GAD is proposed to be the major source through which GABA accumulates in plants after stress. However, GABA is also biosynthesized by other metabolic pathways like the one associated with the catabolism of polyamines or through a part of GABA shunt by the reversible GABA amino transferase reaction. Experiments with soybean cotyledons or Asparagus cell suspension culture suggests that formation of GABA by the metabolism of glutamate is a normal phenomenon and that biosynthesis of GABA is not a response to stress under the conditions studied.
In contrast to the above observation, GABA has also been shown to rapidly accumulate in plants subjected to mechanical stimulation, variation in temperatures like cold or heat shock conditions.

In view of this background, it is seen that significant effort has been devoted to studying GABA synthesis and GAD enzyme activity in plants; however, a direct role for GABA in plants to impart salinity tolerance has not been demonstrated. Therefore, the present invention is a significant advance in this field. Moreover, employing sequence specific nucleases to edit and rewrite the GABA pathway genes has not be achieved at. Therefore, the present invention offer a novel approach for improving plants and other organisms with useful properties for global application.

PRIOR ART

The pathway that converts glutamate to succinate via GABA is called the GABA shunt. The first step of this shunt is the direct and irreversible a-decarboxylation of glutamate by glutamate decarboxylase (GAD, EC 4.1.1.15). The second enzyme involved in the GABA shunt, GABA transaminase (GABA-T; EC 2.6.1.19), catalyzes the reversible conversion of GABA to succinic semialdehyde using either pyruvate or a-ketoglutarate as amino acceptors. The last step of the GABA shunt is catalyzed by succinic semialdehyde dehydrogenase (SSADH; EC 1.2.1.16), irreversibly oxidizing succinic semialdehyde to succinate (Vanderwalle and Olsonn, 1983; Patterson and Graham, 1987; Breitkreuz & Shelp, 1995; Shelp et al, 1985). Glutamate decarboxylase provides a link between intermediary amino acid metabolism and perturbations of cytosolic Ca2+ (Ling et al, 1994, Balm & Fridmann, 1996). Evidence also suggest that GAD accumulation is stimulated plant cells due to changes environmental condition and Ca2+ levels. Thus, GAD ultimately alters the levels of glutamate and Gamma-Amino butyric acid (GABA). These two molecules are important for molecular signaling. Hence, GAD has a direct role stress signalling and its transduction.

In the native state, GAD enzyme exists in the form of a dimer of 120 kilodaltons. After reduction of the disulphide bridges, examination by specific electro immuno analysis (Western blotting) reveals two bands at 65 and 67 kilodalton. It was recently demonstrated that these two monomers correspond to two different proteins encoded by distinct genes (Erlander et al., Neuron 7 (1991) 91). The two molecules, herein after called GAD 67 and GAD 65, differ from an enzymatic point of view, the short form having a lower affinity for pyridoxal phosphate, and by their subcellular localization, the short form being better represented in the neuronal extensions whereas the long form accumulates
in the perikaryon. The long form, by virtue of its lower dependence on the concentration of pyridoxal

phosphate, is therefore the one most capable of being expressed in various types of cells. GAD 65 on the other hand has the feature of becoming rapidly inactivated in the absence of pyridoxal phosphate by becoming converted to an apoenzyme, lacking catalytic activity. The reverse conversion, from apoenzyme to active holoenzyme, is relatively slow.

GABA is a four-carbon non-protein amino acid conserved from bacteria to plants and vertebrates. GABA is a significant component of the free amino acid pool. GABA has an amino group on the gamma-carbon rather than on the alpha carbon, and exists in an unbound form. It is highly soluble in water: structurally it is a flexible molecule that can assume several conformations in solution, including a cyclic structure that is similar to proline. GABA is zwitterionic (carries both a positive and negative charge) at physiological pH values (pK values of 4.03 and 10.56). GABA accumulation is induced in response to sudden decrease in temperature (Wallace, et al, 1984) in response to heat shock (Mayer et al, 1990), mechanical manipulation (Wallace, et al, 1984) and water stress (Rhodes et al, 1986), and due to insect and pest attack (Ramputh & Brown, 1996). The gabaergic pathways represent the main group inhibiting the nervous system in vertebrates. Impairment of the activity of GABAergic, neurons manifests itself immediately in the whole body by dyskinesias or convulsions. Furthermore, the role of cerebral gamma-amino-butyric acid (GABA) is not limited to neurotransmission. A neurotrophic action during development has indeed been attributed to it, in particular in the neuroretina. Moreover, GABA is also present in the beta cells of the islets of Langerhans, where it seems to play a role in the regulation of the production of insulin. The possibility of restoring or installing de novo a GABA synthesis in a precise region of the body therefore has a major therapeutic advantage both for conditions directly linked to the degeneration of GABAergic neurons and for those, which respond to GABA agonists. The present invention thus describes a new approach for treating conditions linked to a GABA deficiency, consisting in inducing the in vivo synthesis of GABA by targeted release of a biologically active enzyme.

GABA is a naturally occurring inhibitory neurotransmitter which has ready access to the nervous system of invertebrates, but not that of vertebrates such as humans, and has been shown to deter insect growth and development (Ramputh and Bown, 1996 Plant Physio. 134l-1349). Typically, GABA levels are low in plants (ranging from 0.03 to 2.00 J.imol/g fresh weight (FW)), but increase several fold in response to many diverse stimuli such as insect walking and feeding (i.e. biotic stress) and temperature shock (i.e. abiotic stress). This result a be attributed to increases in cytosolic H+ or calcium/calmodulin levels which directly affect the activity of the enzyme responsible for the

synthesis of GABA, namely glutamate decarboxylase (GAD) (Shelp et al., 1999 Tr. Plant Sci. 4:446). In particular, calcium/calmodulin binds to a carboxyl-terminal domain on the GAD gene, thereby relieving the autoinhibition of GAD activity. Although the endogenous synthesis of GABA appears to serve as a plant defense mechanism, further increases in GABA levels may lead to corresponding reductions in damage due to invertebrate pests. Transgenic plants which overexpress GAD and thereby cause GABA accumulation have been prepared; however, these plants do not express invertebrate pest-resistant root GABA levels and have questionable utility in that they exhibit severe morphological abnormalities (Baum et al., 1996 EMBO Journal, 15:2988).

There are several genome editing tools discovered and adopted for genome editing, which includes, mega nucleases, ZFN, TALLEN and CRISPR/Cas. Of which CRISPR-Cas (clustered regularly interspaced short palindromic repeat/CRISPR-associated) is preferred technology to edit genomes of prokaryotes and eukaryotes, including plants due to its relative ease. Over the years CRISPR mediated genome editing has been reported in many crops including rice, corn, wheat, and soybean (Banakar et al. 2019, Sci Rep, 9, 1-3; Banakar et al, 2020, Rice, 13, 1-9; Chen et al. 2019, Annu Rev Plant Biol, 70, 667-679; Zhang et al. 2019, Nat Plants, 5, 778-794). Since, CRISPR/Cas has a nuclease property, it can cleave DNA at gRNA/DNA hybridization site. Hence, any given sequence in the genome can be cleaved. Thus, CRISPR editing can lead to production of double strand breaks (DSBs). These DSBs are repaired through either error prone non-homologous end joining (NHEJ), which results in either inframe or out of frame mutations due to indels or through a homology directed repair (HDR) to create gain of function mutation by homology search and copying (Banakar et al. 2019, Sci Rep, 9, 1-3; Banakar et al, 2020, Rice, 13, 1-9; Huang and Puchta, 2019, Plant Cell Rep, 38, 443-453). Therefore, CRISPR/Cas system can be used to edit the GAD gene in plants to create either structural variants with better enzymatic activity or can be used to edit to the expression levels to impart abiotic stress tolerance or pharma properties.

OBJECTS OF THE INVENTION

The present invention relates to a method of increasing salt tolerance in plants (monocotyledons and dicotyledons) with a glutamate decarboxylase gene. A method employing the glutamate decarboxylase gene from rice to increase the salt tolerance of plants has been demonstrated.
Abiotic stress is a complex environmental constraint limiting crop production. A bioengineering stress-signaling pathway to produce stress-tolerant crops is one of the major goals of agricultural

research. Osmotic adjustment is an effective component of such manipulations and accumulation of osmoprotectants (compatible solutes) is a common response observed in plant systems (Penna 2003). Other mechanisms by which compatible solutes protect plants from stress include detoxifying radical oxygen species and stabilizing the quaternary structures of proteins to maintain their function.

Given the complexity of the physiology and the genetics of salt tolerance, it has been a difficult task to generate salt-tolerant crops. There has been only limited success in this direction in the mid-1990s (Flowers and Yeo, 1995) and there has been little progress since then. A variety of approaches have been advocated, including conventional breeding, wide crossing, the use of physiological traits and, more recently, marker-assisted selection and the use of transgenic plants. None of these approaches could be said to offer a universal solution.

SEQUENCE LISTING;

SEQ ID 1 shows the nucleic acid sequence of Oryza sativa glutamate decarboxylase gene. The start and stop codons are written in bold letters.

SEQ ID 2 shows amino acid sequence of Oryza sativa glutamate decarboxylase gene. The start and stop codons are written in bold letters.

SEQ ID 3 shows the nucleotide sequence of Nicotiana tabacum glutamate decarboxylase gene. The start and stop codons are written in bold letters.

SEQ ID 4 shows the amino acid of Nicotiana tabacum glutamate decarboxylase gene. The start and stop codons are written in bold letters.

BRIEF DESCRIPTION OF ACCOMPANYING FIGURES

FIGURE 1 shows the plant transformation vector harboring the glutamate decarboxylase encoding DNA sequence.

FIGURE 2 shows the different stages in the transformation of tobacco leaves with GAD
gene through Agrobacterium mediated gene transfer

FIGURE 3 shows the PCR confirmation of the transformed and regenerated TO seedlings of tobacco with GAD gene with different combination of primers- a) HygR-gene forward and reverse; b) Gene specific forward and reverse primer and c) Gene forward and Nos reverse primer.

FIGURE 4 shows the better performance of T1 GAD transgenic tobacco seedlings (DIA, E2 and H1) under salt stress conditions (200 mM NaCI) grown on agar media in the light room.

FIGURE 5 shows the better performance of T1 GAD transgenic tobacco seedlings (DIA, E2 and Hl) under salt stress conditions (200 mM NaCl) grown on agar media in the light room.

FIGURE 6 shows the better performance of TI GAD transgenic tobacco seedlings (E2 and Hl) under salt stress conditions (300 mM NaCl) grown on hydroponics culture in the green house.

FIGURE 7 shows comparison of plant height between T1 seedlings from GAD transgenics (DIA, E2 and H1), which were positive for Hygromycin and the wild type seedlings when grown in pots in the green house with different levels of salt stress (0,200 & 300 mM NaCl).

FIGURE 8 shows comparison of internodal distance between T1 seedlings from GAD transgenics (DIA, E2 and H1), which were positive for Hygromycin and the wild type seedlings when grown in pots in the green house with different levels of salt stress (0, 200 & 300 mM NaCl).

FIGURE 9 shows comparison of number of leaves between T1 seedlings from GAD transgenics (DIA, E2 and HI), which were positive for Hygromycin and the wild type seedlings when grown in pots in the green house with different levels of salt stress (0,200 & 300 mM NaCl).

FIGURE 10 shows comparison of stem girth or thickness between T1 seedlings from GAD transgenics (DIA, E2 and Hl), which were positive for Hygromycin and the wild type seedlings when grown in pots in the green house with different levels of salt stress (0, 200 & 300 mM NaCI).

FIGURE 11 shows comparison of leaf area between T1 seedlings from GAD transgenics (DIA, E2 and HI), which were positive for Hygromycin and the wild type seedlings when grown in pots in the green house with different levels of salt stress (0, 200 & 300 mM NaCl).

FIGURE 12 shows comparison of total biomass between T1 seedlings from GAD transgenics (DIA, E2 and HI), which were positive for Hygromycin and the wild type seedlings when grown in pots in the green house with different levels of salt stress (0, 200 & 300 mM NaCl).

FIGURE 13 shows comparison of total grain yield between T1 seedlings from GAD transgenics (DIA, E2 and HI), which were positive for Hygromycin and the wild type seedlings when grown in pots in the green house with different levels of salt stress (0, 200 & 300 ml NaCl).

FIGURE 14 shows transgene expression levels determined on primary (T0) transgenic corn events. Red line = expression of GAD transgene, Blue = Endogenous control gene.

FIGURE 15 shows increased GABA in T02069 events. WT = Wild type maize; NS = Pool of non transgenic segregants; TR = pool of 10 transgenic ZmGAD events.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description of the invention is provided to aid those skilled in the art in practicing the present invention. Even so, the following detailed description of the invention should not be construed to unduly limit the present invention as modifications and variations in the embodiments discussed herein may be made by those of ordinary skill in the art without departing from the spirit or scope of the present invention.

This invention relates to a purified and isolated DNA sequence having characteristics of glutamate decarboxylase.

According to the present invention, the purified and isolated DNA sequence usually consists of a glutamate decarboxylase nucleotide sequence or a fragment thereof.

Included in the present invention are as well complementary sequences of the above­
mentioned sequences or fragment, which can be produced by any means.

Encompassed by this present invention variants of the above-mentioned sequences, that is nucleotide sequences that vary from the reference sequence by conservative nucleotide substitutions, whereby one or more nucleotides are substituted by another with same characteristics.

According to the present invention, the above-mentioned nucleotide sequences could be located at both the 5' and the 3' ends of the sequence containing the promoter and the gene of interest in the expression vector.

According to the present invention, corn plants were engineered with rice GAD (Os03g13300) expressed under the rice ubiquitin promoter resulting in the maize lines referred as T01957. Similarly, maize GAD (GRMZM2G017110) expressed under the Cassava Vein Mosaic Virus referred as T02069. Included in the present invention also the enhanced levels of GABA in corn tissues.

Included in the present invention are the use of above-mentioned sequences in increasing the salt tolerance of the plants produced thereof. "salt tolerance" means that after introduction of DNA sequence under suitable conditions into a host plant, the sequence is capable of enhancing the plant’s capacity to withstand high concentrations of salts in the growing environments in the plants as compared to control plants where the plants are not transfected with the said DNA sequence.

Another embodiment of the invention said transgene cassette containing vector is introduced into plant cell by Agrobacterium mediated transformation. Thus, present innovation exploits the natural double strand breaks created in the target genome to insert the expression cassette through non-homologous end joining or illegitimate recombination. Hence, said innovation can be approached by other transformation methods but not limited to transforming plants by biolistics, protoplast transformation and silicon carbide mediated transformation. Therefore, innovation presented here is not limited to one or the transformation methods.

To make the integration of gad transgene more specific in the plant genome, it is of nucleases performing targeted double strand breaks can be exploited. Such nucleases include but not limited to TALLEN (US10400225B2), ZFNs (Zinc Finger Nucleases), engineered homing endonucleases Such as I-SceI (WO9614408) and I-CreI (WO2004067736), MBBBDs (PCT/US2013/051783) and also Cas9/CRISPR systems (Jinek et al., 2012).

As evident from the innovation expression of GAD is important to achieve said innovation. Hence, achieving said innovation through enhanced expression of endogenous GAD gene in or by modifying the functionality of GAD gene is possible through any genome editing/engineering approaches.

The following definitions are used in order to help in understanding the invention. "Chromosome" is organized structure of DNA and proteins found inside the cell.
"Chromatin" is the complex of DNA and protein, found inside the nuclei of eukaryotic cells, which makes up the chromosome.

“DNA” or Deoxyribonucleic Acid contain genetic information. It is made up of different nucleotides.

A "gene" is a deoxyribonucleotide (DNA) sequence coding for a given mature protein. "gene" shall not include untranslated flanking regions such as RNA transcription initiation signals, polyadenylation addition sites, promoters or enhancers.

"Promoter" is a nucleic acid sequence that controls expression of a gene.

"Enhancer" refers to the sequence of gene that acts to initiate the transcription of the gene independent of the position or orientation of the gene.

The definition of "vector" herein refers to a DNA molecule into which foreign fragments of DNA may be inserted. Vectors, usually derived from plasmids, functions like a "molecular carrier", which will carry fragments of DNA into a host cell.

"Plasmid" are small circles of DNA found in bacteria and some other organisms. Plasmids can replicate independently of the host cell chromosome.

"Transcription" refers the synthesis of RNA from a DNA template. "Translation" means the synthesis of a polypeptide from messenger RNA. "Orientation" refers to the order of nucleotides in the DNA sequence.
"Gene amplification" refers to the repeated replication of a certain gene without proportional increase in the copy number of other genes.

"Transformation" means the introduction of a foreign genetic material (DNA) into plant cells by any means of transfer. Different method of transformation includes bombardment with gene gun (biolistic), electroporation, Agrobacterium mediated transformation etc.

"Transformed plant" refers to the plant in which the foreign DNA has been introduced into the said plant. This DNA will be a part of the host chromosome.

"Stable gene expression" means preparation of stable transformed plant that permanently express the gene of interest depends on the stable integration of plasmid into the host chromosome.

"CRISPR" refers to Clustered regularly interspaced short palindromic repeats.
"Cas" refers to CRISPR associated protein.
"gRNA" refers to guide RNA.
"Target" sequence in the genome chosen to edit.

Example 1

Isolation and purification of GAD gene nucleotide sequence from rice and construction of plant transformation vector

The GAD gene is cloned downstream of a CaMV 35s promoter and transcriptional termination is carried out by nos terminator to create functional expression casette.

Plant Material
Oryza sativa Cv. Rasi was used for preparation of nucleic acids. Seeds were germinated, seedlings were grown in hydroponic solution in a culture room. Six days old seedlings were treated with 150 mM NaCl for 7-16 h to induce salt stress conditions.

RNA extraction and EST Library construction
The RNA was extracted from the whole seedlings. An EST library of the salt stressed India rice Cv. Rasi by creating cDNA library. An EST showing identity to glutamate decarboxylase was identified from the EST library.

1. mRNA purification was performed by first, isolating high quality total RNA from 6 day old Rasi seedlings and, subsequently by isolating mRNA from total RNA using oligo (dT) cellulose in a filter syringe by making use of a double purification method.

2. mRNA was converted into first and second strand cDNA followed by San adapter addition, Notl digestion, cDNA vector ligation and transformation was performed to obtain the cDNA library.

3. The superscript™ plasmid system with Gateway™ for cDNA cloning and synthesis was employed throughout.

4. The clones obtained were picked, digested using Notl and San enzymes, to obtain the inserts and these were further sequenced and checked for homology.

5. The sequencing of the selected clones was done on ABI Prism, 377, DNA Sequencer

(Perkin Elmer).

CLONING OF GLUTAMATE DECARBOXYLASE GENE

The Glutamate decarboxylase gene has been cloned into a cloning vector and also into plant transformation vectors (biolistic and binary) under a constitutive promoter.

The cDNA encoding the complete coding sequence of glutamate decarboxylase gene was amplified from the indica rice (cv. Rasi) cDNA using the following pairs of primers tagged with BglII and EcoRI restriction enzyme sites (underlined nucleotide sequences)

Forward: 5'-GCGGATCCATGGTGCTCTCCAAGGCCGTCTC-3' Reverse: 5'-GCGAATTCCTAGCAGACGCCGTTGGTCCTCTTG-3'
Using the following PCR conditions 94°C for 1 min; 94°C for 30 sec; 75°C for 3 min (cycled for five times); 94°C for 30 sec; 68°C for 3 min (cycled for 30 times) with a final extension of 68°C for 7 min.
The amplified cDNA consists of 1479 bp amplicon. Amplicon was gel purified, and the amplified fragment was cloned into pGEMT easy vector.
Bacterial transformation was performed by freeze-thaw method.
From the select clones, plasmid extraction was performed. Using BamHI and EcoRI restriction digestion, clones were verified for 1479 bp insert.

Sanger sequencing of the clones was performed, and sequences were aligned for homology search and BLASTed using NCBI-BLAST tool.

From the pGEMT easy vector, gene was restriction digested using BamHI and EcoRI sites and ligated into a biolistic vector pVl. This biolistic vector was excised at BglII and EcoRI restriction sites (BglII and BamHI enzymes generate compatible ends) to confirm the presence of the gene. The gene was also confirmed by sequencing. The resultant vector (pVl-GAD) has the GAD gene (1.479kb) driven by CaMV 35s promoter and nos terminator along with the ampicillin resistance gene as a selectable marker.

The gene cassette, GAD gene cassette from pVl-GD was restriction digested at HindIII and BamHI sites and the cassette was ligated into pCAMBIA1390 using same enzyme combination. The resultant vector (pAPTV1390-GAD) has the GAD gene (1.479kb) driven by CaMV 35S promoter and terminated by nos terminator. In addition, pAPTV1390-GAD has npt (Kanamycin resistance) for bacterial selection and hpt (hygromycin phosphotransferase to impart selection to antibiotic hygromycin) as selectable markers for plant selection (Fig 1).

Example 2:

Generating plants with an altered GAD gene

Plant Transformations

The Glutamate decarboxylase gene has been transformed via Agrobacterium into tobacco

(model plant) to arrive at the proof of concept for the identified gene. Detailed steps involved in Agrobacterium mediated transformation of tobacco leaf explants with a binary vector harboring GAD gene are as below.
1. The positive colony of Agrobacterium was inoculated in to LB broth with 50mg/L Kanamycin (Kan) and 1Omg/L of Rifamicin (Rif) as vector backbone consists of Kan and Rif resistance gene, which also functions as double selection at one shot.
2. Then the broth was incubated at 28°C on a shaker.

3. The overnight grown colony was inoculated into 50mL LB broth with 50mg/L Kan and 10mg/L of Rifamicin the morning and incubated at 28°C for 3-4 hours and the OD was checked at 600nm and continued to grow till the OD was between 0.6-1.
4. Once the broth reached required OD the broth was centrifuged at 5000rpm for 5min.

5. The supernatant was discarded, and the cell pellet was dissolved in Murashige & Skooge

(MS) liquid medium (Agro-MS broth).

6. The tobacco leaves were cut into small square pieces which served as explants without taking the midrib and care was taken to injure leaf at all four sides without injuring much at the center part of the inoculants.
7. These leaf samples were placed in MS Plain media for two days in a BOD incubator. After

two days of inoculation these leaf samples were infected with transformed Agrobacterium

cells, which are now in Agro-MS broth.

8. The leaf explants were placed in this Agro-MS broth for 30m in and then placed them on co-cultivation media, which consist of MS + lmg/L 6-Benzyl amino purine hydrochloride (BAP) + 0.2mg/L Naphthalene acetic acid (NAA) + 250mg/L Cefotaxime for two days (Fig 2a)

9. After co-cultivation the explants were kept in first selection medium which consist of MS

+ lmg/L BAP + 0.2mg/L NAA + 40mg Hyg + 250mg/L Cefotaxime for 15 days and as the callus started protruding these explants were again sub cultured on to first selection media for callus to mature enough (Fig 2b)
10. Once the callus was found to be matured these callus were inoculated on to second selection medium which consist of MS + lmg/L BAP + 0.2mg/L NAA + 50mg Hyg +250mg/L Cefotaxime. As the concentration of Hygromycin is increased the escapes from first selection get suppressed and only the transformed callus starts surviving on this media.
11. Subsequent sub-cultures on this second selection media were done once in ten days.

12. By this time the plantlets started protruding from the callus. The plantlets from second selection

were taken and placed on to rooting media, which consist of 1/2 MS + 0.2mg/L Indole-3-butyric acid (IBA). Here the plantlets started have protruding roots by 12-15 days. Once the mature roots were formed the plants were subcultured on to rooting media along with 20mg/L of Hygromycin, as escapes can be identified at this stage also (Fig 2c).
13. Plants at this stage were subjected to acclimatization where the caps of bottles were kept open for two days so that plants get adjusted to its growth room environment. Later plants from agar medium were removed and placed on 1/4 MS liquid medium for two days. These plants were further transferred on to vermiculate and watered every day for one week.
14. Depending upon the condition of the plants suitable plants were transferred to green house.
15. Before sending plants to green house during acclimatization period old leaves from the

plants were collected.

16. DNA from respective leaf samples was extracted and PCR with gene specific primers and selection marker gene i.e. Hygromycin primers were performed. PCR confirmed positive plants were further transferred to green house.

Confirmation of plants with introduced GAD Gene

Genomic DNA extraction of GAD tobacco transgenic lines.

Leaf samples of transgenic GAD tobacco plant were collected and genomic DNA was extracted.

Procedure for genomic DNA extraction:

• Around lgm of leaf was collected from each plant.

• The samples were ground using liquid nitrogen in a pestle and mortar.

• 1 ml of extraction buffer Extraction buffer (0.2M Tris Cl pH- 8.0; 2M NaCl; 0.05 M

EDTA; 2% CTAB) was added to the sample and spun at 13000 rpm for 10 min

• Supernatant was collected. RNase [ 3µl (1 mg/mL) for 1ml] was added and incubated at 37°C for 1/2 an hour.
• Equal volumes of chloroform-isoamyl alcohol was then added and spun at 13000 rpm for 10 min.
• Supernatant was collected in fresh tubes and equal volumes of chilled Isopropanol was added and spun at 13000 rpm for 10 min.
• The pellet was washed with 70% alcohol and pellet was dried and dissolved in 30 µl warm autoclaved water.
• 1 µl of DNA was loaded and checked on gel.

The transgenic plants were confirmed by PCR with different combination of primers:

I. PCR with Hygromycin Forward (Hyg F) & Hygromycin Reverse (Hyg R) primers:

Reagent Stock Volume
Template DNA 1 µl
Hyg F 10 pM 0.5 µl
Hyg R 10 pM 0.5 µl
dNTPs 1 0 pM 0.5 µl
Taq DNA polymerase 3 u/ µl 0.3 µl
Taq Buffer A 10X 3 µl
Milli Q water 24.2 µl
Total Volume 30 µl
PCR conditions: (Eppendorf Machine)

Steps Temperature Time Cycle
1 94°C 3 mins
2 94°C 30 sees
3 50°C 50 sees
4 72°C 1min Go to step-2 30X
5 72°C 10 mins
6 l0°C 8

The amplified product was visualized on 0.8% agarose gel shown in Fig 3a.

2. PCR with Gene specific primers GAD Forward (GD F) & GAD Reverse (GD R):

Reagent Stock Volume
Template DNA 2 µl
GD F 10 pM 0.5 µl
GD R 10 pM 0.5 µl
dNTP’s 1 0 pM 0.5 µl
Taq DNA polymerase 3 U/ µl 0.3 µl
Taq Buffer A 10X 2 µl
Milli Q water 14.2 µl
Total Volume 20 µl

PCR conditions: (Eppendorf machine)

Steps Temperature Time Cycle
I 94°C 3 mins
2 94°C 30 sees
3 69°C 50 sees
4 72°C 1.30 min Go to step-2 35X
5 72°C 10 mins
6 10°C 8

The amplified product was visualized on 0.8% agarose gel (Fig 3b)

3. PCR with GD F & Nos MR:

Reagent Stock Volume
Template DNA 2 µl
GD F 10 pM 0.5 µl
Nos MR 10 pM 0.5 µl
dNTP’s 10 mM 0.5 µl
Taq DNA polymerase 3 U/ µl 0.3 µl
Taq Buffer A 10X 2 µl
Milli Q water 14.2 µl
Total Volume 20 µl

PCR conditions: (Eppendorf machine)

Steps Temperature Time Cycle
1 94°C 3 mins
2 94°C 30 sees
3 67°C 50 sees
4 72°C 2 min Go to step-2 35X
5 72°C 10 mins
6 10°C 8

The amplified product was visualized on 0.8% agarose gel shown in Fig 3c.
Primer sequences used in different PCR reactions are listed below: Hyg F : 5'-CTGAACTCACCGCGACGTCT-3'
Hyg R : 5'-CCACTATCGGCGAGTACTTC-3'
GD F : 5'-GCGGATCCATGGTGCTCTCCAAGGCCGTCTC-3'
GD R :5'-GCGAATTCCTAGCAGACGCCGTTGGTCCTCTTG-3' NOS MR: 5'-GATAATCATCGCAAGACCGGCAAC-3'

Example 3

Evidence that plants with altered GAD gene tolerate salt stress at seedling stage
Tolerance of the transgenic plants to salt stress was studied in the Tl generation both at seedling stage and during the adult plant stage encompassing the whole life cycle of the plant. Salt tolerance at seedling stage on media

The salt stress experiments were performed with the wild type and Tl GAD transgenic tobacco seedling. The Tl seeds were surface sterilized by washing twice with sterile water (2-3 min) followed by a wash with 70% alcohol for 2 min and then treated with 70% bleach for 10 min and

finally washing with sterile water for 5-6 times. The seeds were then blot dried and placed on the Y2 MS media plates with different salt concentrations (0, 50 and 200, mM NaCl) and were incubated at 28°C in the dark for germination. After germination they were shifted to light room under 16h light and 8h dark cycle.

Three of the transgenics events- DIA, E2 and HI showed tolerance to 200 mM NaCl as compared to the wild type (Fig 4). The wild type seeds did germinate on 200 mM NaCl but failed to put up a good growth. The presence of high salt concentration in the growth media inhibited the proper growth of the wild type seedlings (plants without the introduced GAD gene) while the presence of high salt did not affect the normal growth of the transgenic seedlings as the introduced GAD gene had rendered them to be tolerant to high salt concentrations in the growth media.
The GAD transgenics performed better than the wild type plants under high salinity conditions for the different agronomic and physiological status of the plants thus indicating the role of GAD gene for the superior performance of the transgenics under salt stress conditions.

Example 4:

Genome Editing of Glutamate decarboxylase from Tobacco.

Glutamate decarboxylase DNA sequence gene from Nicotiana tabacum was PCR amplified using primer pair designed from Sequence ID3.

Sanger sequencing of the amplified PCR product was performed after cloning into pJE1.2.

Next generation sequencing of the amplified product was performed.

Obtained sequence was aligned with the reference sequence to identify possible single nucleotide polymorphism.

Sequence analysis was performed by analyzing chromatograms using SnapGene 5.0 Viewer
(https://www.snapgene.com/snapgene-viewer/),

Alignment of DNA sequences was performed by Clustal Omega with the reference sequence (https://www.ebi.ac.uk/Tools/msa/clustalo/)

NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch search was
performed to check for amount of homology with known sequences in Tobacco.

sgRNA design and vector construction:

Guide RNA were selected by CRISPR genome analysis tool (CGAT, http://cbc.gdcb.iastate.edu/cgat/) developed by Iowa State University, USA.
Selected guides were cloned into CRISPR/Cas binary vector system, in which sgRNA is expressed from Arabidopsis thaliana U6 promoter. For proper expression in tobacco, Cas coding sequence was tobacco codon optimized. Cas expression is driven by CaMV 35s promoter and transcript termination is carried out by Cauliflower Mosaic Virus 35s Terminator (T35s). CRISPR/Cas construct also contain gene for hygromycin phosphotransferase (hpt-to select cells which are transformed), which is driven by CaMV 35s promoter, and transcriptional termination is by T35s. The construct was sequenced to verify sequence homology. This plasmid here onwards will be referred as pRB19. Controls vectors encoding the cassette for hpt (pRB20), Cas (pRB21), and gRNA (pRB22) were also developed.
From an overnight grown bacterial culture, plasmid DNA was prepared using QIAprep Spin Miniprep Kit, Midi prep or Maxi Prep (Qiagen Inc-USA, Germantown, MD), by following the manufacturer’s protocol.
Plasmid prepared was used for the biolistic transformation of tobacco was performed as previously described (Elgabi et al 2011. Plant J, 67, 941-948) with modifications in biolistic parameters.
For Agrobacterium mediated transformation, pRB19, pRB20, pRB21 and pRB22 were transformed into LBA4404 strain of Agrobacterium by freeze thaw method.
Agrobacterium mediated transformation was performed as described in example 2.
Transient editing efficiency at the target site was performed by sacrificing the portion of tissue culture clones for DNA extraction.
Next generation sequencing (NGS) was employed to establish the editing efficiencies.
In the regenerated plants editing efficiencies were established either by amplicon sanger sequencing or by NGS. Biallelic and monoallelic edited events were identified, forwarded to the next generation.

Edited events (biallelic and monoallelic) which did not carry pRB2 were identified for stress tolerance and phenotypic performance.
Plants with stable mutation pattern were established and evaluated in field for stress tolerance.
Transient editing efficiencies will be evaluated.
Transgenic lines were generated as described in Example 2.

DNA extraction was performed from regenerated transgenic plants.
Evaluation of the transgenic plants was be done for editing efficiencies by sanger and NGS sequencing.
Events with required type of genome editing outcomes were forwarded to next generation.
Appropriate next generation genome edited events without the CRISPR-Cas/gRNA constructs were evaluated for GAD expression levels and salt tolerance.
Best performing mutation genotype were forwarded to the next generation to evaluate trait stability.
Field evaluation with GAD mutant genotype will be performed as and when necessary.

Dated this 23 day of July, 2020
Avesthagen Limited

Signature: ____________________________

Name of the Signatory: Dr. Villoo Morawala Patell

To
The Controller of Patents,
The Patent Office at CHENNAI