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JP-7855313-B2 - Genome editing in plants

JP7855313B2JP 7855313 B2JP7855313 B2JP 7855313B2JP-7855313-B2

Inventors

  • ユーロン・チェン
  • アニー・サルタリコス
  • シュー・ジャンピン
  • シュードン・イェ

Assignees

  • モンサント テクノロジー エルエルシー

Dates

Publication Date
20260508
Application Date
20190524
Priority Date
20180524

Claims (20)

  1. A method for editing the genome of a plant, a) Extracting a mature embryo from a dried, mature plant seed, thereby producing an excised mature plant embryo explant; b ) Delivering a recombinant DNA construct, comprising a sequence encoding a site-specific nuclease, wherein the sequence is operably linked to a plant-expressible promoter , to the excised mature plant embryo explant via particle impact ; c) The excised mature plant embryo explant is cultured at approximately 37°C after impact; d ) regenerating a plant from the excised mature plant embryo explant without forming an embryogenetic callus or callus culture from the excised mature plant embryo explant, wherein the regenerated plant contains editing or site-directed integration at or near the target site of the site-specific nuclease in the genome of at least one cell of the regenerated plant; and e) identifying the regenerated plant having at least one cell containing the editing or site-directed integration at or near the target site of the site-specific nuclease, based on phenotype or trait or based on a molecular assay.
  2. The method according to claim 1 , wherein the particles coated with the recombinant DNA construct are delivered to the excised mature plant embryo explant via particle impact.
  3. The method according to claim 2 , wherein the particles are tungsten particles, platinum particles, or gold particles.
  4. The method according to claim 2 or 3 , wherein the particles have a size between 0.5 μm and 1.5 μm.
  5. The method according to claim 4 , wherein the particles have a size of 0.6 μm, 0.7 μm, or 1.3 μm.
  6. The method according to claim 2 , wherein a plurality of particles coated with the recombinant DNA molecule are delivered to the excised mature plant embryo explant via particle impact.
  7. The method according to claim 6 , wherein the amount of particles delivered to the explant is between 50 μg and 5000 μg.
  8. The method according to any one of claims 1 to 7 , wherein the site-specific nuclease is a ribonucleoprotein.
  9. The method according to any one of claims 2 to 7 , wherein the particles are further coated with guide RNA.
  10. The aforementioned site-specific nucleases are Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Csn1, Csx12, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3 The method according to any one of claims 1 to 9, wherein Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, CasZ , or Argonaut protein, or homologs or modified versions thereof.
  11. The method according to any one of claims 1 to 9 , wherein the site-specific nuclease is Cas9 protein.
  12. The method according to claim 1 , wherein the Cas9 protein is derived from Streptococcus pyogenes.
  13. The method according to any one of claims 1 to 9 , wherein the site-specific nuclease is the Cpf1 protein.
  14. The method according to any one of claims 1 to 7 , wherein the site-specific nuclease is not a guide-type nuclease.
  15. The method according to claim 14 , wherein the site-specific nuclease is a meganuclease, a zinc finger nuclease (ZFN), a recombinase, a transposase, or a transcription activator-like effector nuclease (TALEN).
  16. The method according to any one of claims 1 to 15 , wherein the delivery step further comprises delivering a second recombinant DNA construct or molecule to the excised mature plant embryo explant.
  17. The method according to any one of claims 2 to 7 , wherein the particles are further coated with a second recombinant DNA construct or molecule.
  18. The method according to claim 16 or 17 , wherein the second recombinant DNA construct or molecule is a donor template.
  19. The method according to claim 18, wherein the donor template comprises a homologous sequence containing the mutation for introducing the mutation into the genome of the plant via template-mediated repair at or near the target site of the site -specific nuclease.
  20. The method according to claim 18 , wherein the donor template includes the insertion sequence and at least one homologous sequence for incorporating the insertion sequence into the genome of the plant at or near the target site of the site-specific nuclease.

Description

This application, which cross-references related applications, claims the benefit of U.S. Provisional Application No. 62/676,228, filed on 24 May 2018, which is incorporated herein by reference in its entirety. The sequence listing, measured as 4 kilobytes on a Microsoft Windows operating system and contained in the file named "MONS444WO_ST25.txt" generated on May 23, 2019, was filed electronically with this specification and is incorporated herein by reference. Technical field This disclosure relates to compositions for genome editing in plants using DNA molecules encoding genome editing reagents, and methods for using the same. Background: Precise genome editing technology is a powerful tool for manipulating gene expression and function and is expected to have the potential to improve agriculture. There is a continuing need in this field for the development of novel compositions and methods that can be used for the effective and efficient editing of plant genomes. Summary of the Invention This disclosure provides a method for editing a plant genome, comprising delivering a recombinant DNA construct to a mature plant embryo explant, the construct comprising a sequence encoding a site-specific nuclease, the sequence operably linked to a promoter expressible in the plant, and regenerating a plant from the mature plant embryo explant, wherein the regenerated plant includes editing or site-directed integration at or near the target site of the site-specific nuclease in the genome of at least one cell of the regenerated plant. In certain embodiments, the recombinant DNA construct is delivered to the mature plant embryo explant via bacterial-mediated transformation. In other embodiments, the recombinant DNA construct is delivered to the mature plant embryo explant via Agrobacterium-mediated transformation. In some embodiments, a T-DNA transformation vector containing the recombinant DNA construct is delivered to the mature plant embryo explant. In additional embodiments, recombinant DNA constructs are delivered to mature plant embryo explants via particle impact. In some embodiments, particles coated with or treated with recombinant DNA constructs are delivered to mature plant embryo explants via particle impact. In certain embodiments, the particles are tungsten particles, platinum particles, or gold particles. In other embodiments, the particles have a size between about 0.5 μm and about 1.5 μm. In yet another embodiment, the particles have a size of about 0.6 μm, about 0.7 μm, or about 1.3 μm. In some embodiments, multiple particles coated with or treated with recombinant DNA molecules are delivered to mature plant embryo explants via particle impact. In a particular embodiment, the amount of particles delivered to the explant is between approximately 50 μg and approximately 5000 μg, or between approximately 50 μg and approximately 5000 μg, or between approximately 50 μg and approximately 2000 μg, or between approximately 50 μg and approximately 1000 μg, or between approximately 50 μg and approximately 500 μg, or between approximately 100 μg and approximately 500 μg. In certain embodiments, the method further includes identifying regenerated plants having at least one cell containing editing or site-directed integration at or near the target site of a site-specific nuclease. In some embodiments, the identification step includes identifying regenerated plants having editing or site-directed integration based on phenotype or trait. In other embodiments, the identification step includes identifying regenerated plants having editing or site-directed integration based on a molecular assay. In further embodiments, the site-specific nuclease is a guided nuclease (such as a CRISPR-related protein). In other embodiments, the particles are further coated with or further applied with guide nucleic acids. In some embodiments, the guided nucleases are Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Csn1, Csx12, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, C The nautilus is mr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, CasZ, or an Argonaut protein, or a homolog or modified version thereof. In yet another embodiment, the nautilus is the Cas9 protein. In yet another embodiment, the Cas9 protein is from Streptococcus pyogenes. In an additional embodiment, the nautilus is the Cpf1 protein. In a particular embodiment, the site-specific nautilus is not the nautilus. In some embodiments, site-specific nucleases are meganucleases, zinc finger nucleases (ZFNs), recombinases, transposases, or transcription activator-like effector nucleases (TALENs). In certain embodiments, the delivery step further includes delivering a second recombinant DNA construct or molecule to a mature plant embryo explant. In some embodiments, particles are further coated with or applied to the second reco