METHOD FOR PRODUCING WHOLE PLANTS FROM PROTOPLASTS

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.

Claims

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.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0014] FIG. 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 timecourse analysis of genome editing in Arabidopsis protoplasts. (Top) The T7E1 assay. (Bottom) DNA sequences of the wild-type (WT) and mutant sequences.

[0015] FIG. 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.

[0016] FIG. 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.

[0017] 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.

[0018] FIG. 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.

[0019] FIG. 6. Partial nucleotide and amino acid sequences of LsBIN2. Underscored and boxed letters represent the sequences corresponding to degenerate primers and sgRNA, respectively.

[0020] FIG. 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 m). (b) A multicellular colony of protoplast (Bar=100 m). (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)

[0021] 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.

[0022] 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. (f) Root induction onto elongated shoots. (g) Acclimatization of plantlets. (h,i) Regenerated whole plants.

[0023] 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.

BEST MODE FOR CARRYING OUT THE INVENTION

[0024] 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.

[0025] 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.

[0026] In another embodiment, the endogenous gene of the plant may be a gene involved in Brassinosteroid signal transduction of plants.

[0027] 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.

[0028] 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 BIN 2 gene, BKI1 gene, and homolog genes thereof.

[0029] 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.

[0030] 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.

[0031] 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).

[0032] 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.

[0033] 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.

[0034] 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.

[0035] In still another embodiment, the single-chain guide RNA may comprise a part of crRNA and tracrRNA.

[0036] In still another embodiment, the single-chain guide RNA may be in the form of isolated RNA.

[0037] 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.

[0038] In still another embodiment, the Cas protein may be a Cas9 protein or a variant thereof.

[0039] In still another embodiment, the Cas protein may recognize NGG trinucleotide.

[0040] In still another embodiment, the Cas protein may be linked to a protein transduction domain.

[0041] 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.

[0042] In still another embodiment, the amino acid may be alanine.

[0043] In still another embodiment, the nucleic acid encoding a Cas protein or Cas protein may be derived from a microorganism of the genus Streptococcus.

[0044] In still another embodiment, the microorganism of the genus Streptococcus may be Streptococcus pyogenes.

[0045] In still another embodiment, the protoplast may be derived from Lactuca sativa.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] In still another embodiment, the method may further comprise regenerating the protoplast having a knocked-out gene.

[0050] 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.

[0051] Another aspect of the present invention is a plant regenerated from a genome-edited protoplast prepared by the above method.

[0052] 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.

[0053] In still another embodiment, the composition may induce a targeted mutagenesis in a plant cell.

[0054] 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.

[0055] Another aspect of the present invention is a kit for preparing a plant from a protoplast comprising the above composition.

MODE FOR THE INVENTION

[0056] 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

[0057] Cas9 Protein and Guide RNAs.

[0058] 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 1 DNase I reaction buffer. Transcribed sgRNAs were resolved on an 8% denaturing ureapolyacryl amide gel with SYBR gold staining (Invitrogen) for quality control. Transcribed sgRNAs were purified with MG PCR Product Purification SV (Macrogen) and quantified by spectrometry.

TABLE-US-00001 TABLE1 ListofprimersusedforT7E1assay 1.sup.stPCR 2.sup.ndPCR Target Forward(5 to3) Reverse(5 to3) Forward(5 to3) Reverse(5 to3) AOC CGAGCTCAATG GATCAGAATG ATGCAGAGTC AACGTGACC CAGAGTCC CAGCCGT (SEQIDNO:1) AGC(SEQID TAT(SEQIDNO: NO:2) 3) PHYB TGGTTGTTTGC GAAAAGCCTG GCCTCCCCATT CATCACACT AAAGGACGAA TGATTTCTT (SEQIDNO:4) (SEQIDNO:5) (SEQIDNO:6) P450 GGAGCTGAAC CCCAGCACCTG ACCCCAGGCC GGGACAAAGA CACTTCATCC CTTCACTAT AATTCATG TTCATGCAGCA (SEQIDNO:7) (SEQIDNO:8) (SEQIDNO:9) (SEQIDNO:10) DWD1 CCTTTTCTTTG TCCTTCTCCCT ATCTCGTGCCA TGGGGTGTG CTCCTCCTG TCTCCATCC (SEQIDNO:11) (SEQIDNO:12) (SEQIDNO:13) BRI1 ATTTGGGCTGA TGTTGAACACC ACCAATTGGA CCATGCCAAA TCCTTGTTG TGAAACTTTGG AGCTGACTGG ATCTGAAACC (SEQIDNO:14) (SEQIDNO:15) (SEQIDNO:16) (SEQIDNO:17)

TABLE-US-00002 TABLE2 Listofprimersusedfortargeteddeepsequencing(1.sup.stprimers) Target Forward(5 to3) Reverse(5 to3) PHYB-OT1 CCGCATTCAACAGCTCTCTC GCTCAAATCAGGTGGCTAC (SEQIDNO:18) G(SEQIDNO:19) PHYB-OT2 AGGCTGTTCAAAGTCCAGG ATCGCTGGGAGTTCAACAG T(SEQIDNO:20) A(SEQIDNO:21) PHYB-OT3 CCAATGGGCCTGAAAGCTT ACAACCAAAATCCGCAACG T(SEQIDNO:22) A(SEQIDNO:23) BRI1-TS1-OT1 CGCAAGTTGGTCAGAGTGA ACAAGGAGGCTGACGGAAA A(SEQIDNO:24) (SEQIDNO:25) BRI1-TS1-OT2 ACTCGTTACAGGACTCGGT TACAGAGCTGCTTCTGGACC G(SEQIDNO:26) (SEQIDNO:27) BRI1-TS1-OT3 TTACCGTAGCTGGGATCGTC GACTTGTCTCCCTCGCCATA (SEQIDNO:28) (SEQIDNO:29) BRI1-TS1-OT4 GCAAGGACGGATGAGAAAC TGGCATAGTCGCTATTTCGC C(SEQIDNO:30) (SEQIDNO:31) BRI1-TS1-OT5 GTCTCCAAAATCCTCGTCGC GGAAAATTTCTCCCCGCCTC (SEQIDNO:32) (SEQIDNO:33) BRI1-TS1-OT6 TATGGCGGAAGGTGTAGGT TTGCTTGGCTGAAACTCACC C(SEQIDNO:34) (SEQIDNO:35) BRI1-TS2-OT1 CGAGTGCTGATGTGTGTGTT TCTCTTGGTGCAGGGTGAAT (SEQIDNO:36) (SEQIDNO:37) BRI1-TS2-OT2 CCCTCTCAATTGCAGCCATT CGTGTCTTCCTCTGCCATTG (SEQIDNO:38) (SEQIDNO:39) BRI1-TS2-OT3 ACATTTGCTGCATTGGGATC CCAACCCGGCTCAAACTTA T(SEQIDNO:40) C(SEQIDNO:41) BRI1-TS2-OT4 CTCGTCTCAGCCAGGTTAGT ATCAAGAATCCAATGGCGG (SEQIDNO:42) C(SEQIDNO:43)

TABLE-US-00003 TABLE3 Listofprimersusedfortargeteddeepsequencing(2.sup.ndprimers) Sequence(5 to3) AOC-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGAGC TCAATGAACGTGACC(SEQIDNO:44) AOC-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGATC AGAATGCAGAGTCCAGC(SEQIDNO:45) PHYB-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCAAA TGTCAGAGAAACGCG(SEQIDNO:46) PHYB-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATCA GTGCTTAATCCGGTTGA(SEQIDNO:47) P450-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTACCCC AGGCCAATTCATG(SEQIDNO:48) P450-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGCT CTGGTTTCAAGTTAGTACA(SEQIDNO:49) DWD1-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTGCC ACAACCAACGGATC(SEQIDNO:50) DWD1-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGGA TTCAGACCCACCCG(SEQIDNO:51) BRI1-TS1-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTTTGCG GATCTTCTTCAGGCT(SEQIDNO:52) BRI1-TS1-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCTC GTCTCCAACTTTGCAA(SEQIDNO:53) BRI1-TS2-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTTTGCA AAGTTGGAGACGAGC(SEQIDNO:54) BRI1-TS2-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATCT GAAACCCGAGCTTCCA(SEQIDNO:55) BIN2-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGTGG TTTCTTTGAAGCATTGT(SEQIDNO:56) BIN2-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGCC ACTCACAATCACATGT(SEQIDNO:57) PHYB-OT1-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTTTCAT GAAGGTGGCTCAGGT(SEQIDNO:58) PHYB-OT1-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTTC ATTCTCTTGCCGTGGG(SEQIDNO:59) PHYB-OT2-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTGGTGA CAATGTGGCTAATGGT(SEQIDNO:60) PHYB-OT2-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACTC GGCCAATGTTACTCCA(SEQIDNO:61) PHYB-OT3-deepF ACACTCTTTCCCTACACGACGCTCTTCCGATCTGGCTT GTTGGGTGATCTTGA(SEQIDNO:62) PHYB-OT3-deepR GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGACC CACTTCACAGAAAGCA(SEQIDNO:63) BRI1-TS1-OT1- ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCTGC deepF ACGATTCTACCTGACA(SEQIDNO:64) BRI1-TS1-OT1- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCTC deepR CTGTCATGTGTTCCTAAC(SEQIDNO:65) BRI1-TS1-OT2- ACACTCTTTCCCTACACGACGCTCTTCCGATCTTAGCT deepF ATGCCGGTGGAAGTT(SEQIDNO:66) BRI1-TS1-OT2- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACAG deepR AAGTAGCCATTCCGAGA(SEQIDNO:67) BRI1-TS1-OT3- ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGGAG deepF ACCTTTAAGCTTCGC(SEQIDNO:68) BRI1-TS1-OT3- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGCA deepR AAACCATCAGCAGTGG(SEQIDNO:69) BRI1-TS1-OT4- ACACTCTTTCCCTACACGACGCTCTTCCGATCTGTTTG deepF AAGAAGGTGGCCCAG(SEQIDNO:70) BRI1-TS1-OT4- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGTG deepR GGACGATCGAGCTTAT(SEQIDNO:71) BRI1-TS1-OT5- ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGACT deepF AACCGCTTGTCCTCA(SEQIDNO:72) BRI1-TS1-OT5- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACGT deepR TGCCAGTAAAGTTCGC(SEQIDNO:73) BRI1-TS1-OT6- ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTCT deepF CTTACTCGCCTCCTT(SEQIDNO:74) BRI1-TS1-OT6- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCAT deepR CTGAGGTTGGTTCGACA(SEQIDNO:75) BRI1-TS2-OT1- ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCATT deepF CAGCTTTGCCAAACCA(SEQIDNO:76) BRI1-TS2-OT1- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCCG deepR GTGGAATTACTGCTCA(SEQIDNO:77) BRI1-TS2-OT2- ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGTTC deepF ACAATTACTGCCACCA(SEQIDNO:78) BRI1-TS2-OT2- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACTC deepR TCTACGATCGCAACTCT(SEQIDNO:79) BRI1-TS2-OT3- ACACTCTTTCCCTACACGACGCTCTTCCGATCTGGAGA deepF TGGAGGGGATGGAAC(SEQIDNO:80) BRI1-TS2-OT3- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCGGC deepR TCTGAACAGGTCTACA(SEQIDNO:81) BRI1-TS2-OT4- ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGCAA deepF TCAGATGTCCGGTCA(SEQIDNO:82) BRI1-TS2-OT4- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTAC deepR CTCTTCAGCAACCAAGT(SEQIDNO:83)

TABLE-US-00004 TABLE4 Invitrotranscriptiontemplate Sequence(5 to3) AOC-sgF GAAATTAATACGACTCACTATAGCAAAAGACTGTCAATTCCC TGTTTTAGAGCTAGAAATAGCAAG(SEQIDNO:84) PHYB-sgF GAAATTAATACGACTCACTATAGGCACTAGGAGCAACACCCA ACGTTTTAGAGCTAGAAATAGCAAG(SEQIDNO:85) P450-sgF GAAATTAATACGACTCACTATAGGCATATAGTTGGGTCATGG CAGTTTTAGAGCTAGAAATAGCAAG(SEQIDNO:86) DWD1-TS1- GAAATTAATACGACTCACTATAGGTGCATCGTCCAAGCGCAC sgF AGGTTTTAGAGCTAGAAATAGCAAG(SEQIDNO:87) DWD1-TS2- GAAATTAATACGACTCACTATAGGCTACGACGTCAGGTTCTA sgF CCGTTTTAGAGCTAGAAATAGCAAG(SEQIDNO:88) BRI1-TS1- GAAATTAATACGACTCACTATAGGTTTGAAAGATGGAAGCGC sgF GGGTTTTAGAGCTAGAAATAGCAAG(SEQIDNO:89) BRI1-TS2- GAAATTAATACGACTCACTATAGGTGAAACTAAACTGGTCCA sgF CAGTTTTAGAGCTAGAAATAGCAAG(SEQIDNO:90) BIN2-sgF GAAATTAATACGACTCACTATAGATCACAGTGATGCTCGTCA AGTTTTAGAGCTAGAAATAGCAAG(SEQIDNO:91) Universal AAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGG sgR ACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC(SEQID NO:92)

[0059] Protoplast Culture.

[0060] 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 Murashige and Skoog solid medium supplemented with 2% sucrose. The seedlings were grown under a 16 h light (150 mol m.sup.2 s.sup.1) and 8 h dark cycle at 25 C. 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 C. 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 110.sup.6 protoplasts/ml of MMG solution (0.4 M mannitol and 15 mM MgCl.sub.2, 4 mM MES [pH 5.7]).

[0061] Protoplast Transfection.

[0062] PEG-mediated RNP transfections were performed as previously described. Briefly, to introduce DSBs using an RNP complex, 110.sup.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 1NEB buffer 3 and incubated for 10 min at room temperature. A mixture of 110.sup.5 protoplasts (or 510.sup.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.sub.2)), and then incubated at 25 C. for 10 min in darkness. After incubation, 950 L W5 solution (2 mM MES [pH 5.7], 154 mM NaCl, 125 mM CaCl.sub.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 C. for 24-48 h. Cells were analyzed one day after transfection.

[0063] Protoplast Regeneration.

[0064] RNP-transfected protoplasts were re-suspended in B5 culture medium supplemented with 375 mg/l CaCl.sub.2.2H.sub.2O, 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 B5 medium and 2.4% agarose to a culture density of 2.510.sup.5 protoplasts/ml. The protoplasts embedded in agarose were plated onto 6-well plates, overlaid with 2 ml of liquid B5 culture medium, and cultured at 25 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.sup.2 s.sup.1] and 8 h darkness) and cultured at 25 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.

[0065] Rooting, Transfer to Soil and Hardening of Lettuce.

[0066] To regenerate whole plants, proliferated and elongated adventitious shoots were transferred to a fresh regeneration medium and incubated for 4-6 weeks at 25 C. in the light (16 h light [120 mol m.sup.2 s.sup.1] and 8 h darkness). For root induction, approximately 3-5 cm long plantlets were excised and transferred onto a solid hormone-free 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 C. (100-150 mol m.sup.2 s.sup.1 under cool-white fluorescent lamps with a 16-h photoperiod).

[0067] T7E1 Assay.

[0068] 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 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 C. for 20 min and analyzed via agarose gel electrophoresis.

[0069] RGEN-RFLP.

[0070] The RGEN-RFLP assay was performed as previously described. Briefly, PCR products (300-400 ng) were incubated in 1NEB buffer 3 for 60 min at 37 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 C. for 30 min to remove the sgRNA. The reaction was stopped by adding 6 stop solution (30% glycerol, 1.2% SDS, 250 mM EDTA). DNA products were electrophoresed using a 2.5% agarose gel.

[0071] Targeted Deep Sequencing.

[0072] 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

[0073] 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%.

[0074] 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.

[0075] 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.

[0076] 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 bras sinosteroid (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).

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

TABLE-US-00005 TABLE 5 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 1 (on- 0 1 4 27 349 382 sites target) No. of sites with 1 0 1 3 24 72 101 appropriate PCR primers No. of sites amplified 1 0 1 3 22 65 92 successfully

TABLE-US-00006 TABLE6 Indelfrequenciesattheon-targetand91potentialoff-targetsitesin threeregeneratedplantlets.False-positiveindelscausedbysequencing errorsareobservedatfrequenciesthatrangedfrom0%to3.0%. WT T0-20 T0-25 Sitename Sequence Indels(%) Indels(%) Indels(%) On-target ATCACAGTGATGCTCGTCA 0.021 99.912 45.042 AAGG(SEQIDNO:94) OT1 ATCACAGTGcgGCTCGTCAA 0.022 0.039 0 gGG(SEQIDNO:95) OT2 caCACAGTGATGtTCGTCAAg 0 0.014 0.013 GG(SEQIDNO:96) OT3 ATacCAGgGATGCTCGTCAAt 0 0 0 GG(SEQIDNO:97) OT4 ATCAtAGTGATGCTCaTgAAg 0.013 0.03 0 GG(SEQIDNO:98) OT5 ATCACAtTGATGCTCtaCAtAG 0.023 0.033 0.012 G(SEQIDNO:99) OT6 ATaACAGaGAcGaTCGTCAAA 0.029 0.03 0.027 GG(SEQIDNO:100) OT7 ATCACAcTGATGCcCtaCAAA 0.093 0.06 0.109 GG(SEQIDNO:101) OT8 ATCACAtTGAgGCcCGaCAAA GG(SEQIDNO:102) OT9 ATCACAcTGATGCaCtaCAAA 0.057 0.037 0.077 GG(SEQIDNO:103) OT10 caCACAGTGATGtTCaTCAAA 0.635 0.715 0.145 GG(SEQIDNO:104) OT11 ATgACAaTtATGCTCtTCAAA 0.250 0 0 GG(SEQIDNO:105) OT12 ATCAaAGTGcTcCTCGTgAAA 0 0 0 GG(SEQIDNO:106) OT13 taCACAaTGtTGCTCGTCAAcG 0.013 0 0.012 G(SEQIDNO:107) OT14 gcCACAGTGATGaTCGTCgAc 0 0 0.013 GG(SEQIDNO:108) OT15 ATatCAGgGATGCTCGcCAAt 0 0 0 GG(SEQIDNO:109) OT16 AaatCAGTGATcCTCGTCAAc 0 0 0.012 GG(SEQIDNO:110) OT17 ATggCAGTGATGgTCGTgAAg 0 0.045 0.1 GG(SEQIDNO:111) OT18 cTCAgAGTGtTGCTCtTCAAtG 0 0.01 0 G(SEQIDNO:112) OT19 ATCACAGaGATGCTCcaaAAt 0.074 0.033 0.068 GG(SEQIDNO:113) OT20 ATCAagGTtATtCTCGTCAAgG 0 0.009 0 G(SEQIDNO:114) OT21 AgCACAGTGAgGCTtGTCgAg 0 0 0 GG(SEQIDNO:115) OT22 ATatCAagGATGCTCGTCAAtG 0 0 0 G(SEQIDNO:116) OT23 tTCcCAGaGATGCTCtTCAAgG 0.024 0.05 0.035 G(SEQIDNO:117) OT24 gTCACAtTGATGCTCaTCAtgG 0 0 0 G(SEQIDNO:118) OT25 ATCACAGaGATGtTCaTCAtcG 0.022 0 0 G(SEQIDNO:119) OT26 ATCAaAaTGAgGCTCGaCAAc GG(SEQIDNO:120) OT27 ATaACAaTGAaGCTCGTtAAtG 0 0 0 G(SEQIDNO:121) OT28 ATatCAGgGATGCTCaTCAAtG 0 0.011 0.017 G(SEQIDNO:122) OT29 ATCAtAtTGAaGCaCtTCAAAG 0.029 0.019 0.036 G(SEQIDNO:123) OT30 cTCACAtTGATGCaCtaCAAAG 0.069 0.055 0.097 G(SEQIDNO:124) OT31 tcCACAaTGATGCaCtTCAAAG 0.023 0 0.012 G(SEQIDNO:125) OT32 cTCACAaTGtTGCTCtaCAAAG G(SEQIDNO:126) OT33 ATgACAaTGAaGCTCGTaAtA 0 0 0 GG(SEQIDNO:127)

TABLE-US-00007 TABLE7 WT T0-20 T0-24 T0-25 Sitename Sequence Indels(%) Indels(%) Indels(%) Indels(%) On-target ATCACAGTGATGCTCGT 0.021 99.912 99.867 45.042 CAAAGG(SEQIDNO:94) OT34 cTCtCAGTGgTGCTgGTCg 0 0 0 0.029 AAGG(SEQIDNO:128) OT35 ATCACAcTtATaCTCGaCA 0 0 0.054 0.018 gAGG(SEQIDNO:129) OT36 cTCACAGTGAgGCTttTaA 0.16 0.154 0.153 0.082 AAGG(SEQIDNO:130) OT37 ATCACtGTGATGtTCGggA 0 0 0 0.042 gAGG(SEQIDNO:131) OT38 cTCtCgGTGgTGCTgGTCA 0.045 0.061 0.069 0.082 AAGG(SEQIDNO:132) OT39 gTgACAGTcATGCaCGTCc 0.017 0.023 0.013 0.017 AAGG(SEQIDNO:133) OT40 ATCACAcTGATtCcCtaCA 0.051 0.097 0.024 0.077 AAGG(SEQIDNO:134) OT41 ATgAgAGTGATttTCGTtA 0.03 0.017 0 0.05 AAGG(SEQIDNO:135) OT42 ATCACtGTGATGtTtacCAA 0.038 0.035 0.042 0.012 AGG(SEQIDNO:136) OT43 ATCACAGTGATGCTtccac 0 0.02 0.034 0.012 AAGG(SEQIDNO:137) OT44 gTaACAGTGgTGtTCGaCA 0.113 0.209 0.142 0.192 AAGG(SEQIDNO:138) OT45 ATCcCAaTcAgGCTCtTCA 0.022 0.014 0.028 0.023 AAGG(SEQIDNO:139) OT46 cTCACAcTGATGCaCtTCAt 0 0 0 0.01 AGG(SEQIDNO:140) OT47 AaCACAcTGAgGCTCtgCA AAGG(SEQIDNO:141) OT48 ATggCAcTGATGCaCGaCA 0.022 0.014 0.04 0.011 AAGG(SEQIDNO:142) OT49 caCACtGTcATGtTCGTCA 0.34 0.114 0.27 0.054 AAGG(SEQIDNO:143) OT50 tTgACAGTGtTcCTaGTCA 0.017 0.014 0.013 0 AAGG(SEQIDNO:144) OT51 ATCAtAGgtATGtTgGTCA 0 0.016 0.038 0.026 AAGG(SEQIDNO:145) OT52 ATCACAcTGATGCcCtaCA 0.011 0 0 0.021 tAGG(SEQIDNO:146) OT53 ATCACAcTGATtCcCtgCA 0.047 0.036 0.043 0.025 AAGG(SEQIDNO:147) OT54 AaCAtAGcGtTGCTaGTCA 0.049 0.043 0.087 0.119 AAGG(SEQIDNO:148) OT55 ATCACAtgGATcCTCcTgA 0.025 0 0 0 AAGG(SEQIDNO:149) OT56 tTttCAaTGATGCTCaTCAA 0.023 0.015 0.018 0 AGG(SEQIDNO:150) OT57 tTCtCtGTcATGtTCGTCAA 0.027 0.052 0.02 0.019 AGG(SEQIDNO:151) OT58 ATCACAGTatTGgTCcaCA 0.052 0.02 0.044 0.041 AAGG(SEQIDNO:152) OT59 ATgctAGaGATGCTtGTCA 0.029 0.01 0.017 0.078 AAGG(SEQIDNO:153) OT60 ATCACAcTGATGCaCtaCA 0 0 0 0.023 gAGG(SEQIDNO:154) OT61 cTCACAcTGATGCaCtaCA 0.051 0.052 0.061 0.018 AAGG(SEQIDNO:155) OT62 tTgAtAGTGtTcCTCGTCAA AGG(SEQIDNO:156) OT63 ATCACAGatATcaTgGTCA 0.013 0 0.032 0.026 AAGG(SEQIDNO:157) OT64 ATCttAGTcAaGCTaGTCA AAGG(SEQIDNO:158) OT65 ATCAgAtTtATGCTCaTtAA AGG(SEQIDNO:159) OT66 ATCtgAGTGATctTCGTCg 0.033 0.02 0 0.027 AAGG(SEQIDNO:160)

TABLE-US-00008 TABLE8 WT T0-20 T0-24 T0-25 Sitename Sequence Indels(%) Indels(%) Indels(%) Indels(%) On-target ATCACAGTGATGCTCGT 0.021 99.912 99.867 45.042 CAAAGG(SEQIDNO:94) OT67 ATggCAGTGtTcCTaGTCA AAGG(SEQIDNO:161) OT68 ATCACAtTtATGCTtaTCtA 0.019 0.011 0.019 0.023 AGG(SEQIDNO:162) OT69 tcCACAGTGtTcCTaGTCA 0.014 0.024 0.028 0.013 AAGG(SEQIDNO:163) OT70 tTCttAGgGATGgTCGTCA 0.042 0.02 0.024 0.013 AAGG(SEQIDNO:164) OT71 AaCACAGTcATGCTCacC 3.006 2.67 2.831 0.935 AgAGG(SEQIDNO:165) OT72 AaaAgAGTGATGCTtaTCA 0.018 0.012 0.018 0.029 AAGG(SEQIDNO:166) OT73 cTtcCAGTGATGaTaGTCA 0.051 0.021 0.02 0.043 AAGG(SEQIDNO:167) OT74 ATCAaAGTGAgataCGTCA 0.012 0.022 0 0.021 AAGG(SEQIDNO:168) OT75 ATgAtAtTGAcGCTtGTCA 0 0.055 0.02 0.053 AAGG(SEQIDNO:169) OT76 ATCACgcTGATGggCcTCA 0.012 0.016 0 0 AAGG(SEQIDNO:170) OT77 ATagatGTGATGCTtGTCA 0.012 0.02 0 0.022 AAGG(SEQIDNO:171) OT78 gTCcCAtTGATGCaCGaCA 0.017 0.046 0.051 0 AAGG(SEQIDNO:172) OT79 tTgACAaTtATGCTCtTCAA 0.175 0.178 0.18 0.332 AGG(SEQIDNO:173) OT80 ATtAaAaTcATGtTCGTCA 0.082 0.037 0.051 0.025 AAGG(SEQIDNO:174) OT81 caCACAGTcATGtTCcTCA 0 0.022 0.036 0.03 AAGG(SEQIDNO:175) OT82 tTgACAaTcATGCTCtTCA AAGG(SEQIDNO:176) OT83 tTCAtAGTGATGtTttTCAA 0.043 0.059 0.033 0.058 AGG(SEQIDNO:177) OT84 ATCACgcTcATGaTCcTCA 0 0.03 0 0 AAGG(SEQIDNO:178) OT85 ATCACAcTcATGgaCcTCA 0 0.034 0.039 0.01 AAGG(SEQIDNO:179) OT86 ATCAtAtTGAaGCcCtTCA 0.027 0.053 0.079 0.053 AAGG(SEQIDNO:180) OT87 ATCACAaTGATGgTCGgg 0.268 0.358 0.301 0.273 gAAGG(SEQIDNO:181) OT88 ATCAtAaTGAaGCcCtTCA 0.029 0.057 0.085 0.057 AAGG(SEQIDNO:182) OT89 ATgAatGTtATGCTCtTCA 0 0.038 0 0.052 AAGG(SEQIDNO:183) OT90 ATCACAcTGATaCcCtaCA 0.027 0.026 0.053 0.051 AAGG(SEQIDNO:184) OT91 AatAtAaTGATtCTCGTCA 0.022 0.02 0.013 0.036 AAGG(SEQIDNO:185) OT92 ATgACtGTGtTcCTtGTCA 0 0 0.122 0.074 AAGG(SEQIDNO:186) OT93 cTCAaAGTcATGaTCtTCA 0 0.026 0 0.022 AAGG(SEQIDNO:187) OT94 cTCAatGaGATGCTCGaCA 0.053 0.052 0.056 0.057 AAGG(SEQIDNO:188) OT95 ATCACAcTtAaGCTCtTgA 0.201 0.216 0.15 0.161 AAGG(SEQIDNO:189) OT96 gTgACAGTGtTGCTtGTCg 0.012 0.012 0.015 0 AAGG(SEQIDNO:190) OT97 ATaACAacaATGaTCGTCA 0.036 0.016 0.048 0.057 AAGG(SEQIDNO:191) OT98 AaCACtGTGATGtTtGTCA 0 0 0 0 gAGG(SEQIDNO:192) OT99 ATCACgcTGATagTCcTCA 0 0 0 0 AAGG(SEQIDNO:193) OT100 gTgACAaTtATGCTCtTCA 1.201 0.847 1.346 0.61 AAGG(SEQIDNO:194)

[0081] 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.

[0082] 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.