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EP-3469077-B1 - USE OF CPF1 ENDONUCLEASE FOR PLANT GENOME MODIFICATIONS

EP3469077B1EP 3469077 B1EP3469077 B1EP 3469077B1EP-3469077-B1

Inventors

  • CIGAN, Andrew Mark
  • DJUKANOVIC, Vesna
  • YOUNG, JOSHUA K.

Dates

Publication Date
20260513
Application Date
20170531

Claims (15)

  1. A method for modifying a target sequence in the genome of a plant cell, the method comprising: a) introducing into a plant cell a Cpf1 endonuclease protein or a plant-optimized polynucleotide encoding said Cpf1 endonuclease protein, and a guide polynucleotide comprising a variable targeting domain that is substantially complementary to a target sequence in the plant genome or a recombinant DNA expressing said guide polynucleotide; and b) incubating said plant cell at a temperature of 28°C to 37°C for a period of at least about 4 hrs.; wherein said guide polynucleotide and Cpf1 endonuclease are capable of forming a complex that can recognize, bind to, and optionally nick or cleave said target sequence; wherein said method does not comprise an essentially biological process for the production of plants.
  2. The method of claim 1, wherein the plant-optimized polynucleotide is a plant-optimized mRNA encoding said Cpf1 endonuclease protein or a recombinant DNA construct comprising a promoter operably linked to a plant-optimized polynucleotide encoding said Cpf1 endonuclease protein.
  3. The method of claim 1, further comprising identifying at least one plant cell that has a modification at said target sequence, wherein the modification at said target sequence is selected from the group consisting of (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i) - (iii).
  4. The method of claim 3, wherein said modification at said target sequence occurs at an increased frequency when compared to a control method, wherein the plant cell of b) is incubated at a typical plant tissue culture temperature of 28°C.
  5. The method of claim 4, wherein the increased frequency when compared to said control method is increased by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 10-fold, at least 11-fold , at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 30-fold, at least 40-fold, or at least 50-fold.
  6. The method of claim 1, further comprising introducing a donor DNA to the plant cell, wherein said donor DNA comprises a polynucleotide of interest.
  7. The method of claim 6, further comprising identifying at least one plant cell comprising in its genome the polynucleotide of interest integrated into or near said target sequence.
  8. A method for editing a nucleotide sequence in the genome of a plant cell, the method comprising; a) introducing into a plant cell a Cpf1 endonuclease protein or a plant-optimized polynucleotide encoding said Cpf1 endonuclease protein, a guide polynucleotide comprising a variable targeting domain that is substantially complementary to a target sequence in the plant genome, and a polynucleotide modification template, wherein said polynucleotide modification template comprises at least one nucleotide modification of said nucleotide sequence; and, b) incubating said plant cell at a temperature of 28°C to 37°C for a period of at least about 4 hrs.; wherein said guide polynucleotide and Cpf1 endonuclease protein are capable of forming a complex that can recognize, bind to, and optionally nick or cleave all or part of said target sequence; wherein said method does not comprise an essentially biological process for the production of plants.
  9. The method of claim 8, further comprising identifying at least one plant cell comprising in its genome said at least one nucleotide modification of said nucleotide sequence.
  10. The method of claim 1 or 8, wherein said guide polynucleotide is selected from the group consisting of a RNA polynucleotide, a DNA polynucleotide, or a RNA-DNA polynucleotide.
  11. The method of claim 1, wherein the plant cell is a monocot plant cell or a dicot plant cell.
  12. The method of claim 11, wherein the plant cell is selected from the group consisting of maize, rice, sorghum, rye, barley, wheat, millet, oats, sugarcane, turfgrass, or switchgrass, soybean, canola, alfalfa, sunflower, cotton, tobacco, peanut, potato, tobacco, Arabidopsis, and safflower cell.
  13. A method of simultaneously modifying multiple target sequences in the genome of a plant cell, the method comprising: a) introducing into said plant cell a Cpf1 endonuclease protein, or a plant-optimized polynucleotide encoding said Cpf1 endonuclease protein, and a precursor guide RNA transcriptional initiation cassette capable of expressing a single precursor RNA that is processed into a multitude of single guide RNAs, wherein each single guide RNA comprises a variable targeting domain 3' of a Protospacer Adjacent Motif (PAM), wherein said variable targeting domain is complementary to a single target sequence in the plant genome; and b) incubating the plant cell of (a) at a temperature of 28°C to 37°C for a period of at least about 4 hrs.; wherein each of said single guide RNA and said Cpf1 endonuclease protein is capable of forming a ribonucleotide complex that can recognize, bind to, and optionally nick or cleave a target sequence; wherein said method does not comprise an essentially biological process for the production of plants.
  14. The method of claim 13, wherein: a) the multiple target sequences consist of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 target sequences; b) the precursor guide RNA transcriptional initiation cassette comprises a Pol-II promoter operably linked to the precursor guide RNA; or c) the precursor guide RNA transcriptional initiation cassette comprises a Pol-III promoter operably linked to the precursor guide RNA.
  15. A method for modifying a DNA target sequence in the genome of a plant cell, the method comprising: a) introducing into a plant cell a Cpf1 endonuclease protein or a plant-optimized polynucleotide encoding said Cpf1 endonuclease protein, and a guide polynucleotide or a recombinant DNA expressing said guide polynucleotide, capable of forming a Cpf1 complex with the Cpf1 endonuclease protein, the guide polynucleotide comprising a variable targeting domain that is substantially complementary to a target sequence in the plant genome; and b) introducing a polynucleotide modification template comprising at least one region that corresponds to a DNA target sequence adjacent to a PAM sequence recognized by the Cpf1 complex, wherein said method further comprises incubating said plant cell at a temperature of 28°C to 37°C for a period of at least about 4 hrs.; further wherein said Cpf1 complex can recognize, bind to, and optionally nick or cleave said target sequence; wherein said method does not comprise an essentially biological process for the production of plants.

Description

FIELD The disclosure relates to the field of plant molecular biology, in particular to compositions of guide polynucleotide/Cpf1 endonuclease systems and compositions and methods for altering the genome of a plant cell. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named 20170518_7128PCT_ST25.txt created on May 18, 2017 and having a size of 94 kilobytes and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety. BACKGROUND Recombinant DNA technology has made it possible to insert DNA sequences at targeted genomic locations and/or modify specific endogenous chromosomal sequences. Site-specific integration techniques, which employ site-specific recombination systems, as well as other types of recombination technologies, have been used to generate targeted insertions of genes of interest in a variety of organism. Genome-editing techniques such as designer zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), or homing meganucleases, are available for producing targeted genome perturbations, but these systems tends to have a low specificity and employ designed nucleases that need to be redesigned for each target site, which renders them costly and time-consuming to prepare. Although several approaches have been developed to target a specific site for modification in the genome of a plant, there still remains a need for more effective genome engineering technologies that are affordable, easy to set up, scalable, and amenable to targeting multiple positions within the plant genome. SUMMARY OF THE INVENTION The invention provides a method for modifying a target sequence in the genome of a plant cell, the method comprising: a) introducing into a plant cell a Cpf1 endonuclease protein or a plant-optimized polynucleotide encoding said Cpf1 endonuclease protein, and a guide polynucleotide comprising a variable targeting domain that is substantially complementary to a target sequence in the plant genome or a recombinant DNA expressing said guide polynucleotide; andb) incubating said plant cell at a temperature of 28°C to 37°C for a period of at least about 4 hrs.; wherein said guide polynucleotide and Cpf1 endonuclease are capable of forming a complex that can recognize, bind to, and optionally nick or cleave said target sequence;wherein said method does not comprise an essentially biological process for the production of plants. The invention further provides a method for editing a nucleotide sequence in the genome of a plant cell, the method comprising; a) introducing into a plant cell a Cpf1 endonuclease protein or a plant-optimized polynucleotide encoding said Cpf1 endonuclease protein, a guide polynucleotide comprising a variable targeting domain that is substantially complementary to a target sequence in the plant genome, and a polynucleotide modification template, wherein said polynucleotide modification template comprises at least one nucleotide modification of said nucleotide sequence; and,b) incubating said plant cell at a temperature of 28°C to 37°C for a period of at least about 4 hrs.; wherein said guide polynucleotide and Cpf1 endonuclease protein are capable of forming a complex that can recognize, bind to, and optionally nick or cleave all or part of said target sequence;wherein said method does not comprise an essentially biological process for the production of plants. Yet further provided by the invention is a method of simultaneously modifying multiple target sequences in the genome of a plant cell, the method comprising: a) introducing into said plant cell a Cpf1 endonuclease protein, or a plant-optimized polynucleotide encoding said Cpf1 endonuclease protein, and a precursor guide RNA transcriptional initiation cassette capable of expressing a single precursor RNA that is processed into a multitude of single guide RNAs, wherein each single guide RNA comprises a variable targeting domain 3' of a Protospacer Adjacent Motif (PAM), wherein said variable targeting domain is complementary to a single target sequence in the plant genome; andb) incubating the plant cell of (a) at a temperature of 28°C to 37°C for a period of at least about 4 hrs.; wherein each of said single guide RNA and said Cpf1 endonuclease protein is capable of forming a ribonucleotide complex that can recognize, bind to, and optionally nick or cleave a target sequence;wherein said method does not comprise an essentially biological process for the production of plants. The invention further provides a method for modifying a DNA target sequence in the genome of a plant cell, the method comprising: a) introducing into a plant cell a Cpf1 endonuclease protein or a plant-optimized polynucleotide encoding said Cpf1 endonucleas