METHODS FOR ENHANCING GENOME ENGINEERING EFFICIENCY

Abstract

This document relates to methods and materials for genome engineering in eukaryotic cells, and particularly to methods for increasing genome engineering (i.e. transformation or genome editing) efficiency via co-delivery of one or more chemicals, such as protein deacetylase inhibitors, phytohormones and/or regeneration boost genes, with genome engineering components.

Claims

1. A method for genetic modification in a plant cell comprising (a) co-introducing into the plant cell (i) a genome engineering component and (ii) a second compound comprising (ii.1) an epigenetically regulating chemical or an active derivative thereof, in particular a DNA methyltransferase inhibitor or a protein deacetylase inhibitor, preferably histone deacetylase inhibitor (HDACi), and/or (ii.2) a phytohormone or an active derivative thereof, preferably selected from auxins, cytokinins and combinations thereof and/or (ii.3) a protein causing improved plant regeneration from a somatic cell, a callus cell or embryonic cell or an expression cassette comprising a nucleic acid encoding said protein, and (b) cultivating the plant cell under conditions allowing the genetic modification of the genome of said plant cell by activity of the genome engineering component in the presence of the second compound, preferably wherein the genome engineering component (i) and/or the second compound (ii) is transiently active and/or transiently present in the plant cell.

2. The method of claim 1, wherein the genome engineering component comprises a) a double-stranded DNA break (DSB) inducing enzyme or a nucleic acid encoding same, which preferably recognizes a predetermined site in the genome of said cell, and optionally a repair nucleic acid molecule, or b) a single-stranded DNA or RNA break (SSB) inducing enzyme or a nucleic acid encoding same, which preferably recognizes a predetermined site in the genome of said cell, and optionally a repair nucleic acid molecule, or c) a base editor enzyme, optionally fused to a disarmed DSB or SSB inducing enzyme, which preferably recognizes a predetermined site in the genome of said cell, or d) an enzyme effecting DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone sumoylation, histone ribosylation or histone citrullination, optionally fused to a disarmed DSB or SSB inducing enzyme, which preferably recognizes a predetermined site in the genome of said cell.

3. The method of claim 1, wherein the genome engineering component comprises an DSB or SSB inducing enzyme or a variant thereof selected from a CRISPR/Cas endonuclease, preferably a CRISPR/Cas9 endonuclease or a CRISPR/Cpf1 endonuclease, a zinc finger nuclease (ZFN), a homing endonuclease, a meganuclease and a TAL effector nuclease.

4. The method of claim 1, wherein transient activity of the genome engineering component in step b) comprises inducing one or more double-stranded breaks in the genome of the plant cell, one or more single strand breaks in the genome of the plant cell, one or more base editing events in the genome of the plant cell, or one or more DNA methylation, histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone sumoylation, histone ribosylation or histone citrullination in the genome of the plant cell.

5. The method of claim 4, wherein the induction of one or more double-stranded breaks or one or more single strand breaks is followed by non-homologous end joining (NHEJ) and/or by homology directed repair of the break(s) though a homologous recombination mechanism (HDR).

6. The method of claim 1, wherein in step b) the modification of said genome is selected from a) a replacement of at least one nucleotide; b) a deletion of at least one nucleotide; c) an insertion of at least one nucleotide; d) a change of the DNA methylation, e) a change in histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, histone sumoylation, histone ribosylation or histone citrullination or f) any combination of a)-e).

7. The method of claim 1, wherein the protein causing improved plant regeneration from a somatic cell, a callus cell or embryonic cell comprises an amino acid sequence which is selected from a) a sequence as set forth in any of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 or 31, b) a sequence having an identity of at least 60% to the sequence of (a), c) a sequence encoded by a nucleic acid sequence as set forth in any of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 or 32, and d) a sequence encoded by a nucleic acid sequence having an identity of at least 60% to the nucleic acid sequence of (c).

8. The method of claim 1, further comprising a step of pretreatment of the plant cell to be used in step (a), said pretreatment comprising culturing the plant cell or plant material comprising same in a medium containing the epigenetically regulating chemicals or an active derivative thereof, the phytohormone or the active derivative thereof, the protein causing improved plant regeneration, or any combination thereof.

9. A genetically modified plant cell obtained or obtainable according to the method of claim 1.

10. A plant or a plant part comprising the genetically modified plant cell of claim 9.

11. A microparticle coated with at least (i) a genome engineering component and (ii) a second compound comprising (ii.1) an epigenetically regulating chemical or an active derivative thereof, in particular a DNA methyltransferase inhibitor or a protein deacetylase inhibitor, preferably histone deacetylase inhibitor (HDACi), and/or (ii.2) a phytohormone or an active derivative thereof, preferably selected from auxins, cytokinins and combinations thereof and/or (ii.3) a protein causing improved plant regeneration from a somatic cell, a callus cell or embryonic cell or an expression cassette comprising a nucleic acid encoding said protein.

12. A kit for the genetic modification of a plant genome by microprojectile bombardment, comprising (i) one or more microparticles, and (ii) means for coating the microparticles with at least a genome engineering component and a second compound comprising (1) an epigenetically regulating chemical or an active derivative thereof, in particular a DNA methyltransferase inhibitor or a protein deacetylase inhibitor, preferably histone deacetylase inhibitor (HDACi), and/or (2) a phytohormone or an active derivative thereof, preferably selected from auxins, cytokinins and combinations thereof and/or (3) a protein causing improved plant regeneration from a somatic cell, a callus cell or embryonic cell or an expression cassette comprising a nucleic acid encoding said protein.

13. A method for producing a genetically modified plant, comprising the steps: (a) genetically modifying a plant cell according to the method of claim 1, and (b) regenerating a plant from the modified plant cell of step (a), preferably wherein the produced plant does not contain any of the genome engineering component and the second compound, co-introduced in step a).

14. A genetically modified plant or a part thereof obtained or obtainable by the method of claim 13, or a progeny plant thereof.

15. The method of claim 1, comprising the use of an epigenetically regulating chemical or an active derivative thereof, in particular a DNA methyltransferase inhibitor or a protein deacetylase inhibitor, preferably histone deacetylase inhibitor (HDACi), and/or a phytohormone or an active derivative thereof, preferably selected from auxins, cytokinins and combinations thereof, and/or a protein causing improved plant regeneration from a somatic cell, a callus cell or embryonic cell or an expression cassette comprising a nucleic acid encoding said protein.

Description

FIGURES

[0131] FIG. 1: pLH-Pat5077399-70Subi-tDt construct map. tDT defines tdTomato gene.

[0132] FIG. 2: Co-delivery of 15 ng TSA with construct pLH-Pat5077399-70Subi-tDt (FIG. 1) by microprojectile bombardment of 100 μg gold particles (size 0.6 μm). [0133] A: Red fluorescence images showing tdTomato expressing cells in corn Hi II immature embryos 16 hours after bombardment (white spots). The images on the top are taken from control bombardments without TSA (No TSA), while the images on the bottom are taken from the co-bombardments with 15 ng of TSA. [0134] B: Average numbers of red fluorescent cells per embryo 16 hours after the bombardment without (No TSA) or with 15 ng of TSA (15 ng TSA). Error bar=standard deviation.

[0135] FIG. 3: Co-delivery of different amounts of TSA (No TSA, 15 ng, 30 ng, and 45 ng) with construct pLH-Pat5077399-70Subi-tDt (FIG. 1) by microprojectile bombardment of 100 μg gold particles (size 0.6 μm) in Hi II immature embryos. [0136] A: Average numbers of fluorescent cells per field in corn Hi II immature embryos 16 hours after bombardments with different amounts of TSA. [0137] B: Percentage increase in average number of fluorescent cells when co-bombarded with different amounts of TSA. Error bar=standard deviation.

[0138] FIG. 4: pGEP359 construct map. tDT defines tdTomato gene. ZmLpCpf1 defines the maize codon-optimized CDS of the Lachnospiraceae bacterium CRISPR/Cpf1 (LbCpf1) gene.

[0139] FIG. 5: Co-delivery of 15 ng TSA with construct pGEP359 (FIG. 4) by microprojectile bombardment of 100 μg gold particles (size 0.6 μm). [0140] A: Red fluorescence images showing tdTomato expressing cells in corn Hi II type II calluses 16 hours after bombardment. The images on the left were taken from control bombardments without TSA (No TSA), while the images on the right are taken from the co-bombardments with 15 ng of TSA. [0141] B: Average numbers of red fluorescent cells per field 16 hours after bombarded without (No TSA) or with 15 ng of TSA. Error bar=standard deviation.

[0142] FIG. 6: Co-delivery of 15 ng TSA with construct pLH-Pat5077399-70Subi-tDt (FIG. 1) by microprojectile bombardment with 300 μg gold particles (0.6 μm). [0143] A: Red fluorescence images showing tdTomato expressing cells in sugar beet friable calluses 24 hours after bombardment (white spots). The images on the top are taken from control bombardments without TSA (No TSA), while the images on the bottom show the co-bombardments with 15 ng TSA. [0144] B: Average numbers of fluorescent cells per field 24 hours after bombarded without (−TSA) or with 15 ng of TSA (+TSA). Error bar=standard deviation.

[0145] FIG. 7: pGEP284 construct map. tDT defines tdTomato gene. TaCRISPR defines the wheat codon-optimized CDS of a CRISPR nuclease. sgGEP14 defines the guide RNA target to the first exon of maize glossy 2 gene.

[0146] FIG. 8: Co-delivery of different amounts of TSA (No TSA, 15 ng, 30 ng, and 45 ng) with gene-editing construct pGEP284 (FIG. 7) by microprojectile bombardment of 100 μg gold particles (size 0.6 μm). A: Site-specific InDel (insertion and deletion) rates in Hi II embryos 2 days after co-bombardment. B: Percentage changes in InDel rate when different amounts of TSA (No TSA 15 ng, 30 ng, and 45 ng, from left to right) were co-bombarded with a genome-editing construct pGEP284 in corn Hi II embryos.

[0147] FIG. 9: pGEP353 construct map. crGEP46 defines the crRNA46, which target to maize glycerate kinase gene (GLYK).

[0148] FIG. 10: Co-delivery of gene editing constructs pGEP359 (ZmLbCpf1, FIG. 4) and pGEP353 (crRNA46, FIG. 9) with 15 ng of TSA (on the right, 15 ng of TSA) or no TSA (on the left, No TSA) into corn Hi II callus.

[0149] FIG. 11: pGEP362 construct map. mNeonGreen defines mNeonGreen gene, which encodes the brightest monomeric green or yellow fluorescent protein with excitation maximum at 506 nm and emission maximum at 517 nm. ZmLpCpf1 defines the maize codon-optimized CDS of the Lachnospiraceae bacterium CRISPR/Cpf1 (LbCpf1) gene.

[0150] FIG. 12: Co-delivery of 250 ng 2,4-D with construct pGEP362 (FIG. 11) by microprojectile bombardment into corn Hi II immature embryos. [0151] A: Green fluorescence images show mNeonGreen report gene expressing cells in corn Hi II immature embryos 16 hours after bombardment. The images on the top are taken from control bombardments without 2,4-D (No 2,4-D), while the images on the bottom show the co-bombardments with 250 ng of 2,4-D. [0152] B: Average numbers of the green fluorescent cells per embryo 16 hours after the bombardment. Error bar=standard deviation.

[0153] FIG. 13: Co-delivery of different amounts of 2,4-D (0 ng, 125 ng, 250 ng, and 500 ng) with construct pGEP362 (FIG. 11) by microprojectile bombardment of 100 μg gold particles (size 0.6 μm). [0154] A: Green fluorescence images showing mNeonGreen report gene expressing cells in corn Hi II type II callus cells 16 hours after co-bombarded with different amount of 2,4-D (0 ng, 125 ng, 250 ng, and 500 ng). [0155] B: Average numbers of the green fluorescent cells per field 16 hours after the bombardment with different amount of 2,4-D (0 ng, 125 ng, 250 ng, and 500). Error bar=standard deviation.

[0156] FIG. 14: Co-delivery of 2,4-D with construct pGEP359 (FIG. 4) by microprojectile bombardment of 100 μg gold particles (size 0.6 μm) in leaves of corn plants (top: without 2,4-D, bottom: with 250 ng of 2,4-D) (exemplary tdT expression indicated by arrows).

[0157] FIG. 15: Co-delivery of 250 ng 6-BA or zeatin with construct pGEP359 (FIG. 4) by microprojectile bombardment with 100 μg of gold particle size (size 0.6 μm) in corn Hi II type II calluses. [0158] A: red fluorescence images from left to right showing tdTomato report gene expressing cells in corn Hi II type II callus cells 16 hours after bombardment without hormone (no hormone), with 250 ng of 6-BA, or with 250 ng of zeatin. [0159] B: Average numbers of the red fluorescent cells per field 16 hours after the bombardment. Error bar=standard deviation.

[0160] FIG. 16: pABM-BdEF1_ZmPLT5 construct map. Maize PLT5 gene (ZmPLT5) is driven by the strong constitutive EF1 promoter from Brachypodium (BDEF1).

[0161] FIG. 17: pABM-BdEF1_ZmPLT7 construct map. Maize PLT7 gene (ZmPLT7) is driven by the strong constitutive EF1 promoter from Brachypodium (BDEF1).

[0162] FIG. 18: pABM-BdEF1_TaRKD construct map. Wheat RKD gene (TaRKD) is driven by the strong constitutive EF1 promoter from Brachypodium (BDEF1).

[0163] FIG. 19: Co-delivery of 100 ng boost gene construct with construct pGEP359 (FIG. 4) by microprojectile bombardment with 100 μg of gold particle size (size 0.6 μm) into corn Hi II immature embryos. [0164] A: red fluorescence images show tdTomato report gene expressing cells in corn Hi II immature embryos 16 hours after bombardment. The images on the left to right are taken from control bombardments without a boost (tDT only), or with the ZmPLT5 (FIG. 16) (tDT+ZmPLT5) or wheat RKD (TaRKD, FIG. 18) (tDT+TaRKD) boost construct. [0165] B: Average numbers of the red fluorescent cells per embryo 16 hours after the bombardment. Error bar=standard deviation.

[0166] FIG. 20: tdTomato fluorescent embryogenic calluses were observed 12 days after co-bombarded with ZmPLT5 or ZmPLT7 gene construct. Figure shows red fluorescence images showing tdTomato report gene expressing in the embryogenic callus cells induced from the immature embryos 12 days after bombardment. Images from left to right showing the embryos bombarded with tDTomato report gene only (tDT only), or with 100 ng of boost ZmPLT5 (tDT+ZmPLT5), or ZmPLT7 gene construct (tDT+ZmPLT7)

[0167] FIG. 21: Callus induction in A188 immature embryos 17 days after co-bombardment of tdTomato with wheat RKD boost construct. [0168] A: bright field image showing callus induction from the immature embryos bombarded with tDTomato report construct only. [0169] B: bright field image showing callus induction from the immature embryos co-bombarded with tDTomato report and wheat RKD construct.

EXAMPLES

[0170] Example 1: Co-Delivery of Trichostatin a (TSA) with a Construct Containing tdTomato Report Gene (i.e. pLH-Pat5077399-70Subi-tDt) by Microprojectile Bombardment Increased Transient Transformation Efficiency in Corn Immature Embryo without a TSA Pre-Treatment.

[0171] Procedure: Prepare corn immature embryo for bombardment: 8-10 days post pollination, maize ears (i.e. A188 or Hi II) with immature embryos size 0.8 to 1.8 mm were harvested. The ears were sterilized with 70% ethanol for 10-15 minutes. After a brief air-dry in a laminar hood, remove top ˜⅓ of the kernels from the ears with a shark scalpel, and pull the immature embryos out of the kernels carefully with a spatula. The fresh isolated embryos were placed onto the bombardment target area in an osmotic medium plate (see below) with scutellum-side up. Wrap the plates with parafilm and incubated them at 25° C. in dark for 4-20 hours before bombardment.

[0172] The amounts of TSA used for a bombardment with 100 μg of gold particles (approximately, 4.0-5.0×10.sup.7 0.6 micron gold particles) are in range of 0.01 ng to 500 ng, preferred 0.1 to 50 ng. Plasmid DNA and TSA co-coating onto gold particles for bombardment: For 10 shots, 1 mg of gold particle size 0.6 micron (μm) in 50% (v/v) glycerol (100 μg gold particles per shot) in a total volume of 100 microliter (μl) was pipetted into a clear low-retention microcentrifuge tube. Sonicate for 15 seconds to suspend the gold particles. While vortex at a low speed, add the following in order to each 100 μl of gold particles: [0173] Up to 10 μl of DNA (1.0 μg total DNA, 100 ng per shot) [0174] 100 μl of 2.5 M CaCl.sub.2) (pre-cold on ice) [0175] 40 μl of 0.1 M cold spermidine

[0176] Close the lid and vortex the tube for 2-30 minutes at 0-10° C., and spin down the DNA-coated gold particles. After washing in 500 μl of 100% ethanol for two times, the pellet was resuspended in 120 μl of 100% ethanol. Finally, an appropriate amount of TSA (for a bombardment with 100 μg gold particles size 0.6 μm, TSA amount ranging from 0.01 to 500 ng, preferred 0.1-50 ng; TSA was dissolved in DMSO) was added into the re-suspended gold particle solution carefully. While vortexing at a low speed, pipet 10 μl of Plasmid DNA (pLH-Pat5077399-70Subi-tDt construct; FIG. 1) and TSA co-coated gold particles with a wide open 20 μl tip from the tube onto the center of the macrocarrier evenly since the particles tend to form clumps at this point, get the gold particles onto the macrocarriers as soon as possible. Air dry.

[0177] Bombardment was conducted using a Bio-Rad PDS-1000/He particle gun. The bombardment conditions are: 27-28 mm/Hg vacuum, 450 or 650 psi rupture disc, 6 mm gap distance, the specimen platform is in the second position from the bottom in the chamber at a distance of 60 mm. After bombardment the embryos were remained on the osmotic medium for another 16 hours, and then removed onto a type II callus induction medium plate (see below). 16-48 hours after bombardment, transient transformation was examined using a fluorescence microscope for the tdTomato gene expression at excitation maximum 554 nm and emission maximum 581 nm.

[0178] Type II callus induction medium: N6 salt, N6 vitamin, 1.0 mg/L of 2, 4-D, 100 mg/L of Caseine, 2.9 g/L of L-proline, 20 g/L sucrose, 5 g/L of glucose, 5 mg/L of AgNO3, 8 g/L of Bacto-agar, pH 5.8.

[0179] Osmotic medium: N6 vitamin, 1.0 mg/L of 2, 4-D, 100 mg/L of Caseine, 0.7 g/L of L-proline, 0.2 M Mannitol (36.4 g/L), 0.2 M sorbitol (36.4 g/L), 20 g/L sucrose, 15 g/L of Bacto-agar, pH 5.8.

[0180] In FIG. 2, the co-delivery of 15 ng TSA with construct pLH-Pat5077399-70Subi-tDt (FIG. 1) by microprojectile bombardment of 100 μg gold particles (size 0.6 μm) improves the DNA transient transformation in corn Hi II immature embryos. In FIG. 2A the red fluorescence images show tdTomato expressing cells in corn Hi II immature embryos 16 hours after bombardment with 15 ng TSA compared to control bombardments without TSA. The average number of the red fluorescent cells, i.e. positively transient transformed cells, per embryo 16 hours after the bombardment increased by 98.2% by co-delivery of 15 ng of TSA (FIG. 2B).

[0181] This co-delivery experiment has been repeated with different amounts of TSA—no TSA, 15 ng of TSA, 30 ng of TSA, and 45 ng of TSA (FIG. 3). The presence of TSA improves always the transient transformation in corn Hi II immature embryos. The average number of fluorescent cells, i.e. positively transient transformed cells, per field in corn Hi II immature embryos 16 hours showed an optimum around 30 ng of TSA (FIG. 3A). However even lower but also higher concentrations resulted in a significant increase of transient transformed cells (FIG. 3B).

[0182] Example 2: Co-Delivery of Trichostatin a (TSA) with a tdTomato Report Construct pGEP359 (FIG. 4) by Microprojectile Bombardment Promoted Transformation Efficiency in Corn Type II Callus without a TSA Pre-Treatment

[0183] Type II callus induction and selection: Hi II immature embryos size 0.8-1.8 mm were isolated as described in Example 1, and were placed onto type II callus induction medium (see below) immediately with scutellum-side up, in a density of 10-15 embryo per plate (diameter of 100 mm). Wrap the plates with parafilm, and culture the embryos in plate at 27° C. in the dark until type II callus emerged (˜2 weeks). Pick friable type II calluses under a stereoscope, and move them onto type II callus selection medium (see below). Repeat this process for 2-3 more times, and trash the embryos 4 weeks after induction. Select pre-embryo stage of type II callus under a stereoscope carefully based on: friability (highly friable), morphology (no embryo-like structure), color (fresh, white, semi-transparent). Select and subculture type II callus every 1-2 week in callus selection medium (see below) until the callus lines stabilized (about 3-5 rounds of selection). Stable type II callus lines were cultured in type II callus subculture medium (see below) every 1 to 2 weeks.

[0184] Preparation of type II callus for bombardment: Select and transfer highly friable type II callus at pre-embryo stage onto the bombardment target region in an osmotic medium plate (see Example 1) (single layer, no overlapping). Wrap the plates with parafilm and incubated at 25° C. in dark for 4-20 hours (preferred 4 hours) before bombardment.

[0185] Microprojectile bombardment and post-bombardment handlings were conducted using the same procedure as described in Example 1.

[0186] Type II callus induction medium: N6 salt, N6 vitamin, 1.0 mg/L of 2, 4-D, 100 mg/L of Caseine, 2.9 g/L of L-proline, 20 g/L sucrose, 5 g/L of glucose, 5 mg/L of AgNO3, 8 g/L of Bacto-agar, pH 5.8

[0187] Type II callus selection medium: N6 salt, N6 vitamin, 1.0 mg/L of 2, 4-D, 100 mg/L of Caseine, 2.9 g/L of L-proline, 20 g/L sucrose, 2 mg/L of AgNO3, 8 g/L of Bacto-agar, pH 5.8

[0188] Type II callus sub-culture medium: N6 salt, N6 vitamin, 1.0 mg/L of 2, 4-D, 100 mg/L of Caseine, 0.7 g/L of L-proline, 20 g/L sucrose, 8 g/L of Bacto-agar, pH 5.8

[0189] In FIG. 5, the co-delivery of 15 ng TSA with construct pGEP359 by microprojectile bombardment of 100 μg gold particles (size 0.6 μm) increased transient transformation in corn Hi II type II calluses. In FIG. 5A the red fluorescence images show tdTomato expressing cells in corn Hi II type II calluses 16 hours after bombardment with 15 ng TSA compared to control bombardments without TSA. The average number of fluorescent cells, i.e. positively transient transformed cells, per field in corn Hi II type II calluses 16 hours after bombardment increased by 43.3% by co-delivery of 15 ng of TSA (FIG. 5B).

[0190] Example 3: Co-Delivery of Trichostatin a (TSA) with Construct pLH-Pat5077399-70Subi-tDt by Microprojectile Bombardment Improved Transient Transformation in Sugar Beet Friable Callus

[0191] Sugar beet callus induction: young leaves from in vitro cultured sugar beet shoots in shoot culture medium (see below) were cut into small pieces (square, size 3-5 mm) in a laminar hood, and placed them onto callus induction medium (see below), in a density of 10-15 pieces per plate (diameter of 100 mm) with adaxial-side up. Wrap the plates with parafilm, and culture the leaf segments in plate at 23° C. in the dark for 6-8 weeks until callus emerged.

[0192] Preparation of sugar beet callus for bombardment: harvest friable fresh calluses under a stereoscope, and transfer them onto the bombardment target area in a sugar beet osmatic medium (see below) (single layer, no overlapping). Wrap the plates with parafilm and incubated at 25° C. in dark for 4-20 hours before bombardment.

[0193] Microprojectile bombardment and post-bombardment handlings were conducted using the same procedure descripted in Example 1, except for the amount of gold particles used for a bombardment was 300 μg.

[0194] Sugar beet shoot culture medium: MS, 0.25 mg/L of BAP, 30 g/L of sucrose, 8 g/I plant agar, pH 6.0

[0195] Sugar beet callus induction medium: MS, 2.0 mg/L of BAP, 15 g/L of sucrose, 8 g/I plant agar, pH 6.0

[0196] Sugar beet callus osmatic medium: MS, 2.0 mg/L of BAP, 15 g/L of sucrose, 0.2 M Mannitol (36.4 g/L), 0.2 M sorbitol (36.4 g/L), 8 g/I plant agar, pH 6.0

[0197] In FIG. 6, the co-delivery of 15 ng TSA with construct pLH-Pat5077399-70Subi-tDt (FIG. 1) by microprojectile bombardment with 300 μg gold particles (0.6 μm) improved transient transformation in sugar beet friable calluses. In FIG. 6A, the red fluorescence images show tdTomato expressing cells in sugar beet friable calluses 24 hours after bombardment with 15 ng TSA compared to control bombardments without TSA. The average number of fluorescent cells, i.e. positively transient transformed cells, per field 24 hours after bombardment increased by 193.7% by co-delivery of 15 ng of TSA (FIG. 6B).

[0198] Example 4: Co-delivery of trichostatin A (TSA) with gene editing constructs improved genome-editing efficiency in corn immature embryo.

[0199] Embryo isolation, microprojectile bombardment and post-bombardment handlings were performed using the same procedure as described in Example 1.

[0200] Two days after bombardment, the embryos were harvested and used for genomic DNA isolation use Plant DNA Isolation kit from Qiagen (Venlo, Netherlands). NGS (next generation sequencing) was conducted by Miseq platform of Illumina Inc. (San Diego, Calif., USA). InDel (insertion and deletion) rate was analyzed by means of CRISPResso (http://crispresso.rocks/).

[0201] In FIG. 8, the co-bombardment with TSA (No TSA, 15 ng, 30 ng, and 45 ng, from left to right) leads to an improved gene editing efficiency in Hi II embryos 2 days after bombardment. In FIG. 8A, the site-specific InDel rates in the Hi II embryos 2 days after co-bombardment with the gene editing construct pGEP284 (FIG. 7) for different amounts of TSA are shown, wherein the site-specific InDel rate indicates gene editing efficiency. The presence of TSA improves always the frequency of gene editing events in the corn Hi II immature embryos. The rates of InDel events, i.e. positively gene edited embryos, showed an optimum around 30 ng of TSA. However, even lower but also higher concentrations resulted in a significant increase of InDel rates compared to the absence of TSA. The percentage changes in InDel rate when different amounts of TSA were co-bombarded with a gene editing construct pGEP284 in corn Hi II embryos are shown in FIG. 8B).

[0202] Example 5: Co-Delivery of Trichostatin a (TSA) with Gene Editing Constructs pGEP359 (FIG. 4) and pGEP353 (FIG. 9) Improved Genome-Editing Efficiency in Corn Hi II Type II Calluses

[0203] Type II callus culture and microprojectile bombardment and post-bombardment handlings were performed using the same procedure as described in Example 2.

[0204] 2-15 days after bombardment, the calluses were harvested and used for genomic DNA isolation with a Plant DNA Isolation kit from Qiagen. NGS (next generation sequencing) was conducted by Illumina Miseq platform. InDel (insertion and deletion) rate was analyzed by means of CRISPResso.

[0205] In FIG. 10, the co-bombardment of gene editing constructs pGEP359 (ZmLbCpf1, FIG. 4) and pGEP353 (crRNA46, FIG. 9) with 15 ng of TSA (on the right, 15 ng of TSA) or no TSA (on the left, No TSA) in corn Hi II calluses showed 13 days after co-bombardment an increase of the site-specific InDel (insertion and deletion) rate by factor 6.75 or 575%.

[0206] Example 6: Co-Delivery of Auxin 2,4-D with mNeonGreen Report Construct pGEP362 (FIG. 11) by Microprojectile Bombardment Increased its Transient Transformation Efficiency in Corn Immature Embryos

[0207] Embryo isolation and microprojectile bombardment and post-bombardment handlings were performed using the same procedure as described in Example 1.

[0208] The amounts of 2,4-D used for a bombardment with 100 μg of gold particles (approximately, 4.0-5.0×10.sup.7 0.6 μm gold particles) are in range of 1.0 ng to 1000 ng, preferred 10 ng to 500 ng. Plasmid DNA and 2,4-D co-coating onto gold particles for bombardment were conducted as described in Example 1. 2,4-D stock solution (e.g. 1 mg/ml) is prepared in 100% DMSO.

[0209] In FIG. 12, the co-delivery of 250 ng 2,4-D with construct pGEP362 (FIG. 11) by microprojectile bombardment of 100 μg gold particles (size 0.6 μm) improves the DNA transient transformation in corn Hi II immature embryos. In FIG. 12A, the green fluorescence images show mNeonGreen report gene expressing cells in corn Hi II immature embryos 16 hours after bombardment. B: Average numbers of the green fluorescent cells per field 16 hours after the bombarded with 250 ng 2,4-D compared to control bombardments without 2,4-D. The co-bombardment with 250 ng of 2,4-D lead to an increase by 187% in the average number of the fluorescent cells per embryo (FIG. 12B).

[0210] Example 7: Co-Delivery of Auxin 2,4-D with mNeonGreen Report Construct pGEP362 (FIG. 11) by Microprojectile Bombardment Increased its Transient Transformation Efficiency in Corn Hi II Type II Calluses

[0211] Type II callus culture and microprojectile bombardment and post-bombardment handlings were performed using the same procedure as described in Example 2.

[0212] The amounts of 2,4-D used for a bombardment with 100 μg of gold particles (approximately, 4.0-5.0×10.sup.7 0.6 μm gold particles) are in range of 1.0 ng to 1000 ng, preferred 10 ng to 500 ng. Plasmid DNA and 2,4-D co-coating onto gold particles for bombardment were conducted as described in Example 6.

[0213] In FIG. 13, the co-delivery of different amounts of 2,4-D (0 ng, 125 ng, 250 ng, and 500 ng) with construct pGEP362 (FIG. 11) by microprojectile bombardment of 100 μg gold particles (size 0.6 μm) improved the transient transformation in corn Hi II type II callus. The green fluorescence images showing mNeonGreen report gene expressing cells in corn Hi II type II callus cells 16 hours after co-bombarded with different amount of 2,4-D (2,4-D 0 ng, 125 ng, 250 ng, and 500 ng from top left to bottom right) shows a significant increase of fluorescence by the co-bombardment with 2,4-D (FIG. 13A). In FIG. 13B the average numbers of the green fluorescent cells per field 16 hours after the bombarded with different amount of 2,4-D (0 ng, 125 ng, 250 ng, and 500 ng) are shown. By the addition of 2,4-D the average number of the fluorescent cells have been increased by at least 34.8%.

[0214] Example 8: Co-Delivery of Auxin 2,4-D with tDTomato Report Construct pGEP359 (FIG. 4) by Microprojectile Bombardment Increased its Transient Transformation Efficiency in Leaves of Corn Plants

[0215] Corn plants have grown in greenhouse. In stage V8 microprojectile bombardment was conducted using a Bio-Rad PDS-1000/He particle gun. The bombardment conditions are: 27-28 mm/Hg vacuum, 450 or 650 psi rupture disc, 6 mm gap distance. 20 hours after bombardment, transient transformation was examined using a fluorescence microscope for the tdTomato gene expression at excitation maximum 554 nm and emission maximum 581 nm. Plasmid DNA and 2,4-D co-coating onto gold particles for bombardment were conducted as described in Example 1. 2,4-D stock solution (e.g. 25 mg/ml in DMSO).

[0216] In FIG. 14, the co-delivery of 2,4-D with construct pGEP359 (FIG. 4) by microprojectile bombardment improved the transient transformation in corn leaves.

[0217] Example 9: Co-Delivery of Cytokinins Like 6-BA or Zeatin with tDTomato Report Construct pGEP359 (FIG. 4) by Microprojectile Bombardment Increased its Transient Transformation Efficiency in Corn Hi II Type II Calluses

[0218] Type II callus culture and microprojectile bombardment and post-bombardment handlings were performed using the same procedure as described in Example 2.

[0219] The amounts of 6-BA or zeatin used for a bombardment with 100 μg of gold particles (approximately, 4.0-5.0×10.sup.7 0.6 μm gold particles) are in range of 1.0 ng to 10000 ng, preferred 10 ng to 1000 ng. Plasmid DNA and the cytokinin co-coating onto gold particles for bombardment were conducted as described in Example 6.

[0220] In FIG. 15, the Co-delivery of 250 ng 6-BA or zeatin with construct pGEP359 (FIG. 4) by microprojectile bombardment with 100 μg of gold particle size 0.6 μm in corn Hi II type II calluses. The red fluorescence images showing tdTomato report gene expressing cells in corn Hi II type II callus cells 16 hours after bombardment (FIG. 15A), from left to right: control bombardment without hormone (no hormone), with 250 ng of 6-BA, and with 250 ng of zeatin. In FIG. 15B, the average numbers of the red fluorescent cells per field 16 hours after the bombardment are shown. 250 ng 6-BA co-bombardment led to a 35.8% increase and 250 ng zeatin a 31.2% increase in the average number of the fluorescent cells.

[0221] Example 10: Co-Delivery of a Boost Gene with the tDTomato Report Construct (FIG. 4) by Microprojectile Bombardment Increased its Transient Transformation Efficiency in Corn Immature Embryos

[0222] Embryo isolation, microprojectile bombardment and post-bombardment handlings were performed using the same procedure as described in Example 1.

[0223] Boost genes are co-bombarded with a fluorescent report construct (tdTomato gene, FIG. 4). The amounts of a boost gene construct (FIG. 16, FIG. 17, FIG. 18) used for a bombardment with 100 μg of gold particles (approximately, 4.0-5.0×10.sup.7 0.6 μm gold particles) and 100 ng of the tDTomato report construct are in range of 10.0 ng to 1000 ng, preferred 50 ng to 100 ng. Plasmid DNA coating onto gold particles for bombardment were conducted as described in Example 1.

[0224] The boost effect is measured by its capability to increase the transient transformation frequency of the report gene 16-20 after bombardment of corn Hi II immature embryos.

[0225] In FIG. 19, the co-delivery of 100 ng of a boost gene construct with 100 ng of the tDTomato report construct (FIG. 4) by microprojectile bombardment of 100 μg gold particles (size 0.6 μm) improves the tDTomato gene transient transformation in corn Hi II immature embryos.

[0226] In FIG. 19A, the red fluorescence images show tDTomato report gene expressing cells in corn Hi II immature embryos 16 hours after bombardment. FIG. 19B: average numbers of the red fluorescent cells per embryo 16 hours after the bombarded with a boost gene construct compared to control bombardment with the report only (tDT only). The co-bombardment with 100 ng of ZmPLT5 boost gene construct (FIG. 16) (tDT+ZmPLT5) led to an increase by 102%, or with 100 ng of wheat RKD (TaRKD) (FIG. 18) (tDT+TaRKD) resulted into an increase by 144% in the average number of the fluorescent cells per embryo (FIG. 19B).

[0227] Example 11: Transient Over-Expression of Boost Genes Promote Transformation Frequency (TF)

[0228] Embryo isolation, microprojectile co-bombardment, and post-bombardment handlings were performed using the same procedure as described in Example 10. The boost effect on transformation is measured by its capability to increase the transformation frequency of the report gene at 12 days after bombardment of corn Hi II immature embryos (IE) without a selection.

[0229] As shown in Table 1, co-bombardment of tdTomato construct with ZmPLT5 led to an increase of 42.9% of the transformation frequency of tdTomato gene (over 16-fold increase compared to the control), while the co-bombardment with ZmPLT7 gave an increase of 53% of transformation frequency of tdTomato gene (over 16-fold increase compared to the control) 12 days after bombardment without a selection (FIG. 20).

TABLE-US-00001 TABLE 1 tDT transformation frequency (FT) at 12 days after bombardment: FT is defined as the number of embryos with at least one tDT expressing embryogenic structures (No. of tDT positive IEs) from 100 embryos bombarded. tDT only tDT + ZmPLT5 tDT + ZMPLT7 No. of tDT positive 1/40 21/49 26/49 IEs/total IEs tDT TF 2.5% 42.9% 53.1%

[0230] Example 12: Transient Over-Expression of Wheat RKD Boost Gene (SEQ ID NO: 6) Promote Callus Induction in A188 Immature Embryos

[0231] Embryo isolation, microprojectile co-bombardment, and post-bombardment handlings were performed using the same procedure as described in Example 10, and callus induction was conducted as described in Example 2.

[0232] Transient over-expression of wheat RKD gene led to a significant improvement in callus induction, the induction rate increased from 38% without TaRKD to 75% with 100 ng of TaRKD, nearly a doubling of the callus induction rate (FIG. 21).