WO2017061806A1 - Method for producing whole plants from protoplasts - Google Patents

Method for producing whole plants from protoplasts Download PDF

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WO2017061806A1
WO2017061806A1 PCT/KR2016/011217 KR2016011217W WO2017061806A1 WO 2017061806 A1 WO2017061806 A1 WO 2017061806A1 KR 2016011217 W KR2016011217 W KR 2016011217W WO 2017061806 A1 WO2017061806 A1 WO 2017061806A1
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gene
seq
genes
cas protein
protoplast
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PCT/KR2016/011217
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French (fr)
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Jin Soo Kim
Jungeun Kim
Sunghwa Choe
Je Wook Woo
Soon Il Kwon
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Institute For Basic Science
Seoul National University R&Db Foundation
Aict
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Priority to JP2018538510A priority Critical patent/JP6916188B2/en
Priority to MX2018004264A priority patent/MX2018004264A/en
Priority to US15/766,432 priority patent/US11136588B2/en
Priority to CA3001108A priority patent/CA3001108A1/en
Priority to AU2016336566A priority patent/AU2016336566B2/en
Priority to EP16853922.9A priority patent/EP3359674A4/en
Priority to CN201680065399.9A priority patent/CN108368516A/en
Priority to BR112018007070A priority patent/BR112018007070A2/en
Publication of WO2017061806A1 publication Critical patent/WO2017061806A1/en

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Definitions

  • the present invention relates to method for preparing a plant from a protoplast comprising knocking-out or knocking-in one or more the endogenous gene of the protoplast, and the plant regenerated from a genome-modified protoplast prepared by the above method.
  • Programmable nucleases which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided endonucleases (RGENs) repurposed from the type II clustered, regularly-interspaced palindromic repeat (CRISPR)-CRISPR-associated (Cas) adaptive immune system in bacteria and archaea have been successfully used for genome editing in cells and organisms including various plant species, paving the way for novel applications in biomedical research, medicine, and biotechnology (Kim, H. etc., Nat Rev Genet , 2014, 15: 321-334).
  • ZFNs zinc-finger nucleases
  • TALENs transcription activator-like effector nucleases
  • RGENs RNA-guided endonucleases
  • CRISPR RGENs the latest of the trio of nucleases, are rapidly replacing ZFNs and TALENs, owing to their ease of programmability; RGENs that consist of the Cas9 protein derived from Streptococcus pyogenes and guide RNAs (gRNAs) are customized by replacing the RNA component only, sidestepping the labor-intensive and time-consuming protein engineering required for making new TALENs and ZFNs.
  • Programmable nucleases delivered into plant cells via Agrobacterium or transfection of plasmids that encode them, cleave chromosomal target sites in a sequence-dependent manner, producing site-specific DNA double-strand breaks (DSBs). The repair of these DSBs by endogenous systems gives rise to targeted genome modifications.
  • GMO genetically-modified organism
  • Programmable nucleases induce small insertions and deletions (indels) or substitutions at chromosomal target sites that are indistinguishable from naturally-occurring variations. Still, these plants may be considered as GMOs by regulatory authorities in certain countries, hampering widespread use of programmable nucleases in plant biotechnology and agriculture. For example, when Agrobacterium is used, genome-edited plants will contain foreign DNA sequences, including those encoding programmable nucleases in the host genome. Removal of these Agrobacterium -derived DNA sequences by breeding is not feasible in certain plants such as grape, potato, and banana, owing to their asexual reproduction.
  • non-integrating plasmids that encode programmable nucleases can be transfected into plant cells such as protoplasts.
  • transfected plasmids are degraded in cells by endogenous nucleases and that the resulting small DNA fragments can be inserted at the Cas9 on-target and off-target sites, as shown in human cells (Kim, S, etc., Genome research , 2014, 24: 1012-1019).
  • RNPs Cas9 protein-gRNA ribonucleoproteins
  • RFLP restriction fragment length polymorphism
  • Another object of the present invention is to provide a plant regenerated from a genome-edited protoplast prepared by the method for preparing a plant from a protoplast.
  • Still another object of the present invention is to provide a composition for cleaving DNA encoding BIN2 gene in a plant cell, comprising: a guide RNA specific to DNA encoding Brassinosteroid Insensitive 2 ( BIN2 ) gene, BKI1 gene, or homologs thereof, or DNA encoding the guide RNA; and a nucleic acid encoding a Cas protein, or a Cas protein.
  • a guide RNA specific to DNA encoding Brassinosteroid Insensitive 2 ( BIN2 ) gene, BKI1 gene, or homologs thereof, or DNA encoding the guide RNA comprising: a guide RNA specific to DNA encoding Brassinosteroid Insensitive 2 ( BIN2 ) gene, BKI1 gene, or homologs thereof, or DNA encoding the guide RNA; and a nucleic acid encoding a Cas protein, or a Cas protein.
  • Still another object of the present invention is to provide a composition for preparing a plant from a protoplast, comprising: a guide RNA specific to DNA encoding Brassinosteroid Insensitive 2 ( BIN2 ) gene, BKI1 gene, or homologs thereof, or DNA encoding the guide RNA; and a nucleic acid encoding a Cas protein or a Cas protein.
  • a guide RNA specific to DNA encoding Brassinosteroid Insensitive 2 ( BIN2 ) gene, BKI1 gene, or homologs thereof, or DNA encoding the guide RNA comprising: a guide RNA specific to DNA encoding Brassinosteroid Insensitive 2 ( BIN2 ) gene, BKI1 gene, or homologs thereof, or DNA encoding the guide RNA; and a nucleic acid encoding a Cas protein or a Cas protein.
  • Still another object of the present invention is to provide a kit for preparing a plant from a protoplast comprising the composition for preparing a plant from a protoplast.
  • Cas9 protein and guide RNAs into various plant protoplasts, inducing targeted mutagenesis in regenerated plants at frequencies of up to 46%.
  • Cas9 ribonucleoprotein delivery into protoplasts avoided the possibility of inserting foreign DNA in the host genome.
  • the resulting plants contained germline-transmissible, small insertions or deletions at target sites, which are indistinguishable from naturally-occurring variations, possibly bypassing regulatory requirements associated with use of Agrobacterium or plasmids.
  • RGEN RNPs can be delivered into protoplasts derived from various plant species and induce targeted genome modifications in whole plants regenerated from them.
  • FIG. 1 RGEN RNP-mediated gene disruption in various plant protoplasts.
  • Top The T7E1 assay.
  • Bottom DNA sequences of the wild-type (WT) and mutant sequences.
  • FIG. 1 RGEN RNP-mediated gene disruption in bulk population.
  • FIG. 1 Genetic analysis of microcalli derived from a single protoplast treated with RGEN RNP.
  • FIG. 4 Targeted gene knockout in lettuce using an RGEN RNP.
  • (a) The target sequence in the BIN2 gene. The PAM sequence is shown in red.
  • (b) Genotyping of microcalli.
  • (Top) RGEN RFLP analysis.
  • (Bottom) Mutant DNA sequences in microcalli.
  • ( c ) Whole plants regenerated from RGEN RNP-transfected protoplasts.
  • FIG. 7 Regeneration of plantlets from RGEN RNP-transfected protoplast in L. sativa .
  • (b) A multicellular colony of protoplast (Bar 100 ⁇ m).
  • (d) Callus formation from protoplast-derived colonies (e,f) Organogenesis and regenerated shoots from protoplast-derived calli (bar 5 mm)
  • FIG. 8 Targeted deep sequencing of mutant calli. Genotypes of the mutant calli were confirmed by Illumina Miseq. Sequence of each allele and the number of sequencing reads were analyzed. (A1), allele1. (A2), allele2.
  • FIG. 9 Plant regeneration from RGEN RNP-transfected protoplasts in L. sativa .
  • a-c Organogenesis and shoot formation from protoplast-derived calli; wild type (#28), bi-allelic/heterozygote (#24), bi-allelic/homozygote (#30).
  • d In vitro shoot proliferation and development.
  • e Elongation and growth of shoots in MS culture medium free of PGR.
  • FIG. 10 Germline transmission of BIN2 mutant alleles.
  • (a) Bolting and flowering in regenerated plants.
  • (b) RGEN-RFLP analysis for genotyping seeds obtained from a homozygous bi-allelic mutant termed T0-12.
  • (c) DNA sequences of the wild-type, T0-12 mutant, and T1 mutants derived from the T0-12 line. Red triangles indicate an inserted nucleotide.
  • An aspect of the present invention provides a method for preparing a plant from a protoplast comprising knocking-out or knocking-in one or more the endogenous gene of the protoplast.
  • the endogenous gene of the plant may be a gene capable of increasing stress resistance of the plant by knocking-out or knocking-in.
  • the endogenous gene of the plant may be a gene involved in Brassinosteroid signal transduction of plants.
  • the endogenous gene in the knocking-out step, may be one or more genes selected from the group consisting of BIN2 gene, BKI1 gene, and homolog genes thereof; and (ii) in the knocking-in step, the gene being knocked in may be one or more genes selected from the group consisting of BRI1 gene, BSU gene, BZR1 gene, DWF4 gene, CYP85A1, and homolog genes thereof.
  • the knocking-out of genes may be performed by knocking-out one or two alleles of the genes selected from the group consisting of BIN 2 gene, BKI1 gene, and homolog genes thereof.
  • the knocking-out of genes may be performed by gene knock-out and the knocking-in of genes is performed by gene knock-in.
  • the knocking-out of genes may be performed using an engineered nuclease specific to one or more genes selected from the group consisting of BIN 2 gene, BKI1 gene, and homolog genes thereof.
  • the engineered nuclease may be selected from the group consisting of zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and RNA-guided engineered nuclease (RGEN).
  • ZFN zinc finger nuclease
  • TALEN transcription activator-like effector nuclease
  • RGEN RNA-guided engineered nuclease
  • the RGEN may comprise guide RNA, which specifically binds to a specific sequence of one or more genes selected from the group consisting of BIN 2 gene, BKI1 gene, and homolog genes thereof, or DNA encoding the guide RNA; and a nucleic acid encoding a Cas protein, or a Cas protein.
  • the knocking-out of genes may be performed by introducing the guide RNA, which specifically binds to a specific sequence of one or more genes selected from the group consisting of BIN 2 gene, BKI1 gene, and homolog genes thereof, or DNA encoding the guide RNA; and a nucleic acid encoding a Cas protein, or a Cas protein, to the protoplast.
  • the guide RNA may be in the form of a dual RNA or a single-chain guide RNA (sgRNA) comprising crRNA and tracrRNA.
  • sgRNA single-chain guide RNA
  • the single-chain guide RNA may comprise a part of crRNA and tracrRNA.
  • the single-chain guide RNA may be in the form of isolated RNA.
  • the DNA encoding the guide RNA may be encoded by a vector, and the vector is virus vector, plasmid vector, or Agrobacterium vector.
  • the Cas protein may be a Cas9 protein or a variant thereof.
  • the Cas protein may recognize NGG trinucleotide.
  • the Cas protein may be linked to a protein transduction domain.
  • the variant of the Cas9 protein may be in a mutant form of Cas9 protein, wherein the catalytic aspartate residue is substituted with another amino acid.
  • the amino acid may be alanine.
  • nucleic acid encoding a Cas protein or Cas protein may be derived from a microorganism of the genus Streptococcus .
  • the microorganism of the genus Streptococcus may be Streptococcus pyogenes .
  • the protoplast may be derived from Lactuca sativa.
  • the introduction may be performed by co-transfecting or serial-transfecting of a nucleic acid encoding a Cas protein or a Cas protein, and the guide DNA or DNA encoding the guide DNA into a protoplast.
  • the serial-transfection may be performed by firstly transfecting a Cas protein or a nucleic acid encoding a Cas protein followed by secondly transfecting a naked guide RNA.
  • the introduction may be performed by a method selected from the group consisting of microinjection, electroporation, DEAE-dextran treatment, lipofection, nanoparticle-mediated transfection, protein transduction domain-mediated transfection, and PEG-mediated transfection.
  • the method may further comprise regenerating the protoplast having a knocked-out gene.
  • the regeneration may comprise culturing a protoplast having one or more knocked-out genes selected from the group consisting of BIN 2 gene, BKI1 gene, and homolog genes thereof in agarose-containing medium to form callus; and culturing the callus in regeneration medium.
  • Another aspect of the present invention is a plant regenerated from a genome-edited protoplast prepared by the above method.
  • compositions for cleaving DNA encoding BIN2 gene in a plant cell comprising: a guide RNA specific to DNA encoding Brassinosteroid Insensitive 2 ( BIN2 ) gene, BKI1 gene, or homologs thereof, or DNA encoding the guide RNA; and a nucleic acid encoding a Cas protein, or a Cas protein.
  • the composition may induce a targeted mutagenesis in a plant cell.
  • compositions for preparing a plant from a protoplast comprising: a guide RNA specific to DNA encoding Brassinosteroid Insensitive 2 ( BIN2 ) gene, BKI1 gene, or homologs thereof, or DNA encoding the guide RNA; and a nucleic acid encoding a Cas protein or a Cas protein.
  • BIN2 Brassinosteroid Insensitive 2
  • Another aspect of the present invention is a kit for preparing a plant from a protoplast comprising the above composition.
  • Cas9 protein and guide RNAs Cas9 protein and guide RNAs .
  • Cas9 protein tagged with a nuclear localization signal was purchased from ToolGen, Inc. (South Korea). Templates for guide RNA transcription were generated by oligo-extension using Phusion polymerase (Table 1-4). Guide RNAs were in vitro transcribed through run-off reactions using the T7 RNA polymerase (New England Biolabs) according to the manufacturer's protocol. The reaction mixture was treated with DNase I (New England Biolabs) in 1X DNase I reaction buffer. Transcribed sgRNAs were resolved on an 8% denaturing urea-polyacryl amide gel with SYBR gold staining (Invitrogen) for quality control. Transcribed sgRNAs were purified with MG TM PCR Product Purification SV (Macrogen) and quantified by spectrometry.
  • Protoplast culture Protoplasts were isolated as previously described from Arabidopsis, rice, and lettuce. Initially, Arabidopsis ( Arabidopsis thaliana ) ecotype Columbia-0, rice ( Oryza sativa L.) cv. Dongjin, and lettuce ( Lactuca sativa L.) cv Cheongchima seeds were sterilized in a 70 % ethanol, 0.4 % hypochlorite solution for 15 min, washed three times in distilled water, and sown on 1/2X Murashige and Skoog solid medium supplemented with 2% sucrose.
  • the seedlings were grown under a 16 h light (150 ⁇ mol m -2 s -1 ) and 8 h dark cycle at 25 o C in a growth room.
  • the leaves of 14 d Arabidopsis seedlings, the stem and sheath of 14 d rice seedlings, and the cotyledons of 7 d lettuce seedlings were digested with enzyme solution (1.0 % cellulase R10, 0.5 % macerozyme R10, 0.45 M mannitol, 20 mM MES [pH 5.7], CPW solution) during incubation with shaking (40 rpm) for 12 h at 25 o C in darkness and then diluted with an equal volume of W5 solution.
  • enzyme solution 1.0 % cellulase R10, 0.5 % macerozyme R10, 0.45 M mannitol, 20 mM MES [pH 5.7], CPW solution
  • Protoplast transfection PEG-mediated RNP transfections were performed as previously described. Briefly, to introduce DSBs using an RNP complex, 1x10 5 protoplast cells were transfected with Cas9 protein (10-60 ⁇ g) premixed with in vitro transcribed sgRNA (20-120 ⁇ g). Prior to transfection, Cas9 protein in storage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 1 mM DTT, and 10% glycerol) was mixed with sgRNA in 1X NEB buffer 3 and incubated for 10 min at room temperature.
  • a mixture of 1x10 5 protoplasts (or 5x10 5 protoplasts in the case of lettuce) re-suspended in 200 ⁇ L MMG solution was gently mixed with 5-20 ⁇ L of RNP complex and 210 ⁇ L of freshly prepared PEG solution (40 % [w/v] PEG 4000; Sigma No. 95904, 0.2 M mannitol and 0.1 M CaCl 2 ), and then incubated at 25 o C for 10 min in darkness. After incubation, 950 ⁇ L W5 solution (2 mM MES [pH 5.7], 154 mM NaCl, 125 mM CaCl 2 and 5 mM KCl) were added slowly. The resulting solution was mixed well by inverting the tube.
  • Protoplasts were pelleted by centrifugation at 100 g for 3 min and re-suspended gently in 1 ml WI solution (0.5 M mannitol, 20 mM KCl and 4 mM MES at pH 5.7). Finally, the protoplasts were transferred into multi-well plates and cultured under dark conditions at 25 o C for 24-48 h. Cells were analyzed one day after transfection.
  • Protoplast regeneration RNP-transfected protoplasts were re-suspended in 1/2X B5 culture medium supplemented with 375 mg/l CaCl 2' 2H 2 O, 18.35 mg/l NaFe-EDTA, 270 mg/l sodium succinate, 103 g/l sucrose, 0.2 mg/l 2,4 dichlorophenoxyacetic acid (2,4-D), 0.3 mg/l 6-benzylaminopurine (BAP), and 0.1 g/l MES.
  • the protoplasts were mixed with a 1:1 solution of 1/2X B5 medium and 2.4 % agarose to a culture density of 2.5 X 10 5 protoplasts/ml.
  • the protoplasts embedded in agarose were plated onto 6-well plates, overlaid with 2 ml of liquid 1/2X B5 culture medium, and cultured at 25 o C in darkness. After 7 days, the liquid medium replaced with fresh culture medium. The cultures were transferred to the light (16 h light [30 ⁇ mol m -2 s -1 ] and 8 h darkness) and cultured at 25 o C. After 3 weeks of culture, micro-calli were grown to a few mm in diameter and transferred to MS regeneration medium supplemented with 30 g/l sucrose, 0.6 % plant agar, 0.1 mg/l ⁇ -naphthalaneacetic acid (NAA), 0.5 mg/l BAP. Induction of multiple lettuce shoots was observed after about 4 weeks on regeneration medium.
  • NAA 0.1 mg/l ⁇ -naphthalaneacetic acid
  • Rooting, transfer to soil and hardening of lettuce To regenerate whole plants, proliferated and elongated adventitious shoots were transferred to a fresh regeneration medium and incubated for 4-6 weeks at 25 o C in the light (16 h light [120 ⁇ mol m -2 s -1 ] and 8 h darkness).
  • 25 o C For root induction, approximately 3-5 cm long plantlets were excised and transferred onto a solid hormone-free 1/2X MS medium in Magenta vessels. Plantlets developed from adventitious shoots were subjected to acclimation, transplanted to potting soil, and maintained in a growth chamber at 25 o C (100-150 ⁇ mol m -2 s -1 under cool-white fluorescent lamps with a 16-h photoperiod).
  • T7E1 assay Genomic DNA was isolated from protoplasts or calli using DNeasy Plant Mini Kit (Qiagen). The target DNA region was amplified and subjected to the T7E1 assay as described previously. In brief, PCR products were denatured at 95 o C and cooled down to a room temperature slowly using a thermal cycler. Annealed PCR products were incubated with T7 endonuclease I (ToolGen, Inc.) at 37 o C for 20 min and analyzed via agarose gel electrophoresis.
  • ToolGen, Inc. T7 endonuclease I
  • RGEN - RFLP The RGEN-RFLP assay was performed as previously described. Briefly, PCR products (300-400 ng) were incubated in 1X NEB buffer 3 for 60 min at 37 o C with Cas9 protein (1 ⁇ g) and sgRNA (750 ng) in a reaction volume of 10 ⁇ l. RNase A (4 ⁇ g) was then added to the reaction mixture and incubated at 37 o C for 30 min to remove the sgRNA. The reaction was stopped by adding 6X stop solution (30 % glycerol, 1.2 % SDS, 250 mM EDTA). DNA products were electrophoresed using a 2.5 % agarose gel.
  • Purified Cas9 protein was mixed with two to 10 fold molar excess of gRNAs targeting four genes in three plant species in vitro to form preassembled RNPs.
  • the RGEN RNPs were then incubated with protoplasts derived from Arabidopsis ( A. thaliana ), a wild type of tobacco ( N. attenuate ), and rice ( O. sativa ) in the presence of polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • T7E1 assay was used both the T7 endonuclease I (T7E1) assay and targeted deep sequencing to measure mutation frequencies in transfected cells (Fig. 1a,b). Indels were detected at the expected position, that is, 3 nucleotide (nt) upstream of a NGG protospacer-adjacent motif (PAM), with frequencies that ranged from 8.4% to 44%.
  • RGEN target site SEQ ID NO: 93
  • BIN2 BRASSINOSTEROID INSENSITIVE 2
  • BR brassinosteroid
  • Indels were detected at the expected position, that is, 3 nucleotide (nt) upstream of NGG protospacer-adjacent motif (PAM), with frequencies that ranged from 8.3% to 11% (9.0% on average) using T7E1 assay and 3.2% to 5.7% (4.3% on average) using NGS assay (Fig. 2b, c).
  • nt 3 nucleotide upstream of NGG protospacer-adjacent motif
  • RGEN-RFLP assay distinguishes mono-allelic mutant clones (50% cleavage) from heterozygous bi-allelic mutant clones (no cleavage) and homozygous bi-allelic mutant clones (no cleavage) from wild-type clones (100 %) cleavage.
  • BIN2 gene disruption showed no morphological changes but, some stress-tolerant phenotypes in rice.
  • up-regulation of BR signaling caused by knocking out the BIN2 gene may facilitate the overall rate of cell proliferation and growth and give advantages to calli standing the stressful regeneration process.
  • this analysis distinguishes mono-allelic mutant clones (50% cleavage) from heterozygous bi-allelic mutant clones (no cleavage) and homozygous bi-allelic mutant clones (no cleavage) from wild-type clones (100% cleavage).
  • the RGEN-RFLP assay is not limited by sequence polymorphisms near the nuclease target site that may exist in the lettuce genome. This assay showed that two of 35 (5.7%) calli contained mono-allelic mutations and 14 of 35 (40%) calli contained bi-allelic mutations at the target site (Fig. 3, Fig 4b), demonstrating that RGEN-induced mutations were stably maintained after regeneration.
  • the mutation frequency in lettuce calli was 46%.
  • the number of base pairs deleted or inserted at the target site ranged from -9 to +1, consistent with the mutagenic patterns observed in human cells. No apparent mosaicism was detected in these clones (Fig. 8), suggesting that the RGEN RNP cleaved the target site immediately after transfection and induced indels before cell division.
  • RGEN RNPs were successfully delivered into plant protoplasts and induced targeted genome modifications in 6 genes in 4 different plant species. Importantly, RGEN-induced mutations were stably maintained in whole plants regenerated from the protoplasts and transmitted to germlines. Because no recombinant DNA is used in this process, the resulting genome-edited plants could be exempted from current GMO regulations, paving the way for the widespread use of RNA-guided genome editing in plant biotechnology and agriculture.

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Abstract

The present invention relates to method for preparing a plant from a protoplast comprising knocking-out or knocking-in one or more the endogenous gene of the protoplast, and the plant regenerated from a genome-modified protoplast prepared by the above method.

Description

METHOD FOR PRODUCING WHOLE PLANTS FROM PROTOPLASTS
The present invention relates to method for preparing a plant from a protoplast comprising knocking-out or knocking-in one or more the endogenous gene of the protoplast, and the plant regenerated from a genome-modified protoplast prepared by the above method.
Programmable nucleases, which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided endonucleases (RGENs) repurposed from the type II clustered, regularly-interspaced palindromic repeat (CRISPR)-CRISPR-associated (Cas) adaptive immune system in bacteria and archaea have been successfully used for genome editing in cells and organisms including various plant species, paving the way for novel applications in biomedical research, medicine, and biotechnology (Kim, H. etc., Nat Rev Genet, 2014, 15: 321-334). Among these nucleases, CRISPR RGENs, the latest of the trio of nucleases, are rapidly replacing ZFNs and TALENs, owing to their ease of programmability; RGENs that consist of the Cas9 protein derived from Streptococcus pyogenes and guide RNAs (gRNAs) are customized by replacing the RNA component only, sidestepping the labor-intensive and time-consuming protein engineering required for making new TALENs and ZFNs. Programmable nucleases, delivered into plant cells via Agrobacterium or transfection of plasmids that encode them, cleave chromosomal target sites in a sequence-dependent manner, producing site-specific DNA double-strand breaks (DSBs). The repair of these DSBs by endogenous systems gives rise to targeted genome modifications.
It remains unclear whether the resulting genome-edited plants will be regulated by genetically-modified organism (GMO) legislation in the EU and other countries (Jones, H.D., Nature Plants, 2015, 1: 14011). Programmable nucleases induce small insertions and deletions (indels) or substitutions at chromosomal target sites that are indistinguishable from naturally-occurring variations. Still, these plants may be considered as GMOs by regulatory authorities in certain countries, hampering widespread use of programmable nucleases in plant biotechnology and agriculture. For example, when Agrobacterium is used, genome-edited plants will contain foreign DNA sequences, including those encoding programmable nucleases in the host genome. Removal of these Agrobacterium-derived DNA sequences by breeding is not feasible in certain plants such as grape, potato, and banana, owing to their asexual reproduction.
Alternatively, non-integrating plasmids that encode programmable nucleases can be transfected into plant cells such as protoplasts. We note, however, that transfected plasmids are degraded in cells by endogenous nucleases and that the resulting small DNA fragments can be inserted at the Cas9 on-target and off-target sites, as shown in human cells (Kim, S, etc., Genome research, 2014, 24: 1012-1019).
Delivery of preassembled Cas9 protein-gRNA ribonucleoproteins (RNPs) rather than plasmids encoding these components into plant cells could avoid the possibility of inserting recombinant DNA in the host genome. Furthermore, as shown in cultured human cells, RGEN RNPs cleave chromosomal target sites immediately after transfection and are degraded rapidly by endogenous proteases in cells, potentially reducing mosaicism and off-target effects in regenerated whole plants. Preassembled RGEN RNPs can be used broadly across plant species without prior optimization of codon usage and promoters to express Cas9 and gRNAs in each species. In addition, RGEN RNPs enable pre-screening in vitro to choose highly active gRNAs and genotyping of mutant clones via restriction fragment length polymorphism (RFLP) analysis.
To the best of our knowledge, however, RGEN RNPs have never been used in any plant species.
It is an object of the present invention to provide a method for preparing a plant from a protoplast comprising knocking-out or knocking-in one or more endogenous genes of the protoplast.
Another object of the present invention is to provide a plant regenerated from a genome-edited protoplast prepared by the method for preparing a plant from a protoplast.
Still another object of the present invention is to provide a composition for cleaving DNA encoding BIN2 gene in a plant cell, comprising: a guide RNA specific to DNA encoding Brassinosteroid Insensitive 2 (BIN2) gene, BKI1 gene, or homologs thereof, or DNA encoding the guide RNA; and a nucleic acid encoding a Cas protein, or a Cas protein.
Still another object of the present invention is to provide a composition for preparing a plant from a protoplast, comprising: a guide RNA specific to DNA encoding Brassinosteroid Insensitive 2 (BIN2) gene, BKI1 gene, or homologs thereof, or DNA encoding the guide RNA; and a nucleic acid encoding a Cas protein or a Cas protein.
Still another object of the present invention is to provide a kit for preparing a plant from a protoplast comprising the composition for preparing a plant from a protoplast.
We transfected purified Cas9 protein and guide RNAs into various plant protoplasts, inducing targeted mutagenesis in regenerated plants at frequencies of up to 46%. Cas9 ribonucleoprotein delivery into protoplasts avoided the possibility of inserting foreign DNA in the host genome. The resulting plants contained germline-transmissible, small insertions or deletions at target sites, which are indistinguishable from naturally-occurring variations, possibly bypassing regulatory requirements associated with use of Agrobacterium or plasmids.
In the present invention, we showed that RGEN RNPs can be delivered into protoplasts derived from various plant species and induce targeted genome modifications in whole plants regenerated from them.
Figure 1. RGEN RNP-mediated gene disruption in various plant protoplasts. (a) Mutation frequencies measured by the T7E1 assay and targeted deep sequencing. (b) Mutant DNA sequences induced by RGEN RNPs in plant cells. The PAM sequences are shown in red. Inserted nucleotides are shown in blue. WT, wild-type. (c) A time-course analysis of genome editing in Arabidopsis protoplasts. (Top) The T7E1 assay. (Bottom) DNA sequences of the wild-type (WT) and mutant sequences.
Figure 2. RGEN RNP-mediated gene disruption in bulk population. (a) The target sequence in the BIN2 gene. The PAM sequence is shown in red. (b) Mutation frequencies measured by the T7E1 assay and targeted deep sequencing in bulk population. (c) Mutant DNA sequences induced by RGEN RNPs in plant cells. The PAM sequences are shown in red. Inserted nucleotides are shown in blue. WT, wild-type.
Figure 3. Genetic analysis of microcalli derived from a single protoplast treated with RGEN RNP. (a) Genotyping of microcalli. (Top) RGEN RFLP analysis. (Bottom) Mutant DNA sequences in microcalli. (b) Summary of genetic analysis of BIN2 gene in T0 generation.
Figure 4. Targeted gene knockout in lettuce using an RGEN RNP. (a) The target sequence in the BIN2 gene. The PAM sequence is shown in red. (b) Genotyping of microcalli. (Top) RGEN RFLP analysis. (Bottom) Mutant DNA sequences in microcalli. (c) Whole plants regenerated from RGEN RNP-transfected protoplasts.
Figure 5. Analysis of off-target effects. Mutation frequencies at on-target and potential off-target sites of the PHYB and BRI1 gene-specific sgRNAs were measured by targeted deep sequencing. About ~80,000 paired-end reads per site were obtained to calculate the indel rate.
Figure 6. Partial nucleotide and amino acid sequences of LsBIN2. Underscored and boxed letters represent the sequences corresponding to degenerate primers and sgRNA, respectively.
Figure 7. Regeneration of plantlets from RGEN RNP-transfected protoplast in L. sativa. Protoplast division, callus formation and shoot regeneration from RGEN RNP-transfected protoplasts in the lettuce. (a) Cell division after 5 days of protoplast culture (Bar = 100 ㎛). (b) A multicellular colony of protoplast (Bar = 100 ㎛). (c) Agarose-embedded colonies after 4 weeks of protoplast culture. (d) Callus formation from protoplast-derived colonies (e,f) Organogenesis and regenerated shoots from protoplast-derived calli (bar = 5 mm)
Figure 8. Targeted deep sequencing of mutant calli. Genotypes of the mutant calli were confirmed by Illumina Miseq. Sequence of each allele and the number of sequencing reads were analyzed. (A1), allele1. (A2), allele2.
Figure 9. Plant regeneration from RGEN RNP-transfected protoplasts in L. sativa. (a-c) Organogenesis and shoot formation from protoplast-derived calli; wild type (#28), bi-allelic/heterozygote (#24), bi-allelic/homozygote (#30). (d) In vitro shoot proliferation and development. (e) Elongation and growth of shoots in MS culture medium free of PGR. (f) Root induction onto elongated shoots. (g) Acclimatization of plantlets. (h,i) Regenerated whole plants.
Figure 10. Germline transmission of BIN2 mutant alleles. (a) Bolting and flowering in regenerated plants. (b) RGEN-RFLP analysis for genotyping seeds obtained from a homozygous bi-allelic mutant termed T0-12. (c) DNA sequences of the wild-type, T0-12 mutant, and T1 mutants derived from the T0-12 line. Red triangles indicate an inserted nucleotide.
An aspect of the present invention provides a method for preparing a plant from a protoplast comprising knocking-out or knocking-in one or more the endogenous gene of the protoplast.
In one embodiment, the endogenous gene of the plant may be a gene capable of increasing stress resistance of the plant by knocking-out or knocking-in.
In another embodiment, the endogenous gene of the plant may be a gene involved in Brassinosteroid signal transduction of plants.
In still another embodiment, (i) in the knocking-out step, the endogenous gene may be one or more genes selected from the group consisting of BIN2 gene, BKI1 gene, and homolog genes thereof; and (ii) in the knocking-in step, the gene being knocked in may be one or more genes selected from the group consisting of BRI1 gene, BSU gene, BZR1 gene, DWF4 gene, CYP85A1, and homolog genes thereof.
In still another embodiment, the knocking-out of genes may be performed by knocking-out one or two alleles of the genes selected from the group consisting of BIN2 gene, BKI1 gene, and homolog genes thereof.
In still another embodiment, the knocking-out of genes may be performed by gene knock-out and the knocking-in of genes is performed by gene knock-in.
In still another embodiment, the knocking-out of genes may be performed using an engineered nuclease specific to one or more genes selected from the group consisting of BIN2 gene, BKI1 gene, and homolog genes thereof.
In still another embodiment, the engineered nuclease may be selected from the group consisting of zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and RNA-guided engineered nuclease (RGEN).
In still another embodiment, the RGEN may comprise guide RNA, which specifically binds to a specific sequence of one or more genes selected from the group consisting of BIN2 gene, BKI1 gene, and homolog genes thereof, or DNA encoding the guide RNA; and a nucleic acid encoding a Cas protein, or a Cas protein.
In still another embodiment, the knocking-out of genes may be performed by introducing the guide RNA, which specifically binds to a specific sequence of one or more genes selected from the group consisting of BIN2 gene, BKI1 gene, and homolog genes thereof, or DNA encoding the guide RNA; and a nucleic acid encoding a Cas protein, or a Cas protein, to the protoplast.
In still another embodiment, the guide RNA may be in the form of a dual RNA or a single-chain guide RNA (sgRNA) comprising crRNA and tracrRNA.
In still another embodiment, the single-chain guide RNA may comprise a part of crRNA and tracrRNA.
In still another embodiment, the single-chain guide RNA may be in the form of isolated RNA.
In still another embodiment, the DNA encoding the guide RNA may be encoded by a vector, and the vector is virus vector, plasmid vector, or Agrobacterium vector.
In still another embodiment, the Cas protein may be a Cas9 protein or a variant thereof.
In still another embodiment, the Cas protein may recognize NGG trinucleotide.
In still another embodiment, the Cas protein may be linked to a protein transduction domain.
In still another embodiment, the variant of the Cas9 protein may be in a mutant form of Cas9 protein, wherein the catalytic aspartate residue is substituted with another amino acid.
In still another embodiment, the amino acid may be alanine.
In still another embodiment, the nucleic acid encoding a Cas protein or Cas protein may be derived from a microorganism of the genus Streptococcus.
In still another embodiment, the microorganism of the genus Streptococcus may be Streptococcus pyogenes.
In still another embodiment, the protoplast may be derived from Lactuca sativa.
In still another embodiment, the introduction may be performed by co-transfecting or serial-transfecting of a nucleic acid encoding a Cas protein or a Cas protein, and the guide DNA or DNA encoding the guide DNA into a protoplast.
In still another embodiment, the serial-transfection may be performed by firstly transfecting a Cas protein or a nucleic acid encoding a Cas protein followed by secondly transfecting a naked guide RNA.
In still another embodiment, the introduction may be performed by a method selected from the group consisting of microinjection, electroporation, DEAE-dextran treatment, lipofection, nanoparticle-mediated transfection, protein transduction domain-mediated transfection, and PEG-mediated transfection.
In still another embodiment, the method may further comprise regenerating the protoplast having a knocked-out gene.
In still another embodiment, the regeneration may comprise culturing a protoplast having one or more knocked-out genes selected from the group consisting of BIN2 gene, BKI1 gene, and homolog genes thereof in agarose-containing medium to form callus; and culturing the callus in regeneration medium.
Another aspect of the present invention is a plant regenerated from a genome-edited protoplast prepared by the above method.
Another aspect of the present invention is a composition for cleaving DNA encoding BIN2 gene in a plant cell, comprising: a guide RNA specific to DNA encoding Brassinosteroid Insensitive 2 (BIN2) gene, BKI1 gene, or homologs thereof, or DNA encoding the guide RNA; and a nucleic acid encoding a Cas protein, or a Cas protein.
In still another embodiment, the composition may induce a targeted mutagenesis in a plant cell.
Another aspect of the present invention is a composition for preparing a plant from a protoplast, comprising: a guide RNA specific to DNA encoding Brassinosteroid Insensitive 2 (BIN2) gene, BKI1 gene, or homologs thereof, or DNA encoding the guide RNA; and a nucleic acid encoding a Cas protein or a Cas protein.
Another aspect of the present invention is a kit for preparing a plant from a protoplast comprising the above composition.
Hereinafter, the present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
METHODS
Cas9 protein and guide RNAs . Cas9 protein tagged with a nuclear localization signal was purchased from ToolGen, Inc. (South Korea). Templates for guide RNA transcription were generated by oligo-extension using Phusion polymerase (Table 1-4). Guide RNAs were in vitro transcribed through run-off reactions using the T7 RNA polymerase (New England Biolabs) according to the manufacturer's protocol. The reaction mixture was treated with DNase I (New England Biolabs) in 1X DNase I reaction buffer. Transcribed sgRNAs were resolved on an 8% denaturing urea-polyacryl amide gel with SYBR gold staining (Invitrogen) for quality control. Transcribed sgRNAs were purified with MGTM PCR Product Purification SV (Macrogen) and quantified by spectrometry.
List of primers used for T7E1 assay
1st PCR 2nd PCR
Target Forward (5' to 3') Reverse (5' to 3') Forward (5' to 3') Reverse (5' to 3')
AOC CGAGCTCAATGAACGTGACC(SEQ ID NO: 1) GATCAGAATGCAGAGTCC AGC(SEQ ID NO: 2) ATGCAGAGTCCAGCCGT TAT(SEQ ID NO: 3)
PHYB TGGTTGTTTGCCATCACACT(SEQ ID NO: 4) GAAAAGCCTGAAAGGACGAA(SEQ ID NO: 5) GCCTCCCCATTTGATTTCTT(SEQ ID NO: 6)
P450 GGAGCTGAACCACTTCATCC(SEQ ID NO: 7) CCCAGCACCTGCTTCACTAT(SEQ ID NO: 8) ACCCCAGGCCAATTCATG(SEQ ID NO: 9) GGGACAAAGATTCATGCAGCA(SEQ ID NO: 10)
DWD1 CCTTTTCTTTGTGGGGTGTG(SEQ ID NO: 11) TCCTTCTCCCTCTCCTCCTG(SEQ ID NO: 12) ATCTCGTGCCATCTCCATCC(SEQ ID NO: 13)
BRI1 ATTTGGGCTGATCCTTGTTG(SEQ ID NO: 14) TGTTGAACACCTGAAACTTTGG(SEQ ID NO: 15) ACCAATTGGAAGCTGACTGG(SEQ ID NO: 16) CCATGCCAAAATCTGAAACC(SEQ ID NO: 17)
List of primers used for targeted deep sequencing (1st primers)
Target Forward (5' to 3') Reverse (5' to 3')
PHYB-OT1 CCGCATTCAACAGCTCTCTC(SEQ ID NO: 18) GCTCAAATCAGGTGGCTACG(SEQ ID NO: 19)
PHYB-OT2 AGGCTGTTCAAAGTCCAGGT(SEQ ID NO: 20) ATCGCTGGGAGTTCAACAGA(SEQ ID NO: 21)
PHYB-OT3 CCAATGGGCCTGAAAGCTTT(SEQ ID NO: 22) ACAACCAAAATCCGCAACGA(SEQ ID NO: 23)
BRI1-TS1-OT1 CGCAAGTTGGTCAGAGTGAA(SEQ ID NO: 24) ACAAGGAGGCTGACGGAAA(SEQ ID NO: 25)
BRI1-TS1-OT2 ACTCGTTACAGGACTCGGTG(SEQ ID NO: 26) TACAGAGCTGCTTCTGGACC(SEQ ID NO: 27)
BRI1-TS1-OT3 TTACCGTAGCTGGGATCGTC(SEQ ID NO: 28) GACTTGTCTCCCTCGCCATA(SEQ ID NO: 29)
BRI1-TS1-OT4 GCAAGGACGGATGAGAAACC(SEQ ID NO: 30) TGGCATAGTCGCTATTTCGC(SEQ ID NO: 31)
BRI1-TS1-OT5 GTCTCCAAAATCCTCGTCGC(SEQ ID NO: 32) GGAAAATTTCTCCCCGCCTC(SEQ ID NO: 33)
BRI1-TS1-OT6 TATGGCGGAAGGTGTAGGTC(SEQ ID NO: 34) TTGCTTGGCTGAAACTCACC(SEQ ID NO: 35)
BRI1-TS2-OT1 CGAGTGCTGATGTGTGTGTT(SEQ ID NO: 36) TCTCTTGGTGCAGGGTGAAT(SEQ ID NO: 37)
BRI1-TS2-OT2 CCCTCTCAATTGCAGCCATT(SEQ ID NO: 38) CGTGTCTTCCTCTGCCATTG(SEQ ID NO: 39)
BRI1-TS2-OT3 ACATTTGCTGCATTGGGATCT(SEQ ID NO: 40) CCAACCCGGCTCAAACTTAC(SEQ ID NO: 41)
BRI1-TS2-OT4 CTCGTCTCAGCCAGGTTAGT(SEQ ID NO: 42) ATCAAGAATCCAATGGCGGC(SEQ ID NO: 43)
List of primers used for targeted deep sequencing (2nd primers)
Sequence (5' to 3')
AOC-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGAGCTCAATGAACGTGACC(SEQ ID NO: 44)
AOC-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGATCAGAATGCAGAGTCCAGC(SEQ ID NO: 45)
PHYB-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCAAATGTCAGAGAAACGCG(SEQ ID NO: 46)
PHYB-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATCAGTGCTTAATCCGGTTGA(SEQ ID NO: 47)
P450-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTACCCCAGGCCAATTCATG(SEQ ID NO: 48)
P450-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGCTCTGGTTTCAAGTTAGTACA(SEQ ID NO: 49)
DWD1-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTGCCACAACCAACGGATC(SEQ ID NO: 50)
DWD1-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGGATTCAGACCCACCCG(SEQ ID NO: 51)
BRI1-TS1-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTTTGCGGATCTTCTTCAGGCT(SEQ ID NO: 52)
BRI1-TS1-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCTCGTCTCCAACTTTGCAA(SEQ ID NO: 53)
BRI1-TS2-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTTTGCAAAGTTGGAGACGAGC(SEQ ID NO: 54)
BRI1-TS2-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATCTGAAACCCGAGCTTCCA(SEQ ID NO: 55)
BIN2-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGTGGTTTCTTTGAAGCATTGT(SEQ ID NO: 56)
BIN2-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGCCACTCACAATCACATGT(SEQ ID NO: 57)
PHYB-OT1-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTTTCATGAAGGTGGCTCAGGT(SEQ ID NO: 58)
PHYB-OT1-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTTCATTCTCTTGCCGTGGG(SEQ ID NO: 59)
PHYB-OT2-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTGGTGACAATGTGGCTAATGGT(SEQ ID NO: 60)
PHYB-OT2-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACTCGGCCAATGTTACTCCA(SEQ ID NO: 61)
PHYB-OT3-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTGGCTTGTTGGGTGATCTTGA(SEQ ID NO: 62)
PHYB-OT3-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGACCCACTTCACAGAAAGCA(SEQ ID NO: 63)
BRI1-TS1-OT1-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCTGCACGATTCTACCTGACA(SEQ ID NO: 64)
BRI1-TS1-OT1-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCTCCTGTCATGTGTTCCTAAC(SEQ ID NO: 65)
BRI1-TS1-OT2-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTTAGCTATGCCGGTGGAAGTT(SEQ ID NO: 66)
BRI1-TS1-OT2-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACAGAAGTAGCCATTCCGAGA(SEQ ID NO: 67)
BRI1-TS1-OT3-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGGAGACCTTTAAGCTTCGC(SEQ ID NO: 68)
BRI1-TS1-OT3-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGCAAAACCATCAGCAGTGG(SEQ ID NO: 69)
BRI1-TS1-OT4-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTGTTTGAAGAAGGTGGCCCAG(SEQ ID NO: 70)
BRI1-TS1-OT4-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGTGGGACGATCGAGCTTAT(SEQ ID NO: 71)
BRI1-TS1-OT5-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGACTAACCGCTTGTCCTCA(SEQ ID NO: 72)
BRI1-TS1-OT5-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACGTTGCCAGTAAAGTTCGC(SEQ ID NO: 73)
BRI1-TS1-OT6-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTCTCTTACTCGCCTCCTT(SEQ ID NO: 74)
BRI1-TS1-OT6-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCATCTGAGGTTGGTTCGACA(SEQ ID NO:75)
BRI1-TS2-OT1-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCATTCAGCTTTGCCAAACCA(SEQ ID NO: 76)
BRI1-TS2-OT1-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCCGGTGGAATTACTGCTCA(SEQ ID NO: 77)
BRI1-TS2-OT2-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGTTCACAATTACTGCCACCA(SEQ ID NO: 78)
BRI1-TS2-OT2-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACTCTCTACGATCGCAACTCT(SEQ ID NO: 79)
BRI1-TS2-OT3-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTGGAGATGGAGGGGATGGAAC(SEQ ID NO: 80)
BRI1-TS2-OT3-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCGGCTCTGAACAGGTCTACA(SEQ ID NO: 81)
BRI1-TS2-OT4-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGCAATCAGATGTCCGGTCA(SEQ ID NO: 82)
BRI1-TS2-OT4-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTACCTCTTCAGCAACCAAGT(SEQ ID NO: 83)
In vitro transcription template
Sequence (5' to 3')
AOC-sgF GAAATTAATACGACTCACTATAG CAAAAGACTGTCAATTCCCTGTTTTAGAGCTAGAAATAGCAAG(SEQ ID NO: 84)
PHYB-sgF GAAATTAATACGACTCACTATAGG CACTAGGAGCAACACCCAACGTTTTAGAGCTAGAAATAGCAAG(SEQ ID NO: 85)
P450-sgF GAAATTAATACGACTCACTATAGG CATATAGTTGGGTCATGGCAGTTTTAGAGCTAGAAATAGCAAG(SEQ ID NO: 86)
DWD1-TS1-sgF GAAATTAATACGACTCACTATAGG TGCATCGTCCAAGCGCACAGGTTTTAGAGCTAGAAATAGCAAG(SEQ ID NO: 87)
DWD1-TS2-sgF GAAATTAATACGACTCACTATAGG CTACGACGTCAGGTTCTACCGTTTTAGAGCTAGAAATAGCAAG(SEQ ID NO: 88)
BRI1-TS1-sgF GAAATTAATACGACTCACTATAGG TTTGAAAGATGGAAGCGCGGGTTTTAGAGCTAGAAATAGCAAG(SEQ ID NO: 89)
BRI1-TS2-sgF GAAATTAATACGACTCACTATAGG TGAAACTAAACTGGTCCACAGTTTTAGAGCTAGAAATAGCAAG(SEQ ID NO: 90)
BIN2-sgF GAAATTAATACGACTCACTATAG ATCACAGTGATGCTCGTCAAGTTTTAGAGCTAGAAATAGCAAG(SEQ ID NO: 91)
Universal sgR AAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC(SEQ ID NO: 92)
Protoplast culture. Protoplasts were isolated as previously described from Arabidopsis, rice, and lettuce. Initially, Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0, rice (Oryza sativa L.) cv. Dongjin, and lettuce (Lactuca sativa L.) cv Cheongchima seeds were sterilized in a 70 % ethanol, 0.4 % hypochlorite solution for 15 min, washed three times in distilled water, and sown on 1/2X Murashige and Skoog solid medium supplemented with 2% sucrose. The seedlings were grown under a 16 h light (150 μmol m-2 s-1) and 8 h dark cycle at 25 oC in a growth room. For protoplast isolation, the leaves of 14 d Arabidopsis seedlings, the stem and sheath of 14 d rice seedlings, and the cotyledons of 7 d lettuce seedlings were digested with enzyme solution (1.0 % cellulase R10, 0.5 % macerozyme R10, 0.45 M mannitol, 20 mM MES [pH 5.7], CPW solution) during incubation with shaking (40 rpm) for 12 h at 25 oC in darkness and then diluted with an equal volume of W5 solution. The mixture was filtered before protoplasts were collected by centrifugation at 100 g in a round-bottomed tube for 5 min. Re-suspended protoplasts were purified by floating on a CPW 21S (21% [w/v] sucrose in CPW solution, pH 5.8) solution followed by centrifugation at 80 g for 7 min. The purified protoplasts were washed with W5 solution and pelleted by centrifugation at 70 g for 5 min. Finally, protoplasts were re-suspended in W5 solution and counted under the microscope using a hemocytometer. Protoplasts were diluted to a density of 1x106 protoplasts/ml of MMG solution (0.4 M mannitol and 15 mM MgCl2, 4 mM MES [pH 5.7]).
Protoplast transfection . PEG-mediated RNP transfections were performed as previously described. Briefly, to introduce DSBs using an RNP complex, 1x105 protoplast cells were transfected with Cas9 protein (10-60 ㎍) premixed with in vitro transcribed sgRNA (20-120 ㎍). Prior to transfection, Cas9 protein in storage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 1 mM DTT, and 10% glycerol) was mixed with sgRNA in 1X NEB buffer 3 and incubated for 10 min at room temperature. A mixture of 1x105 protoplasts (or 5x105 protoplasts in the case of lettuce) re-suspended in 200 μL MMG solution was gently mixed with 5-20 μL of RNP complex and 210 μL of freshly prepared PEG solution (40 % [w/v] PEG 4000; Sigma No. 95904, 0.2 M mannitol and 0.1 M CaCl2), and then incubated at 25 oC for 10 min in darkness. After incubation, 950 μL W5 solution (2 mM MES [pH 5.7], 154 mM NaCl, 125 mM CaCl2 and 5 mM KCl) were added slowly. The resulting solution was mixed well by inverting the tube. Protoplasts were pelleted by centrifugation at 100 g for 3 min and re-suspended gently in 1 ml WI solution (0.5 M mannitol, 20 mM KCl and 4 mM MES at pH 5.7). Finally, the protoplasts were transferred into multi-well plates and cultured under dark conditions at 25 oC for 24-48 h. Cells were analyzed one day after transfection.
Protoplast regeneration. RNP-transfected protoplasts were re-suspended in 1/2X B5 culture medium supplemented with 375 mg/l CaCl2'2H2O, 18.35 mg/l NaFe-EDTA, 270 mg/l sodium succinate, 103 g/l sucrose, 0.2 mg/ l 2,4 dichlorophenoxyacetic acid (2,4-D), 0.3 mg/l 6-benzylaminopurine (BAP), and 0.1 g/l MES. The protoplasts were mixed with a 1:1 solution of 1/2X B5 medium and 2.4 % agarose to a culture density of 2.5 X 105 protoplasts/ml. The protoplasts embedded in agarose were plated onto 6-well plates, overlaid with 2 ml of liquid 1/2X B5 culture medium, and cultured at 25 oC in darkness. After 7 days, the liquid medium replaced with fresh culture medium. The cultures were transferred to the light (16 h light [30 μmol m-2 s-1] and 8 h darkness) and cultured at 25 oC. After 3 weeks of culture, micro-calli were grown to a few mm in diameter and transferred to MS regeneration medium supplemented with 30 g/l sucrose, 0.6 % plant agar, 0.1 mg/l α-naphthalaneacetic acid (NAA), 0.5 mg/l BAP. Induction of multiple lettuce shoots was observed after about 4 weeks on regeneration medium.
Rooting, transfer to soil and hardening of lettuce. To regenerate whole plants, proliferated and elongated adventitious shoots were transferred to a fresh regeneration medium and incubated for 4-6 weeks at 25 oC in the light (16 h light [120 μmol m-2 s-1] and 8 h darkness). For root induction, approximately 3-5 cm long plantlets were excised and transferred onto a solid hormone-free 1/2X MS medium in Magenta vessels. Plantlets developed from adventitious shoots were subjected to acclimation, transplanted to potting soil, and maintained in a growth chamber at 25 oC (100-150 μmol m-2 s-1 under cool-white fluorescent lamps with a 16-h photoperiod).
T7E1 assay. Genomic DNA was isolated from protoplasts or calli using DNeasy Plant Mini Kit (Qiagen). The target DNA region was amplified and subjected to the T7E1 assay as described previously. In brief, PCR products were denatured at 95 oC and cooled down to a room temperature slowly using a thermal cycler. Annealed PCR products were incubated with T7 endonuclease I (ToolGen, Inc.) at 37 oC for 20 min and analyzed via agarose gel electrophoresis.
RGEN - RFLP . The RGEN-RFLP assay was performed as previously described. Briefly, PCR products (300-400 ng) were incubated in 1X NEB buffer 3 for 60 min at 37 oC with Cas9 protein (1 μg) and sgRNA (750 ng) in a reaction volume of 10 μl. RNase A (4 μg) was then added to the reaction mixture and incubated at 37 oC for 30 min to remove the sgRNA. The reaction was stopped by adding 6X stop solution (30 % glycerol, 1.2 % SDS, 250 mM EDTA). DNA products were electrophoresed using a 2.5 % agarose gel.
Targeted deep sequencing. The on-target and potential off-target sites were amplified from genomic DNA. Indices and sequencing adaptors were added by additional PCR. High-throughput sequencing was performed using Illumina Miseq (v2, 300 cycle).
RESULT
Purified Cas9 protein was mixed with two to 10 fold molar excess of gRNAs targeting four genes in three plant species in vitro to form preassembled RNPs. The RGEN RNPs were then incubated with protoplasts derived from Arabidopsis (A. thaliana), a wild type of tobacco (N. attenuate), and rice (O. sativa) in the presence of polyethylene glycol (PEG). We used both the T7 endonuclease I (T7E1) assay and targeted deep sequencing to measure mutation frequencies in transfected cells (Fig. 1a,b). Indels were detected at the expected position, that is, 3 nucleotide (nt) upstream of a NGG protospacer-adjacent motif (PAM), with frequencies that ranged from 8.4% to 44%.
We also co-transfected two gRNAs whose target sites were separated by 201 base pairs (bps) in another gene in Arabidopsis to investigate whether the repair of two concurrent DSBs would give rise to targeted deletion of the intervening sequence, as shown in human cells. Sanger sequencing showed that 223 bp DNA sequences were deleted in the protoplasts (Fig. 1c). Notably, RGEN-induced mutations were detected 24 hours post-transfection, suggesting that RGENs cut target sites immediately after transfection and induce mutations before a full cycle of cell division.
Next, we investigated whether RGEN RNPs can induce off-target mutations at sites highly homologous to on-target sites. We searched for potential off-target sites of the PHYTOCHROME B (PHYB) and BRASSINOSTEROID INSENSITIVE 1 (BRI1) gene-specific sgRNAs in the Arabidopsis genome using the Cas-OFFinder program and used targeted deep sequencing to measure mutation frequencies (Fig. 5). Indels were not detected at any of these sites that differed from on-target sites by two to five nucleotides, in line with our previous results in human cells.
We designed an RGEN target site (SEQ ID NO: 93) to disrupt the BRASSINOSTEROID INSENSITIVE 2 (BIN2) gene, which encodes a negative regulator in a brassinosteroid (BR) signaling pathway (Fig. 2a). We transfected the RGEN RNP in the presence of polyethylene glycol (PEG) and measured the targeted gene modification efficiencies caused by RGEN using both the T7 endonuclease 1 (T7E1) assay and targeted deep sequencing. Indels were detected at the expected position, that is, 3 nucleotide (nt) upstream of NGG protospacer-adjacent motif (PAM), with frequencies that ranged from 8.3% to 11% (9.0% on average) using T7E1 assay and 3.2% to 5.7% (4.3% on average) using NGS assay (Fig. 2b, c).
We performed the regeneration process to produce whole plants which contain the BIN2 mutant alleles from RGEN-RNP treated protoplasts. Only a fraction (<0.5%) of protoplasts could be cultured to form calli. Among these, 35 of fast-growing lines were used to perform further analyses (Fig. 3). We performed the RGEN-RFLP assay and targeted deep sequencing to genotype the lettuce microcalli. RGEN-RFLP assay distinguishes mono-allelic mutant clones (50% cleavage) from heterozygous bi-allelic mutant clones (no cleavage) and homozygous bi-allelic mutant clones (no cleavage) from wild-type clones (100 %) cleavage. Remarkably, these analyses showed that two of 35 (5.7%) calli contained mono-allelic mutations and 14 of 35 (40%) calli contained bi-allelic mutations at the target site. Thus, the mutation frequency in regenerated calli was 42.9% (= 30 mutant alleles/70 alleles), showing up to 10-fold increase from that in protoplasts. Note that we have obtained genome-edited lettuce at a frequency of 43% without any selection, an extremely high frequency compared to the mutation frequency in bulk populations, suggesting that RGEN-induced mutations in the BIN2 gene were stably maintained and enriched during regeneration process.
BIN2 gene disruption showed no morphological changes but, some stress-tolerant phenotypes in rice. We propose that up-regulation of BR signaling caused by knocking out the BIN2 gene may facilitate the overall rate of cell proliferation and growth and give advantages to calli standing the stressful regeneration process.
Finally, we transfected an RGEN RNP to disrupt the lettuce (Lactuca sativa) homolog of Arabidopsis BRASSINOSTEROID INSENSITIVE 2 (BIN2) gene (Fig. 6), which encodes a negative regulator in a brassinosteroid (BR) signaling pathway, in lettuce protoplasts and obtained microcalli regenerated from the RNP-transfected cells (Fig. 2-4 and Fig. 7). We used the same RGEN RNP in a RFLP analysis to genotype the lettuce microcalli. Unlike the T7E1 assay, this analysis distinguishes mono-allelic mutant clones (50% cleavage) from heterozygous bi-allelic mutant clones (no cleavage) and homozygous bi-allelic mutant clones (no cleavage) from wild-type clones (100% cleavage). Furthermore, the RGEN-RFLP assay is not limited by sequence polymorphisms near the nuclease target site that may exist in the lettuce genome. This assay showed that two of 35 (5.7%) calli contained mono-allelic mutations and 14 of 35 (40%) calli contained bi-allelic mutations at the target site (Fig. 3, Fig 4b), demonstrating that RGEN-induced mutations were stably maintained after regeneration. Thus, the mutation frequency in lettuce calli was 46%. We also used targeted deep sequencing to confirm these genotypes in the 16 mutant calli. The number of base pairs deleted or inserted at the target site ranged from -9 to +1, consistent with the mutagenic patterns observed in human cells. No apparent mosaicism was detected in these clones (Fig. 8), suggesting that the RGEN RNP cleaved the target site immediately after transfection and induced indels before cell division.
We then determined whether the BIN2-specific RGEN induced collateral damage in the lettuce genome using high-throughput sequencing. No off-target mutations were induced at 91 homologous sites that differed by one to 5 nucleotides from the on-target site in three BIN2-mutated plantlets (Tables 5-8), consistent with our findings in human cells : Off-target mutations induced by CRISPR RGENs are rarely found in a single cell-derived clone.
Number of potential off-target sites in the lettuce genome. Potential RGEN off-target sites were identified in the lettuce genome using Cas-OFFinder (www.regenome.net). We used the Legassy_V2 database (Genebank: AFSA00000000.1) as the reference genome and identified homologous sequences that differed from on-target sequences by up to 5 nt. We chose a total of 92 sites and performed targeted deep sequencing. Some sites were excluded in this analysis because PCR primers couldn't be designed owing to a poor quality of reference genome data or because no amplicons were obtained using PCR.
No. of mismatches to on-target site
0 1 2 3 4 5 Total
No. of potential off-target sites 1(on-target) 0 1 4 27 349 382
No. of sites with appropriate PCR primers 1 0 1 3 24 72 101
No. of sites amplified successfully 1 0 1 3 22 65 92
Indel frequencies at the on-target and 91 potential off-target sites in three regenerated plantlets. False-positive indels caused by sequencing errors are observed at frequencies that ranged from 0% to 3.0%.
WT T0-20 T0-24 T0-25
Site name Sequence Indels ( % ) Indels ( % ) Indels ( % ) Indels ( % )
On-target ATCACAGTGATGCTCGTCAAAGG(SEQ ID NO: 94) 0.021 99.912 99.867 45.042
OT1 ATCACAGTGcgGCTCGTCAAgGG(SEQ ID NO: 95) 0.022 0.039 0 0
OT2 caCACAGTGATGtTCGTCAAgGG(SEQ ID NO: 96) 0 0.014 0.024 0.013
OT3 ATacCAGgGATGCTCGTCAAtGG(SEQ ID NO: 97) 0 0 0 0
OT4 ATCAtAGTGATGCTCaTgAAgGG(SEQ ID NO: 98) 0.013 0.03 0.019 0
OT5 ATCACAtTGATGCTCtaCAtAGG(SEQ ID NO: 99) 0.023 0.033 0.029 0.012
OT6 ATaACAGaGAcGaTCGTCAAAGG(SEQ ID NO: 100) 0.029 0.03 0.014 0.027
OT7 ATCACAcTGATGCcCtaCAAAGG(SEQ ID NO: 101) 0.093 0.06 0.07 0.109
OT8 ATCACAtTGAgGCcCGaCAAAGG(SEQ ID NO: 102) - - - -
OT9 ATCACAcTGATGCaCtaCAAAGG(SEQ ID NO: 103) 0.057 0.037 0.023 0.077
OT10 caCACAGTGATGtTCaTCAAAGG(SEQ ID NO: 104) 0.635 0.715 0.663 0.145
OT11 ATgACAaTtATGCTCtTCAAAGG(SEQ ID NO: 105) 0.250 0 0.102 0
OT12 ATCAaAGTGcTcCTCGTgAAAGG(SEQ ID NO: 106) 0 0 0 0
OT13 taCACAaTGtTGCTCGTCAAcGG(SEQ ID NO: 107) 0.013 0 0 0.012
OT14 gcCACAGTGATGaTCGTCgAcGG(SEQ ID NO: 108) 0 0 0 0.013
OT15 ATatCAGgGATGCTCGcCAAtGG(SEQ ID NO: 109) 0 0 0 0
OT16 AaatCAGTGATcCTCGTCAAcGG(SEQ ID NO: 110) 0 0 0 0.012
OT17 ATggCAGTGATGgTCGTgAAgGG(SEQ ID NO: 111) 0 0.045 0.08 0.1
OT18 cTCAgAGTGtTGCTCtTCAAtGG(SEQ ID NO: 112) 0 0.01 0 0
OT19 ATCACAGaGATGCTCcaaAAtGG(SEQ ID NO: 113) 0.074 0.033 0.068 0.068
OT20 ATCAagGTtATtCTCGTCAAgGG(SEQ ID NO: 114) 0 0.009 0 0
OT21 AgCACAGTGAgGCTtGTCgAgGG(SEQ ID NO: 115) 0 0 0 0
OT22 ATatCAagGATGCTCGTCAAtGG(SEQ ID NO: 116) 0 0 0 0
OT23 tTCcCAGaGATGCTCtTCAAgGG(SEQ ID NO: 117) 0.024 0.05 0 0.035
OT24 gTCACAtTGATGCTCaTCAtgGG(SEQ ID NO: 118) 0 0 0.157 0
OT25 ATCACAGaGATGtTCaTCAtcGG(SEQ ID NO: 119) 0.022 0 0.013 0
OT26 ATCAaAaTGAgGCTCGaCAAcGG(SEQ ID NO: 120) - - - -
OT27 ATaACAaTGAaGCTCGTtAAtGG(SEQ ID NO: 121) 0 0 0 0
OT28 ATatCAGgGATGCTCaTCAAtGG(SEQ ID NO: 122) 0 0.011 0 0.017
OT29 ATCAtAtTGAaGCaCtTCAAAGG(SEQ ID NO: 123) 0.029 0.019 0 0.036
OT30 cTCACAtTGATGCaCtaCAAAGG(SEQ ID NO: 124) 0.069 0.055 0.073 0.097
OT31 tcCACAaTGATGCaCtTCAAAGG(SEQ ID NO: 125) 0.023 0 0 0.012
OT32 cTCACAaTGtTGCTCtaCAAAGG(SEQ ID NO: 126) - - - -
OT33 ATgACAaTGAaGCTCGTaAtAGG(SEQ ID NO: 127) 0 0 0.018 0
WT T0-20 T0-24 T0-25
Site name Sequence Indels ( % ) Indels ( % ) Indels ( % ) Indels ( % )
On-target ATCACAGTGATGCTCGTCAAAGG(SEQ ID NO: 94) 0.021 99.912 99.867 45.042
OT34 cTCtCAGTGgTGCTgGTCgAAGG(SEQ ID NO: 128) 0 0 0 0.029
OT35 ATCACAcTtATaCTCGaCAgAGG(SEQ ID NO: 129) 0 0 0.054 0.018
OT36 cTCACAGTGAgGCTttTaAAAGG(SEQ ID NO: 130) 0.16 0.154 0.153 0.082
OT37 ATCACtGTGATGtTCGggAgAGG(SEQ ID NO: 131) 0 0 0 0.042
OT38 cTCtCgGTGgTGCTgGTCAAAGG(SEQ ID NO: 132) 0.045 0.061 0.069 0.082
OT39 gTgACAGTcATGCaCGTCcAAGG(SEQ ID NO: 133) 0.017 0.023 0.013 0.017
OT40 ATCACAcTGATtCcCtaCAAAGG(SEQ ID NO: 134) 0.051 0.097 0.024 0.077
OT41 ATgAgAGTGATttTCGTtAAAGG(SEQ ID NO: 135) 0.03 0.017 0 0.05
OT42 ATCACtGTGATGtTtacCAAAGG(SEQ ID NO: 136) 0.038 0.035 0.042 0.012
OT43 ATCACAGTGATGCTtccacAAGG(SEQ ID NO: 137) 0 0.02 0.034 0.012
OT44 gTaACAGTGgTGtTCGaCAAAGG(SEQ ID NO: 138) 0.113 0.209 0.142 0.192
OT45 ATCcCAaTcAgGCTCtTCAAAGG(SEQ ID NO: 139) 0.022 0.014 0.028 0.023
OT46 cTCACAcTGATGCaCtTCAtAGG(SEQ ID NO: 140) 0 0 0 0.01
OT47 AaCACAcTGAgGCTCtgCAAAGG(SEQ ID NO: 141) - - - -
OT48 ATggCAcTGATGCaCGaCAAAGG(SEQ ID NO: 142) 0.022 0.014 0.04 0.011
OT49 caCACtGTcATGtTCGTCAAAGG(SEQ ID NO: 143) 0.34 0.114 0.27 0.054
OT50 tTgACAGTGtTcCTaGTCAAAGG(SEQ ID NO: 144) 0.017 0.014 0.013 0
OT51 ATCAtAGgtATGtTgGTCAAAGG(SEQ ID NO: 145) 0 0.016 0.038 0.026
OT52 ATCACAcTGATGCcCtaCAtAGG(SEQ ID NO: 146) 0.011 0 0 0.021
OT53 ATCACAcTGATtCcCtgCAAAGG(SEQ ID NO: 147) 0.047 0.036 0.043 0.025
OT54 AaCAtAGcGtTGCTaGTCAAAGG(SEQ ID NO: 148) 0.049 0.043 0.087 0.119
OT55 ATCACAtgGATcCTCcTgAAAGG(SEQ ID NO: 149) 0.025 0 0 0
OT56 tTttCAaTGATGCTCaTCAAAGG(SEQ ID NO: 150) 0.023 0.015 0.018 0
OT57 tTCtCtGTcATGtTCGTCAAAGG(SEQ ID NO: 151) 0.027 0.052 0.02 0.019
OT58 ATCACAGTatTGgTCcaCAAAGG(SEQ ID NO: 152) 0.052 0.02 0.044 0.041
OT59 ATgctAGaGATGCTtGTCAAAGG(SEQ ID NO: 153) 0.029 0.01 0.017 0.078
OT60 ATCACAcTGATGCaCtaCAgAGG(SEQ ID NO: 154) 0 0 0 0.023
OT61 cTCACAcTGATGCaCtaCAAAGG(SEQ ID NO: 155) 0.051 0.052 0.061 0.018
OT62 tTgAtAGTGtTcCTCGTCAAAGG(SEQ ID NO: 156) - - - -
OT63 ATCACAGatATcaTgGTCAAAGG(SEQ ID NO: 157) 0.013 0 0.032 0.026
OT64 ATCttAGTcAaGCTaGTCAAAGG(SEQ ID NO: 158) - - - -
OT65 ATCAgAtTtATGCTCaTtAAAGG(SEQ ID NO: 159) - - - -
OT66 ATCtgAGTGATctTCGTCgAAGG(SEQ ID NO: 160) 0.033 0.02 0 0.027
WT T0-20 T0-24 T0-25
Site name Sequence Indels ( % ) Indels ( % ) Indels ( % ) Indels ( % )
On-target ATCACAGTGATGCTCGTCAAAGG(SEQ ID NO: 94) 0.021 99.912 99.867 45.042
OT67 ATggCAGTGtTcCTaGTCAAAGG(SEQ ID NO: 161) - - - -
OT68 ATCACAtTtATGCTtaTCtAAGG(SEQ ID NO: 162) 0.019 0.011 0.019 0.023
OT69 tcCACAGTGtTcCTaGTCAAAGG(SEQ ID NO: 163) 0.014 0.024 0.028 0.013
OT70 tTCttAGgGATGgTCGTCAAAGG(SEQ ID NO: 164) 0.042 0.02 0.024 0.013
OT71 AaCACAGTcATGCTCacCAgAGG(SEQ ID NO: 165) 3.006 2.67 2.831 0.935
OT72 AaaAgAGTGATGCTtaTCAAAGG(SEQ ID NO: 166) 0.018 0.012 0.018 0.029
OT73 cTtcCAGTGATGaTaGTCAAAGG(SEQ ID NO: 167) 0.051 0.021 0.02 0.043
OT74 ATCAaAGTGAgataCGTCAAAGG(SEQ ID NO: 168) 0.012 0.022 0 0.021
OT75 ATgAtAtTGAcGCTtGTCAAAGG(SEQ ID NO: 169) 0 0.055 0.02 0.053
OT76 ATCACgcTGATGggCcTCAAAGG(SEQ ID NO: 170) 0.012 0.016 0 0
OT77 ATagatGTGATGCTtGTCAAAGG(SEQ ID NO: 171) 0.012 0.02 0 0.022
OT78 gTCcCAtTGATGCaCGaCAAAGG(SEQ ID NO: 172) 0.017 0.046 0.051 0
OT79 tTgACAaTtATGCTCtTCAAAGG(SEQ ID NO: 173) 0.175 0.178 0.18 0.332
OT80 ATtAaAaTcATGtTCGTCAAAGG(SEQ ID NO: 174) 0.082 0.037 0.051 0.025
OT81 caCACAGTcATGtTCcTCAAAGG(SEQ ID NO: 175) 0 0.022 0.036 0.03
OT82 tTgACAaTcATGCTCtTCAAAGG(SEQ ID NO: 176) - - - -
OT83 tTCAtAGTGATGtTttTCAAAGG(SEQ ID NO: 177) 0.043 0.059 0.033 0.058
OT84 ATCACgcTcATGaTCcTCAAAGG(SEQ ID NO: 178) 0 0.03 0 0
OT85 ATCACAcTcATGgaCcTCAAAGG(SEQ ID NO: 179) 0 0.034 0.039 0.01
OT86 ATCAtAtTGAaGCcCtTCAAAGG(SEQ ID NO: 180) 0.027 0.053 0.079 0.053
OT87 ATCACAaTGATGgTCGgggAAGG(SEQ ID NO: 181) 0.268 0.358 0.301 0.273
OT88 ATCAtAaTGAaGCcCtTCAAAGG(SEQ ID NO: 182) 0.029 0.057 0.085 0.057
OT89 ATgAatGTtATGCTCtTCAAAGG(SEQ ID NO: 183) 0 0.038 0 0.052
OT90 ATCACAcTGATaCcCtaCAAAGG(SEQ ID NO: 184) 0.027 0.026 0.053 0.051
OT91 AatAtAaTGATtCTCGTCAAAGG(SEQ ID NO: 185) 0.022 0.02 0.013 0.036
OT92 ATgACtGTGtTcCTtGTCAAAGG(SEQ ID NO: 186) 0 0 0.122 0.074
OT93 cTCAaAGTcATGaTCtTCAAAGG(SEQ ID NO: 187) 0 0.026 0 0.022
OT94 cTCAatGaGATGCTCGaCAAAGG(SEQ ID NO: 188) 0.053 0.052 0.056 0.057
OT95 ATCACAcTtAaGCTCtTgAAAGG(SEQ ID NO: 189) 0.201 0.216 0.15 0.161
OT96 gTgACAGTGtTGCTtGTCgAAGG(SEQ ID NO: 190) 0.012 0.012 0.015 0
OT97 ATaACAacaATGaTCGTCAAAGG(SEQ ID NO: 191) 0.036 0.016 0.048 0.057
OT98 AaCACtGTGATGtTtGTCAgAGG(SEQ ID NO: 192) 0 0 0 0
OT99 ATCACgcTGATagTCcTCAAAGG(SEQ ID NO: 193) 0 0 0 0
OT100 gTgACAaTtATGCTCtTCAAAGG(SEQ ID NO: 194) 1.201 0.847 1.346 0.61
Subsequently, whole plants were successfully regenerated from these genome-edited calli and grown in soil (Fig. 4c and Fig. 9). Seeds were obtained from a fully-grown homozygous bi-allelic mutant. As expected, the mutant allele was transmitted to the seeds (Fig. 10). Further studies are warranted to test whether the BIN2-disrupted lettuce displays enhanced BR signaling.
In summary, RGEN RNPs were successfully delivered into plant protoplasts and induced targeted genome modifications in 6 genes in 4 different plant species. Importantly, RGEN-induced mutations were stably maintained in whole plants regenerated from the protoplasts and transmitted to germlines. Because no recombinant DNA is used in this process, the resulting genome-edited plants could be exempted from current GMO regulations, paving the way for the widespread use of RNA-guided genome editing in plant biotechnology and agriculture.

Claims (32)

  1. Method for preparing a plant from a protoplast comprising knocking-out or knocking-in one or more the endogenous gene of the protoplast.
  2. The method of claim 1, wherein the endogenous gene of the plant is a gene capable of increasing stress resistance of the plant by knocking-out or knocking-in.
  3. The method of claim 1, wherein the endogenous gene of the plant is a gene involved in Brassinosteroid signal transduction of plants.
  4. The method of claim 1, wherein:
    (i) in the knocking-out step, the endogenous gene is one or more genes selected from the group consisting of BIN2 gene, BKI1 gene, and homolog genes thereof; and
    (ii) in the knocking-in step, the gene being knocked in is one or more genes selected from the group consisting of BRI1 gene, BSU gene, BZR1 gene, DWF4 gene, CYP85A1, and homolog genes thereof.
  5. The method of claim 1, wherein the knocking-out of genes is performed by knocking-out one or two alleles of the genes selected from the group consisting of BIN2 gene, BKI1 gene, and homolog genes thereof.
  6. The method of claim 1, wherein the knocking-out of genes is performed by gene knock-out and the knocking-in of genes is performed by gene knock-in.
  7. The method of claim 1, wherein the knocking-out of genes is performed using an engineered nuclease specific to one or more genes selected from the group consisting of BIN2 gene, BKI1 gene, and homolog genes thereof.
  8. The method of claim 7, wherein the engineered nuclease is selected from the group consisting of zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and RNA-guided engineered nuclease (RGEN).
  9. The method of claim 8, wherein the RGEN comprises guide RNA, which specifically binds to a specific sequence of one or more genes selected from the group consisting of BIN2 gene, BKI1 gene, and homolog genes thereof, or DNA encoding the guide RNA; and a nucleic acid encoding a Cas protein, or a Cas protein.
  10. The method of claim 1, wherein the knocking-out of genes is performed by introducing the guide RNA, which specifically binds to a specific sequence of one or more genes selected from the group consisting of BIN2 gene, BKI1 gene, and homolog genes thereof, or DNA encoding the guide RNA; and a nucleic acid encoding a Cas protein, or a Cas protein, to the protoplast.
  11. The method of claim 10, wherein the guide RNA is in the form of a dual RNA or a single-chain guide RNA (sgRNA) comprising crRNA and tracrRNA.
  12. The method of claim 11, wherein the single-chain guide RNA comprises a part of crRNA and tracrRNA.
  13. The method of claim 10, wherein the single-chain guide RNA is in the form of isolated RNA.
  14. The method of claim 10, wherein the DNA encoding the guide RNA is encoded by a vector, and the vector is virus vector, plasmid vector, or Agrobacterium vector.
  15. The method of claim 10, wherein the Cas protein is a Cas9 protein or a variant thereof.
  16. The method of claim 10, wherein the Cas protein recognizes NGG trinucleotide.
  17. The method of claim 10, wherein the Cas protein is linked to a protein transduction domain.
  18. The method of claim 15, wherein the variant of the Cas9 protein is in a mutant form of Cas9 protein, wherein the catalytic aspartate residue is substituted with another amino acid.
  19. The method of claim 18, wherein the amino acid is alanine.
  20. The method of claim 10, wherein the nucleic acid encoding a Cas protein or Cas protein is derived from a microorganism of the genus Streptococcus.
  21. The method of claim 20, wherein the microorganism of the genus Streptococcus is Streptococcus pyogenes.
  22. The method of claim 1, wherein the protoplast is derived from Lactuca sativa.
  23. The method of claim 10, wherein the introduction is performed by co-transfecting or serial-transfecting of a nucleic acid encoding a Cas protein or a Cas protein, and the guide DNA or DNA encoding the guide DNA into a protoplast.
  24. The method of claim 23, wherein the serial-transfection is performed by firstly transfecting a Cas protein or a nucleic acid encoding a Cas protein followed by secondly transfecting a naked guide RNA.
  25. The method of claim 10, wherein the introduction is performed by a method selected from the group consisting of microinjection, electroporation, DEAE-dextran treatment, lipofection, nanoparticle-mediated transfection, protein transduction domain-mediated transfection, and PEG-mediated transfection.
  26. The method of claim 1, further comprising regenerating the protoplast having a knocked-out gene.
  27. The method of claim 26, wherein the regeneration comprises culturing a protoplast having one or more knocked-out genes selected from the group consisting of BIN2 gene, BKI1 gene, and homolog genes thereof in agarose-containing medium to form callus; and culturing the callus in regeneration medium.
  28. A plant regenerated from a genome-edited protoplast prepared by a method according to any of claims 1 to 27.
  29. A composition for cleaving DNA encoding BIN2 gene in a plant cell, comprising:
    a guide RNA specific to DNA encoding Brassinosteroid Insensitive 2 (BIN2) gene, BKI1 gene, or homologs thereof, or DNA encoding the guide RNA; and
    a nucleic acid encoding a Cas protein, or a Cas protein.
  30. The composition of claim 29, wherein the composition induces a targeted mutagenesis in a plant cell.
  31. A composition for preparing a plant from a protoplast, comprising:
    a guide RNA specific to DNA encoding Brassinosteroid Insensitive 2 (BIN2) gene, BKI1 gene, or homologs thereof, or DNA encoding the guide RNA; and
    a nucleic acid encoding a Cas protein or a Cas protein.
  32. A kit for preparing a plant from a protoplast comprising the composition according to any of claims 29 to 31.
PCT/KR2016/011217 2015-10-06 2016-10-06 Method for producing whole plants from protoplasts WO2017061806A1 (en)

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CN201680065399.9A CN108368516A (en) 2015-10-06 2016-10-06 Method for generating entire plant from protoplast
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