WO2014186686A2

WO2014186686A2 - Targeted mutagenesis and genome engineering in plants using rna-guided cas nucleases

Info

Publication number: WO2014186686A2
Application number: PCT/US2014/038359
Authority: WO
Inventors: Jonathan D.G. Jones; Sophien KAMOUN; Vladimir NEKRASOV
Original assignee: Two Blades Foundation
Current assignee: Two Blades Foundation
Priority date: 2013-05-17
Filing date: 2014-05-16
Publication date: 2014-11-20
Anticipated expiration: 2015-11-17
Also published as: WO2014186686A3

Abstract

Methods for modifying the genome of plants at a target nucleic acid sequence are provided. Such methods involve producing a plant cell comprising a Cas protein and a single guide RNA (sgRNA) that is capable of recognizing the target nucleic acid sequence in the plant cell. Further provided are methods for targeting fusion proteins to target nucleic acid sequences in the genome of plant. Such methods involve producing a plant cell comprising a fusion protein and an sgRNA designed to recognize the target nucleic acid sequence. The fusion protein is capable of recognizing the target sequence in the presence of the sgRNA. The fusion protein comprises a Cas protein domain that is defective in nuclease activity and an additional domain. Also provided are methods for testing components of the Cas system in plants, modified plants and plant cells, fusion proteins, and nucleic acid molecules encoding such fusion proteins.

Description

TARGETED MUTAGENESIS AND GENOME ENGINEERING IN PLANTS USING

RNA-GUIDED CAS NUCLEASES

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS WEB

The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named 446220SEQLIST.TXT, created on May 13, 2014, and having a size of 62.3 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 OF THE INVENTION

Sustainable intensification of crop production is essential to ensure food demand is matched by supply as the human population continues to increase¹. This will require high yielding crop varieties that can be grown sustainably with fewer inputs on less land. Both plant breeding and GM methods make valuable contributions to varietal improvement, but targeted genome engineering promises to be critical to elevating future yields. Most such methods require targeting DNA breaks to defined locations followed by either non-homologous end joining (NHEJ) or homologous recombination . Zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs) can be engineered to create such breaks, but the recombinant DNA work is complicated by the necessity to create two different DNA binding proteins flanking a sequence of interest, each with a C- terminal Fokl nuclease module. Therefore, new methods for targeted mutagenesis genome engineering that are less complicated and costly than existing methods are desired. BRIEF SUMMARY OF THE INVENTION

The present invention is drawn to methods for targeted mutagenesis and genome engineering in plants using RNA-guided Cas nucleases and assay methods of the rapid testing of the components of the Cas system in plants. The invention is based in part on the discovery that the genome of a plant cell can be modified at or in vicinity of a target nucleic acid sequence by introducing into the plant cell a Cas nuclease, particularly a Cas9 nuclease and an engineered, single guide RNA (sgRNA) that is designed for recognition of the target nucleic acid sequence. The methods and compositions of the present invention find use in the genome engineering of plants, particularly in the genome engineering of agricultural plants for the development of improved agricultural plant varieties. This discovery and the methods and compositions of the present invention are further described below.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1. Targeted mutagenesis in planta using the Cas9 RNA-guided

endonuclease. (A) Scheme illustrating the assay. (B) DNA gel with PCR bands obtained upon amplification using primers flanking the target site within the phytoene desaturase (PDS) gene of Nicotiana benthamiana. In lanes 1-3 the template genomic DNA was pre-digested with Mlyl, while in lane 4 non-digested genomic DNA was used. (C) Alignment of reads with Cas9-induced indels in the PDS gene obtained from lane 1 of FIG. IB. The wild-type sequence is shown at the top. The sgRNA target sequence is shaded in grey. Deleted DNA residues are shown as asterisks. Insertions and substitutions are shown as underlined capitals. The change in length and sequence are shown to the right. PAM, the protospacer-adjacent motif, was selected to follow the consensus sequence NGG. The change in length and sequence are shown to the right. Three additional replicates of this experiment are presented in FIG. 5.

FIG. 2. Measurement of the mutation rate induced by Cas9 RNA-guided endonuclease in N. benthamiana. The PDS locus was amplified using non-digested genomic DNA from leaf tissue expressing Cas9 and sgRNA targeting PDS as well as from negative controls (Cas9 plus a sgRNA targeting GFP, and Cas9 on its own). To measure the mutation rate, the intensity of the uncut band was divided by intensity of all bands in the lane. The arrowhead indicates the Mlyl- resistant band; the asterisk indicates the band resulting from star activity of Mlyl. Three additional replicates of this experiment are presented in FIG. 8.

FIG. 3. Cas9 localises to plant nuclei. GFP-Cas9 was expressed in N.

benthamiana leaf tissue and visualised 2 days post in:iltration. (a) GFP. (b) Plastids auto:luorescence. (c) Bright field, (d) Overlay of (a), (b) and (c).

FIG. 4. Description of the U6p: :sgRNA construct. (A) Schematic of Arabidopsis U6p::sgRNA construct. (B) Arabidopsis U6 promoter sequence is shown in regular uppercase; the sgRNA transcription start nucleotide is shown in bold lowercase (g); the guide sequence is shown in underlined uppercase; the sgRNA backbone sequence is shown in lowercase italics; the RNA polymerase III terminator sequence is shown in underlined lowercase italics.

FIG. 5. Replicates of the experiment presented in Figure 1. The PCR

amplification was done with undigested genomic DNA (a) or Mlyl-digested genomic DNA (b). In the case of the pre-digested genomic DNA, co-expression of both Cas9 and sgRNA results in markedly increased levels of the amplicon. A total of three independent replicates (1-3) are shown.

FIG. 6. PCR products amplified on genomic DNA partially digested with Mlyl are resistant to Mlyl digestion only when Cas9 and sgRNA are co-expressed in plant tissue. The arrow head indicates the Mlyl- resistant band; the asterisk indicates the band resulting from Mlyl star activity. A total of three independent replicates (1-3) are shown.

FIG. 7. Representative sequence chromatograms of Cas9- induced indels in the Nicotiana benthamiana genome. Regions highlighted in black belong to the target site within the PDS gene.

FIG. 8. Mutation rate replicates. The PCR was done with the undigested genomic DNA as a template. Resulting amplicons were then digested with Mlyl. The band intensity was quantised with the ImageJ software. The arrowhead indicates the Mlyl% resistant band; the asterisk indicates the band resulting from the star activity of Mlyl.

FIG. 9. Examples of restriction site loss assays with potential off- targets. Analysis of examples of potential off-targets from groups 1 (a), 2 (b)

and 3 (c) (Table 1). The PDS positive control is shown in (d). FIG. 10. A large chromosomal deletion can be generated by targeting two adjacent sequences within the PDS locus of Nicotiana benthamiana using transient expression. A. Schematic representation explaining setup of the

experiment. B. Detection of deletion mutations using the AFLP analysis.

Agarose gel shows PCR bands amplified across targets 1 and 2 using genomic

DNA extracted from respective leaf samples. Cas9, sgR Al and 2 were

expressed in N. benthamiana leaf tissue using the standard agroinfiltration

protocol. In lane 2, Cas9, sgRNAl, and sgRNA2 were expressed from three

separate plasmids, while in lane 4 they were expressed from a single plasmid. C.

Types of deletion mutations identified. Bottom PCR bands from lanes 2 and 4 were cloned into a high copy vector and 15 individual clones were sequenced.

All clones contained deletions that can be grouped in three different types (ml- 3).

FIG. 11. Large chromosomal deletions can be generated by targeting

two adjacent sequences within the Mlol locus of tomato. (A) Schematic

representation explaining the setup of the experiment. (B) Detection of deletion mutations by the AFLP analysis using transient expression in tomato. Cas9 and sgRNAs were expressed in tomato leaf tissue using the standard agro- infiltration protocol. (C) TO tomato transgenic plants genotyped for the presence of CRISPR/Cas-induced deletions. Plants with encircled numbers carry

deletions. Transgenic plants were produced by agro-transformation of the callus tissue and selection on kanamycin. Agarose gels show PCR bands amplified

across respective targets using genomic DNA extracted from tomato leaf

samples. In all cases, Cas9 and both sgRNAs were expressed from a single

plasmid.

FIG. 12. Sequences of sgRNA constructs of Example 2. The AtU6p

sequence is in italics, the guide sequence is underlined, and the sgRNA

backbone is in bold typeface. SEQUENCE LISTING

The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three-letter code for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5' end of the sequence and proceeding forward (i.e., from left to right in each line) to the 3' end. Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand. The amino acid sequences follow the standard convention of beginning at the amino terminus of the sequence and proceeding forward (i.e., from left to right in each line) to the carboxy terminus. As used herein and in the accompanying sequence listing, an "N" in a DNA sequence is intended to mean A, G, C, or T, an "N" in an RNA sequence is intended to mean A, G, C, or U, and an "N" an amino acid sequence is intended to mean any amino acid, preferably any naturally occurring amino acid in a plant, unless stated otherwise or otherwise apparent from the context of usage.

SEQ ID NO: 1 sets forth a nucleotide sequence of a human-codon optimized Cas9 (hCas9) coding sequence.

SEQ ID NO: 2 sets forth the amino acid sequence of the human Cas9 protein encoded by the nucleotide sequence set forth in SEQ ID NO: 1.

SEQ ID NO: 3 sets forth a nucleotide sequence of the human-codon optimized GFP-hCas9 fusion protein coding seqeuence.

SEQ ID NO: 4 sets forth the amino acid sequence of the GFP-hCas9 fusion protein encoded by the nucleotide sequence set forth in SEQ ID NO: 3.

SEQ ID NO: 5 sets forth the nucleotide sequence of the U6p::sgRNA nucleic acid expression construct.

SEQ ID NO: 6 sets forth an RNA sequence of an sgRNA.

DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described more fully hereinafter with reference to the accompanying FIG.s, in which some, but not all embodiments of the inventions are shown. Indeed, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the invention set forth herein will come to mind to one skilled in the art to which these invention pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Embodiments of the invention include, but are not limited to, the following embodiments. 1. A method for modifying the genome of a plant, plant part, or plant cell, the method comprising producing at least one plant cell comprising a Cas protein and at least one engineered, single guide RNA (sgRNA) that is capable of recognizing a target nucleic acid sequence, wherein the plant, plant part, plant cell, or descent thereof, comprises in its genome the target nucleic acid sequence.

2. The method of embodiment 1, wherein the Cas protein is Cas9 or a mutant or derivative of Cas9.

3. The method of embodiment 2, wherein the mutant or derivative is naturally occurring or engineered.

4. The method of any one of embodiments 1-3, wherein the Cas protein is capable of causing a double-strand break in DNA.

5. The method of any one of embodiments 1-3, wherein the Cas protein is capable of causing a single-strand break in DNA.

6. The method of embodiment 2 or 3, wherein the mutant or derivative comprises the DIOA amino acid substitution or the H841A amino acid substitution, and wherein the mutant or derivative is capable of causing a single-strand break in DNA.

7. The method of any one of embodiments 1-6, wherein producing at least one plant cell comprising a Cas protein and an engineered sgRNA, comprises:

(a) introducing into the at least one plant cell or a progenitor cell thereof a nucleic acid construct for expression of the sgRNA in the plant cell, whereby the sgRNA is expressed in the at least one plant cell;

(b) introducing into the at least one a plant cell or a progenitor cell thereof a nucleic acid construct for expression of the Cas protein in the plant cell, whereby the Cas protein is expressed in the at least one plant cell; or (c) both (a) and (b).

8. The method of any one of embodiments 1-7, wherein the sgR A, the Cas protein, or both are transiently expressed in the plant cell.

9. The method of embodiment 7 or 8, wherein the nucleic acid construct for expression of the sgRNA, the nucleic acid construct for expression of the Cas protein, or both are stably incorporated in the genome of the plant cell.

10. The method of any one of embodiments 1-9, wherein producing at least one plant cell comprising a Cas protein and an engineered sgRNA, comprises:

(i) directly introducing an engineered sgRNA into a plant cell and optionally further comprises virus-mediated delivery of the sgRNA into the plant cell;

(ii) directly introducing the Cas protein into the plant cell; or

(iii) both (i) and (ii).

11. The method of any one of embodiments 1-10, wherein the Cas protein or the nucleic acid construct for expression of the Cas protein is introduced into the plant cell or the progenitor, before, concurrently with, or after introducing the sgRNA or the nucleic acid construct for expression of the sgRNA.

12. The method of any one of embodiments 1-11, wherein at least one modification is introduced into the genome of the plant, plant part, or plant cell, or descendant thereof.

13. The method of embodiment 12, wherein the modification occurs within the target nucleic acid sequence.

14. The method of embodiment 12 or 13, wherein the modification comprises the insertion of at least one base pair in the genome of the plant, plant part, plant cell, or descendant thereof.

15. The method of embodiment 12 or 13, wherein the modification comprises the deletion of at least one base pair in the genome of the plant, plant part, plant cell, or descendant thereof.

16. The method of embodiment 12 or 13, wherein the modification comprises the insertion of at least one base pair in the genome of the plant, plant part, plant cell, or descendant thereof and the deletion of at least one base pair in the genome of the plant, plant part, plant cell, or descendant thereof.

17. The method of any one of embodiments 1-16, wherein the target nucleic acid sequence is within a gene or wherein the target nucleic acid sequence affects the level of expression of a gene. 18. The method of embodiment 17, wherein the gene is selected from the group consisting of a disease resistance gene, a disease susceptibility gene, a gene encoding an enzyme, a gene encoding a structural protein, a gene encoding a receptor, a gene encoding a membrane channel protein, a gene encoding a transcription factor, a gene that is associated with herbicide tolerance, a gene that affects development of male or female reproductive structures, and a gene that affects self-incompatibility.

19. The method of embodiment 17 or 18, wherein modification is a mutation in the gene that results in an alteration or loss of the wild-type function of the gene and/or the protein encoded thereby.

20. The method of embodiment 19, wherein the mutation is a loss-of- function mutation.

21. The method of any one of embodiments 1-20, further comprising introducing into the plant, plant part, plant cell, or descendant thereof a donor nucleic acid .

22. The method of embodiment 21 , wherein the donor nucleic acid molecule or part thereof is incorporated into the genome of the plant, plant part, or plant cell or descendant thereof.

23. The method of embodiment 21 or 22, wherein the donor nucleic acid molecule or part thereof is incorporated into the genome of the plant, plant part, plant cell, or descendant thereof within the target nucleic acid sequence.

24. The method of any one of embodiments 21-23, wherein the donor nucleic acid molecule comprises a nucleic acid sequence which comprises in a 5' to 3' order: a first homologous region, a donor region, and a second homologous region.

25. The method of embodiment 25, wherein the first homologous region comprises nucleic acid sequence homology to a first genomic region and the second homologous region comprises nucleic acid sequence homology to a second genomic region.

26. The method of embodiment 26, wherein the first genomic region and the second genomic region are on opposite sides of the site of incorporation of the donor nucleic acid molecule or part thereof into the genome of the plant, plant part, plant cell, or descendant thereof.

27. The method of any one of embodiments 21-26, wherein the donor region comprises a coding and/or a non-coding nucleic acid sequence.

28. The method of any one of embodiments 21-27, wherein the donor region comprises at least one transgene. 29. The method of embodiment 28, wherein the transgene comprises a promoter operably linked to a nucleic acid sequence, and wherein the promoter is capable of driving expression of the operably linked nucleic acid in a plant cell.

30. The method of embodiment 28 or 29, where the transgene encodes a protein. 31. The method of embodiment 30, wherein the protein is native to the plant, plant part, plant cell.

32. The method of embodiment 30, wherein the protein is a foreign protein that is not native the plant, plant part, or plant cell.

33. The method of any one of embodiments 21-32, wherein the donor nucleic acid comprises or further comprises at least one selectable marker gene.

34. The method of any one of embodiments 22-33, wherein incorporation of the donor nucleic acid into the genome of the plant, plant part, or plant cell or descendant thereof produces a selectable marker gene in the genome of the plant, plant part, or plant cell or descendant thereof.

35. The method of embodiment 34, wherein the donor nucleic acid comprises the entire, at least a portion of, or none of, the coding region of a protein encoded by the selectable marker gene.

36. The method of any one of embodiments 21-35, wherein the donor nucleic acid molecule is single stranded or double stranded.

37. The method of any one of embodiments 21-36, wherein the donor nucleic acid molecule is a linear nucleic acid molecular or a circular nucleic acid molecule.

38. The method of any one of embodiments 7-37, wherein the nucleic acid construct for expression of the sgR A comprises a promoter operably linked to a nucleic acid sequence encoding the sgRNA and wherein the promoter is capable of driving expression of a nucleic acid sequence in a plant cell.

39. The method of embodiment 38, wherein the promoter is an RNA polymerase III promoter.

40. The method of embodiment 38 or 39, wherein the promoter is the Arabidopsis U6 promoter.

41. The method of any one of embodiments 38-40, wherein the promoter comprises the Arabidopsis U6 promoter-derived synthetic nucleic acid sequence set forth in FIG. 4B. 42. The method of any one of embodiments 7-41, wherein nucleic acid construct for expression of the sgR A comprises the nucleic acid sequence of the U6::sgRNA construct set forth in FIG. 4B.

43. The method of any one of embodiments 7-42, wherein the nucleic acid construct for expression of the Cas protein comprises a promoter operably linked to a nucleic acid sequence encoding the Cas protein and optionally an operably linked eukaryotic nuclear localization signal sequence, wherein the promoter is capable of driving expression of a nucleic acid sequence in a plant cell.

44. The method of embodiment 43, wherein the promoter is an RNA polymerase II promoter.

45. The method of embodiments 43 or 44, wherein the promoter is a constitutive promoter, a tissue-specific promoter or an inducible promoter.

46. The method of any one of embodiments 43-45, wherein the promoter is selected from the group consisting of the CaMV 35S promoter, a ubiquitin promoter, an estradiol- inducible promoter, a dexamethasone-inducible promoter, an alcohol-inducible promoter, a heat shock promoter, an inflorescence-specific promoter, the Arabidopsis Apetala2 promoter, and the Arabidopsis Agamous promoter.

47. The method of any one of embodiments 1-46, wherein the target nucleic acid sequence comprises or consists of 19 bases.

48. The method of any one of embodiments 1-47, wherein immediately following the 3' end of the target nucleic acid sequence in the genome of the plant, plant part, or plant cells is a protospacer-adjacent motif (PAM).

49. The method of embodiment 48, wherein the PAM is NGG, wherein N can be A, C, G, or T.

50. The method of embodiment 47, wherein the Cas protein recognizes a PAM that is not NGG, wherein N can be A, C, G, or T.

51. The method of any one of embodiments 1-50, wherein the sgRNA comprises a guide nucleic acid sequence that is capable of binding to or recognizing the target nucleic acid or its complement.

52. The method of embodiment 51 , wherein the guide nucleic acid sequence is identical to the target nucleic acid sequence or its complement, when a U in the guide nucleic acid sequence is equivalent to a T in the target nucleic acid sequence or its complement. 53. The method of embodiment 51 , wherein the guide nucleic acid sequence is not identical to the target nucleic acid sequence or its complement, when a U in the guide nucleic acid sequence is equivalent to a T in the target nucleic acid sequence or its complement.

54. The method of embodiment 51 or 53, wherein there are one or more mismatched bases between the guide nucleic acid sequence and the target nucleic acid sequence or its complement.

55. The method of embodiment 54, wherein the one or more mismatches occur at a position or positions in the target nucleic acid sequence that are located within the first 8, 9, 10, 11, or 12 bases beginning from the 5' end of the target nucleic acid sequence.

56. The method of any of embodiments 1-55, further comprising regenerating the plant cell or descendent thereof into a plant comprising at least one modification in its genome or in the genome of a progeny plant or descendent of the regenerated plant, wherein at least one modification in the genome of a progeny plant or descendent is due to the presence of the sgRNA and the Cas protein in the progeny plant or descendent.

57. A plant, plant part, seed, or plant cell produced by the method of any one of embodiments 1-56 and 132, wherein the genome of the plant, plant part, seed, or plant cell is modified when compared to the genome of a control plant cell, and wherein said control plant cell is plant cell before its genome has been modified by the method.

58. A plant, plant part, seed, or plant cell comprising a modification in its genome that was introduced into the genome according to the method of any one of embodiments 1-56 and 132.

59. Use of the plant, plant part, or seed of embodiment 57 or 58 in agriculture and/or in the production of a human and/or animal food product.

60. Use of the plant, plant part, or seed of embodiment 57 or 58 in a plant breeding method and/or in the production of hybrid seed.

61. A method for targeting a fusion protein to a target nucleic acid sequence in the genome of a plant, plant part, or plant cell, producing at least one plant cell comprising a fusion protein and an engineered, single guide RNA (sgRNA), wherein the fusion protein comprises a Cas protein domain that is completely or partially defective in nuclease activity and an additional domain, wherein the sgRNA is capable of recognizing the target nucleic acid sequence, whereby the fusion protein is capable of binding to or recognizing the target sequence when in the presence of the sgRNA. 62. The method of embodiment 61, wherein the Cas protein domain is a Cas9 protein domain or mutant or dervitive of Cas9 protein domain.

63. The method of embodiment 62, wherein Cas9 protein domain comprises the DIOA amino acid substitution, the H841A amino acid substitution, or both the DIOA and H841A amino acid substitutions.

64. The method of any one of embodiments 61-63, wherein producing the at least one plant cell comprising the fusion protein and the engineered, single guide R A, comprises:

(b) introducing into the at least one a plant cell or a progenitor cell thereof a nucleic acid construct for expression of the fusion protein in the plant cell, whereby the fusion protein is expressed in the at least one plant cell; or

(c) both (a) and (b).

65. The method of embodiment of embodiment 64, wherein the sgRNA, the fusion protein, or both are transiently expressed in the plant cell.

66. The method of embodiment 64 or 65, wherein the nucleic acid construct for expression of the sgRNA, the nucleic acid construct for expression of the fusion protein, or both are stably incorporated in the genome of the plant cell.

67. The method of any one of embodiments 61-66, wherein producing the at least one plant cell comprising the fusion protein and the sgRNA, comprises:

(ii) directly introducing the fusion protein into the plant cell; or

(iii) both (i) and (ii).

68. The method of any one of embodiments 61-67, wherein the fusion protein or the nucleic acid construct for expression of the fusion protein is introduced into the plant cell or the progenitor, before, concurrently with, or after introducing the sgRNA or the nucleic acid construct for expression of the sgRNA.

69. The method of any one of embodiments 61-68, wherein the additional domain is selected from the group consisting of a transcriptional activator domain, a transcriptional repressor or co-repressor domain, a protein domain known to elevate the local mutagenesis rate, and a protein that can elevate the local recombination rate.

70. The method of any one of embodiments 61-69, wherein the additional domain is selected from the group consisting of an EAR domain, a SRDX domain, a TOPLESS domain, a cytidine deaminase or other DNA or histone modifying enzyme domain, a MAGI domain, and a RAD54 domain.

71. The method of any one of embodiments 61-70, wherein the target nucleic acid sequence is within a gene.

72. The method of any one of embodiments 61-71, wherein the target nucleic acid sequence is within a promoter.

73. The method of any one of embodiments 64-72, wherein the nucleic acid construct for expression of the fusion protein comprises a promoter operably linked to a nucleic acid sequence encoding the fusion protein and optionally an operably linked eukaryotic nuclear localization signal sequence, wherein the promoter is capable of driving expression of a nucleic acid sequence in a plant cell.

74. The method of embodiment 71, wherein the promoter is an RNA polymerase II promoter.

75. The method of embodiment 73 or 74, wherein the promoter is a constitutive promoter, an inducible promoter, a developmentally regulated promoter, a tissue-specific promoter, and a seed-specific promoter.

76. The method of any one of embodiments 73-75, wherein the promoter is selected from the group consisting of the CaMV 35S promoter, a ubiquitin promoter, an estradiol- inducible promoter, a dexamethasone-inducible promoter, an alcohol-inducible promoter, a heat shock promoter, an inflorescence-specific promoter, the Arabidopsis Apetala2 promoter, and the Arabidopsis Agamous promoter.

77. The method of any one of embodiments 64-76, wherein the nucleic acid construct for expression of the sgRNA comprises a promoter operably linked to a nucleic acid sequence encoding the sgRNA, wherein the promoter is capable of driving expression of a nucleic acid sequence in a plant cell.

78. The method of embodiment 77, wherein the promoter is an RNA polymerase III promoter.

79. The method of embodiment 77 or 78, wherein the promoter is the Arabidopsis U6 promoter. 80. The method of any one of embodiments 77-79, wherein the promoter comprises the Arabidopsis U6 promoter-derived synthetic nucleic acid sequence set forth in FIG. 4B.

81. The method of any one of embodiments 77-80, wherein nucleic acid construct for expression of the sgR A comprises the nucleic acid sequence of the U6: :sgRNA construct set forth in FIG. 4B.

82. The method of any one of embodiments 61-81, wherein the target nucleic acid sequence comprises or consists of 19 bases.

83. The method of any one of embodiments 61-82, wherein immediately following the 3' end of the target nucleic acid sequence in the genome of the plant, plant part, or plant cells is a protospacer-adjacent motif (PAM).

84. The method of embodiment 83, wherein the PAM is NGG, wherein N can be A, C, G, or T.

85. The method of embodiment 83, wherein the Cas protein recognizes a PAM that is not NGG, wherein N can be A, C, G, or T.

86. The method of any one of embodiments 61-85, wherein the sgRNA comprises a guide nucleic acid sequence that is capable of binding to or recognizing the target nucleic acid or its complement.

87. The method of embodiment 86, wherein the guide nucleic acid sequence is identical to the target nucleic acid sequence or its complement, when a U in the guide nucleic acid sequence is equivalent to a T in the target nucleic acid sequence or its complement.

88. The method of embodiment 86, wherein the guide nucleic acid sequence is not identical to the target nucleic acid sequence or its complement, when a U in the guide nucleic acid sequence is equivalent to a T in the target nucleic acid sequence or its complement.

89. The method of embodiment 86 or 88, wherein there are one or more mismatched bases between the guide nucleic acid sequence and the target nucleic acid sequence or its complement.

90. The method of embodiment 89, wherein the one or more mismatches occur at a position or positions in the target nucleic acid sequence that are located within the first 8, 9, 10, 11, or 12 bases beginning from the 5' end of the target nucleic acid sequence. 91. The method of any one of embodiments 61-90, further comprising introducing at least one additional engineered sgRNA, wherein the sgRNA is capable of recognizing an additional target nucleic acid sequence.

92. The method of embodiment 91, wherein the fusion protein is also capable of binding to or recognizing the additional target sequence when in the presence of the additional sgRNA.

93. The method of any of embodiments 61-92, further comprising regenerating the plant cell or descendent thereof into a plant comprising the fusion protein and/or the sgRNA and/or at least one modification in the genome of the plant cell or descendent that is due the fusion protein and the sgRNA.

94. A plant, plant part, seed, or plant cell produced by the method of any one of embodiments 61-93, wherein the plant, plant part, seed, or plant cell comprises the fusion protein and the engineered sgRNA.

95. A plant, plant part, seed, or plant cell comprising the fusion protein and/or sgRNA introduced into the genome according to the method of any one of embodiments 61-93 or comprising at least one modification in its genome that is due to the fusion protein and the sgRNA according to the method of any one of embodiments 61-93.

96. Use of the plant, plant part, or seed of embodiment 94 or 95 in agriculture and/or in the production of a human and/or animal food product.

97. Use of the plant, plant part, or seed of embodiment 94 or 95 in a plant breeding method and/or in the production of hybrid seed.

98. A fusion protein according to any of embodiments 61-93 or a nucleic acid molecule encoding the fusion protein.

99. A transient assay method for use in testing components of the Cas system in plants, the method comprising the steps of:

(a) contacting a plant tissue with one or more cells of a first bacterial strain comprising a first nucleic acid construct, wherein the first nucleic acid construct comprises an RNA polymerase II promoter that is expressible in a plant cell operably linked to nucleic acid sequence encoding a Cas protein, and wherein a cell of the first bacterial strain is capable of transferring a nucleic acid molecule to a plant cell;

(b) contacting the plant tissue with one or more cells of a second bacterial strain comprising a second nucleic acid construct, wherein the second nucleic acid construct comprises an RNA polymerase III promoter that is expressible in a plant cell operably linked to nucleic acid sequence encoding an engineered, single guide R A (sgRNA) that is capable of recognizing a target nucleic acid sequence in the genome of plant tissue, and wherein a cell of the second bacterial strain is capable of transferring a nucleic acid molecule to a plant cell; and

(c) monitoring the plant tissue for a modification of the genome within the target site.

100. The method of embodiment 99, wherein the Cas protein is Cas9 or a mutant or derivative of Cas9.

101. The method of embodiment 100, wherein the mutant or derivative is naturally ocurring or engineered.

102. The method of any one of embodiments 99- 101, wherein the Cas protein is capable of causing a double-strand break in DNA.

103. The method of any one of embodiments 99- 101, wherein the Cas protein is capable of causing a single-strand break in DNA.

104. The method of any one of embodiments 100, 101, and 103, wherein the mutant or derivative comprises the D10A amino acid substitution or the H841A amino acid substitution, and wherein the mutant or derivative is capable of causing a single-strand break in DNA.

105. The method of any one of embodiments 99-104, wherein at least one of the first and the second bacterial strains is a strain from a bacterial species that is selected from the group consisting of Agrobacterium tumefaciens, Agrobacterium rhizogenes, Ensifer adhaerens and other Ensifer spp., Sinorhizobium meliloti, and Mesorhizobium loti.

106. The method of any one of embodiments 99-105, wherein at least one of the first and the second bacterial strains is selected from the group consisting of Agrobacterium tumefaciens strain AGL1 and Ensifer adhaerens strain OV14.

107. The method of any one of embodiments 99-106, wherein at least one of the first and the second nucleic acid constructs is contained in a Ti plasmid or an Agrobacterium T-DNA border-containing broad host range binary vector..

108. The method of any one of embodiments 99-107, wherein the contacting of step (a) comprises infiltration of the plant tissue with one or more cells of the first bacterial strain and/or the contacting of step (b) comprises infiltration of the plant tissue with one or more cells of the second bacterial strain. 109. The method of embodiment 108, wherein infiltration comprises use of a syringe to deliver the cells to the plant tissue.

110. The method of any one of embodiments 99-109, wherein steps (a) and (b) are performed at the same time.

111. The method of embodiment 110, wherein the plant tissue is infiltrated with a mixture comprising cells of the first bacterial strain and the second bacterial strain.

112. The method of any one of embodiments 99-111, wherein the plant tissue is from a plant selected from the group consisting of Nicotiana benthamiana, Nicotiana tabacum, Lactuca sativa, Brassica rapa, and any other plant species in which transient expression of genes after delivery of DNA either from bacteria or by other means can be achieved.

113. The method of any one of embodiments 99-112, wherein the plant tissue is leaf tissue.

114. The method of embodiment 112, wherein the leaf tissue is, or is part of, a whole leaf.

115. The method of any one of embodiments 99-114, wherein the plant tissue is attached to a plant or is detached from a plant.

116. The method of any one of embodiments 99-115, wherein the target nucleic acid sequence comprises or consists of 19 bases.

117. The method of any one of embodiments 99-116, wherein immediately following the 3' end of the target nucleic acid sequence in the genome of the plant, plant part, or plant cells is the protospacer-adjacent motif (PAM).

118. The method of embodiment 117, wherein the PAM is NGG, wherein N can be A, C, G, or T.

119. The method of embodiment 117, wherein the Cas protein recognizes a PAM that is not NGG, wherein N can be A, C, G, or T.

120. The method of any one of embodiments 99-119, wherein the sgRNA comprises a guide nucleic acid sequence that is capable of binding to or recognizing the target nucleic acid or its complement.

121. The method of embodiment 120, wherein the guide nucleic acid sequence is identical to the target nucleic acid sequence or its complement, when a U in the guide nucleic acid sequence is equivalent to a T in the target nucleic acid sequence or its complement. 122. The method of embodiment 120, wherein the guide nucleic acid sequence is not identical to the target nucleic acid sequence or its complement, when a U in the guide nucleic acid sequence is equivalent to a T in the target nucleic acid sequence or its complement.

123. The method of embodiment 120 or 123, wherein there are one or more mismatched bases between the guide nucleic acid sequence and the target nucleic acid sequence or its complement.

124. The method of embodiment 123, wherein the one or more mismatches occur at a position or positions in the target nucleic acid sequence that are located within the first 8, 9, 10, 11, or 12 bases beginning from the 5' end of the target nucleic acid sequence.

125. The method of any one of embodiments 99-124, wherein the target nucleic acid sequence is:

(i) native to the genome,

(ii) a foreign nucleic acid sequence that was introduced and stably incorporated into the genome of a plant from which the plant tissue was obtained,

(iii) a foreign nucleic acid sequence that was introduced and stably incorporated into the genome of a progenitor of the plant from which the plant tissue was obtained, or

(iv) co-delivered with the Cas gene and the sgR A.

126. The method of any one of embodiments 99-125, wherein monitoring the plant tissue for a modification of the genome within the target site comprises determining the nucleotide sequence of all or a part of the target nucleic acid sequence.

127. The method of any one of embodiments 99-126, wherein the target nucleic acid sequence comprises a restriction enzyme recognition sequence.

128. The method of embodiment 127, wherein step (c) comprises:

extracting genomic DNA from the plant tissue contacted by the first and second Agrobacterium strains:

digesting the genomic DNA with a restriction enzyme that recognizes the restriction enzyme recognition sequence; amplifying the digested DNA by polymerase chain reaction using primers designed to amplify across the target nucleic acid sequence so as to produce PCR products comprising one or more modifications within target nucleic sequence;

cloning the PCR products; and

determining the nucleic acid sequence of the cloned PCR products.

129. The method of embodiment 127 or 128, wherein the restriction enzyme recognition sequence is a Mlyl restriction enzyme recognition sequence.

130. The method of embodiment 128 or 129, wherein the DNA is extracted from plant tissue harvested at about 1-48 hours after the later of the contacting steps.

131. The method of embodiment 128 or 129, wherein the DNA is extracted from leaves harvested at about 6, 12, 18, 24, 30, 36, 42, or 48 hours after the later of the contacting steps.

132. The method of any one of embodiments 1-55, wherein the method comprises producing at least one plant cell comprising a Cas protein and at least two engineered, sgRNAs that are each capable of recognizing a different target nucleic acid sequence.

133. A method for modifying the genome of a plant, plant part, or plant cell, the method comprising producing at least one plant cell comprising a Cas protein and at least two engineered, sgRNAs that are each capable of recognizing a different target nucleic acid sequence, wherein the plant, plant part, plant cell, or descent thereof, comprises in its genome the target nucleic acid sequences.

134. The method of embodiment 133, wherein a first sgRNA is capable of recognizing a first target sequence and a second sgRNA is capable of recognizing a second target sequence.

135. The method of embodiment 134, wherein the first target sequence and the second target sequence are on the same chromosome.

136. The method of embodiment 134 or 135, wherein the first target sequence and the second target sequence are within the same gene.

137. The method of any one of embodiments 134-136, wherein the first target sequence and the second target sequence are separated by at least 10, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500, 1000, 1500, 2000, 3000 or more nucleotides. 138. The method of any one of embodiments 134-137, wherein the modification comprises the deletion of at least one base pair in the genome of the plant, plant part, plant cell, or descendant thereof.

139. The method embodiment 138, wherein the deletion occurs between the first target sequence and the second target sequence.

140. The method of any one of embodiments 134-139, wherein at least one of the Cas protein, the first sgR A, and the second sgR A are transiently expressed in the plant cell.

It is an object of the present invention to provide new methods for targeted mutagenesis and genome engineering that are less complicated and costly than existing methods such as, for example, those methods involving zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs).

The present invention provides new methods for targeted mutagenesis and genome engineering that do not depend on the use of a ZFNs or a TALENs but instead involve the use of a Cas protein and engineered, single guide RNA (sgRNA) that specifies a targeted nucleic acid sequence . In the examples below, the Cas protein, Cas9 is used for purposes of illustration. The present invention is not limited to the use of Cas9. Mutants and derivatives of Cas9 as well as other Cas proteins can be used in the methods disclosed herein. Preferably, such other Cas proteins are able to recognize a target nucleic acid sequence when in a plant cell in the presence of an sgRNA that is engineered for recognition of the target sequence. In some embodiments of the invention, the Cas proteins will additional comprise a double-strand endonuclease activity or a single strand endonuclease activity (i.e., a nickase activity). In other embodiments, the Cas protein will not comprise a double-strand endonuclease activity and/or single strand endonuclease activity. In yet other embodiments, a Cas protein domain will be operably linked with at least one additional protein domain to form a fusion protein. Such a fusion protein a can comprise a double-strand endonuclease activity or a single strand endonuclease activity or no endonuclease at all but instead can comprise an additional activity that is due to the additional domain.

Fragments and variants of the disclosed nucleic acid molecules and proteins encoded thereby are also encompassed by the present invention. Such fragments and variants of the disclosed nucleic acid molecules and proteins include, for example, nucleic acid molecules encoding mutants and dervivaties of the Cas9 protein and the proteins encoded thereby. The nucleic acid and amino acid sequences of Cas9 are disclosed elsewhere herein or otherwise known in the art. By "fragment" is intended a portion of the nucleic acid molecule or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of a nucleic acid molecule comprising coding sequences may encode protein fragments that retain biological activity of the native protein and hence DNA recognition or binding activity to a target DNA sequence as herein described. Alternatively, fragments of a nucleic acid molecule that are useful as hybridization probes generally do not encode proteins that retain biological activity or do not retain promoter activity. Thus, fragments of a nucleic acid molecule may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length nucleic acid molecule of the invention.

"Variants" is intended to mean substantially similar sequences. For nucleic acid molecules, a variant comprises a nucleic acid molecule having deletions (i.e., truncations) at the 5' and/or 3' end; deletion and/or addition of one or more nucleotides at one or more internal sites in the native polynucleotide; and/or substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a "native" nucleic acid molecule or polypeptide comprises a naturally occurring nucleiotide sequence or amino acid sequence, respectively. For nucleic acid molecules, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the polypeptides of the invention. Variant nucleic acid molecules also include synthetically derived nucleic acid molucules, such as those generated, for example, by using site-directed mutagenesis but which still encode a protein of the invention. Generally, variants of a particular nucleic acid molecule of the invention will have at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%), 96%o, 97%), 98%>, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters as described elsewhere herein.

Variants of a particular nucleic acid molecule of the invention (i.e., the reference DNA sequence) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant nucleic acid molecule and the polypeptide encoded by the reference nucleic acid molecule. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. Where any given pair of nucleic acid molecule of the invention is evaluated by comparison of the percent sequence identity shared by the two polypeptides that they encode, the percent sequence identity between the two encoded polypeptides is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

"Variant" protein is intended to mean a protein derived from the native protein by deletion (so-called truncation) of one or more amino acids at the N-terminal and/or C- terminal end of the native protein; deletion and/or addition of one or more amino acids at one or more internal sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein as described herein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a protein of the invention will have at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs and parameters described elsewhere herein. A biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

The proteins or polypeptides of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants and fragments of the proteins can be prepared by mutations in the DNA.

Techniques to specifically modify DNA sequences in order to obtain a specified codon for a specific amino acid are well known in the art. Methods for mutagenesis and polynucleotide alterations are also well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in

Enzymol. 154:367-382; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be optimal .

The deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays as described elsewhere herein or known in the art.

Variant nucleic acid molecule and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91 : 10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391 :288-291; and U.S. Patent Nos. 5,605,793 and

5,837,458.

In a PCR approaches, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding nucleic acid molecules from cDNA or genomic DNA extracted from any organism of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (1989)

Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York). See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers, and the like.

It is recognized that the nucleic acid molecules and proteins of the invention encompass polynucleotide molecules and proteins comprising a nucleotide or an amino acid sequence that is sufficiently identical to the nucleotide sequences or to the amino acid sequence disclosed herein. The term "sufficiently identical" is used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of identical or equivalent (e.g., with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain and/or common functional activity. For example, amino acid or nucleotide sequences that contain a common structural domain having at least about 70% identity, preferably 75% identity, more preferably 85%, 90%, 95%, 96%, 97%, 98% or 99% identity are defined herein as sufficiently identical.

To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical positions/total number of positions (e.g., overlapping positions) x 100). In one embodiment, the two sequences are the same length. The percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.

The determination of percent identity between two sequences can be

accomplished using a mathematical algorithm. A preferred, nonlimiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the polynucleotide molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and

NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another preferred, non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Alignment may also be performed manually by inspection. Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using the full-length sequences of the invention and using multiple alignment by mean of the algorithm Clustal W (Nucleic Acid Research, 22(22):4673- 4680, 1994) using the program AlignX included in the software package Vector NTI Suite Version 7 (InforMax, Inc., Bethesda, MD, USA) using the default parameters; or any equivalent program thereof. By "equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by

CLUSTALW (Version 1.83) using default parameters (available at the European

Bioinformatics Institute website: http://www.ebi . ac .uk/Too 1 s/c lustalw/inde . html

The methods of the invention involve introducing a polypeptide or

polynucleotide into a plant. "Introducing" is intended to mean presenting to the plant the polynucleotide or polypeptide in such a manner that the sequence gains access to the interior of a cell of the plant. The methods of the invention do not depend on a particular method for introducing a sequence into a plant, only that the polynucleotide or polypeptides gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotide or polypeptides into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

"Stable transformation" is intended to mean that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof. "Transient transformation" is intended to mean that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant or a polypeptide is introduced into a plant.

Transformation protocols as well as protocols for introducing polypeptides or polynucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing polypeptides and polynucleotides into plant cells include microinjection (Crossway et al.

(1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad.

Sci. USA 83:5602-5606, Agrobacterium-mediatGd transformation (U.S. Patent No.

5,563,055 and U.S. Patent No. 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, U.S. Patent

Nos. 4,945,050; U.S. Patent No. 5,879,918; U.S. Patent No. 5,886,244; and, 5,932,782;

Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed.

Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology

6:923-926); and Lecl transformation (WO 00/28058). Also see Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology

5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro

Cell Dev. Biol. 27P: 175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-

324 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988) Biotechnology

6:559-563 (maize); U.S. Patent Nos. 5,240,855; 5,322,783; and, 5,324,646; Klein et al.

(1988) Plant Physiol. 91 :440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839

(maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311 :763-764; U.S.

Patent No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA

84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of

Ovule Tissues, ed. Chapman et al. (Longman, New York), pp. 197-209 (pollen);

Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor.

Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992)

Plant Cell 4: 1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996)

Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.

In specific embodiments, the nucleic acid molecules and constructs of the invention can be provided to a plant using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, the introduction of a Cas protein and/or one or more sgRNAs directly into the plant or the introduction into the plant of a Cas protein transcript and/or one or more transcripts encoding sgRNAs.

Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway et al. (1986) Mol Gen. Genet. 202: 179-185; Nomura et al. (1986) Plant Sci. 44:53-58; Hepler et al. (1994) Proc. Natl. Acad. Sci. 91 : 2176-2180 and Hush et al. (1994) The Journal of Cell Science 707:775-784, all of which are herein incorporated by reference. Alternatively, the polynucleotide can be transiently transformed into the plant using techniques known in the art. Such techniques include viral vector system and the precipitation of the polynucleotide in a manner that precludes subsequent release of the DNA. Thus, the transcription from the particle-bound DNA can occur, but the frequency with which its released to become integrated into the genome is greatly reduced. Such methods include the use particles coated with polyethylimine (PEI; Sigma #P3143).

In other embodiments, the polynucleotide of the invention may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a nucleotide construct of the invention within a viral DNA or RNA molecule. It is recognized that the an nucleic acid molecule of the invention may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired recombinant protein. Further, it is recognized that promoters of the invention also encompass promoters utilized for transcription by viral RNA polymerases. Methods for introducing polynucleotides into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, for example, U.S. Patent Nos. 5,889,191,

5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta et al. (1996) Molecular

Biotechnology 5:209-221; herein incorporated by reference.

Methods are known in the art for the targeted insertion of a polynucleotide at a specific location in the plant genome. In one embodiment, the insertion of the polynucleotide at a desired genomic location is achieved using a site-specific

recombination system. See, for example, W099/25821, W099/25854, WO99/25840, W099/25855, and W099/25853, all of which are herein incorporated by reference. Briefly, the polynucleotide of the invention can be contained in transfer cassette flanked by two non-recombinogenic recombination sites. The transfer cassette is introduced into a plant having stably incorporated into its genome a target site which is flanked by two non-recombinogenic recombination sites that correspond to the sites of the transfer cassette. An appropriate recombinase is provided and the transfer cassette is integrated at the target site. The polynucleotide of interest is thereby integrated at a specific chromosomal position in the plant genome.

The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting progeny having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the present invention provides transformed seed (also referred to as "transgenic seed") having a polynucleotide of the invention, for example, an expression cassette of the invention, stably incorporated into their genome.

As used herein, the term plant also includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like. Grain is intended to mean the mature seed produced by

commercial growers for purposes other than growing or reproducing the species.

Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced

polynucleotides.

The present invention may be used for any plant species of interest, including, but not limited to, monocots and dicots. Examples of plant species of interest include, but are not limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B.juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolid), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C sativus), cantaloupe (C. cantalupensis), and musk melon (C melo). Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum.

Conifers that may be employed in practicing the present invention include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).

A "subject plant or plant cell" is one in which genetic alteration, such as transformation, has been effected as to a gene of interest, or is a plant or plant cell which is descended from a plant or cell so altered and which comprises the alteration. A "control" or "control plant" or "control plant cell" provides a reference point for measuring changes in phenotype of the subject plant or plant cell.

A control plant or plant cell may comprise, for example: (a) a wild-type plant or cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the subject plant or cell; (b) a plant or plant cell of the same genotype as the starting material but which has been transformed with a null construct (i.e. with a construct which has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) a plant or plant cell which is a non-transformed segregant among progeny of a subject plant or plant cell; (d) a plant or plant cell genetically identical to the subject plant or plant cell but which is not exposed to conditions or stimuli that would induce expression of the gene of interest; or (e) the subject plant or plant cell itself, under conditions in which the gene of interest is not expressed.

The following examples are offered by way of illustration and not by way of limitation.

EXAMPLE 1

Targeted Mutagenesis of Nicotiana benthamiana To test the potential of the Cas9 system to induce gene knock-outs (i.e., loss-of- function mutations) in plants, we took advantage of Agrobacterium turne/adens-mediated transient expression assays (agroinfiltration) to co-express a Cas9 variant with a eukaryotic nuclear localization signal (nls) and an sgRNA in the model plant Nicotiana benthamiana⁴. First, we constructed a GFP-tagged version of Cas9 using the clone described in Mali et al.⁵. We expressed GFP-Cas9 in N. benthamiana leaf tissue using standard agroinfiltration protocols⁶ and observed a clear nuclear localization (FIG. 3) consistent with the nuclear localization previously observed in human cells⁷. We then generated an sgRNA with the guide sequence matching a 19 bp region within the phytoene desaturase (PDS) gene in the Nicotiana benthamiana (FIG. la). The sgRNA was placed under an Arabidopsis U6 promoter (FIG. 4). Both GFP-Cas9 and sgRNA were co-expressed in N. benthamiana leaf tissue using A. tumefaciens as a vector. The tissue was harvested 2 days later and DNA was extracted. In order to easily detect sgRNA-guided, Cas9-induced mutations at the PDS locus, we used the restriction enzyme site loss method . As the target sequence within the PDS gene overlaps with an Mlyl restriction site, we digested the genomic DNA with Mlyl and then performed a polymerase chain reaction (PCR) with primers flanking the target site. By doing so we greatly reduced unaltered wild type DNA in the sample and enriched for DNA molecules carrying mutations that remove the Mlyl site. As illustrated in FIG. lb, the presence of both Cas9 and the sgRNA resulted in increased levels of the PCR product (lane 1) compared to negative control treatments (lanes 2-3). Non-digested N benthamiana genomic DNA was used as a positive control (lane 4). The assay was robust and reproducible because we detected Mlyl-resistant amplicons in three additional independent experiments using different plants (FIG. 5 and FIG. 6). The PCR products from FIG. lb lanes 1 and 4 were cloned into a high copy vector and individual clones sequenced. Sequence analysis of 20 clones derived from the PCR product in lane 1 revealed the presence of indels in 17 of them. The indels can be grouped into nine different types ranging from 1-9 bp deletions to 1 bp insertions (FIG. lc and FIG. 7). All recovered indels abolish the Mlyl restriction site within the target region. Sequences of the 8 clones derived from the control PCR product shown in lane 4 were all wild type.

To estimate the efficiency of targeted mutagenesis, we amplified non-digested genomic DNA from Cas9/sgRNA expressing N. benthamiana leaves and negative controls, and subsequently digested the amplicons with Mlyl and subjected them to gel electrophoresis. We then measured the intensity of the uncut band relative to the intensity of all detectable bands in a gel lane as described in Qi et al. (FIG. 2 and FIG. 8). We estimated the mutation rate to be in the range of 1.8 to 2.4% (2.1% average) based on four independent experiments.

Given that the target sequence is 19 bp, the Cas9/sgRNA system may not be as specific as TALEN-induced mutagenesis. We examined 18 off-target sites in the N. benthamiana genome that have 14 to 16 out of 19 bp identity to the targeted PDS sequence and overlap a Mlyl restriction site. None of 18 amplicons showed unambiguous evidence of Cas9/sgRNA dependent Mlyl restriction site loss as observed with the PDS target sequence (Table 1 and FIG. 9). We therefore conclude that any off-target nuclease activity is much lower than activity against the specified target.

These data clearly indicate that Cas9 and an engineered sgRNA can direct DNA breaks at defined chromosomal locations in plants. The rapid and robust transient assay we developed will enable plant-specific optimization of the Cas9 system. Relative to other methods of plant genome engineering, the Cas9 system has the potential to simplify the process of plant genome engineering and editing since only a short fragment in the sgRNA needs to be designed to target a new locus. This creates a valuable new tool for plant biologists and breeders, and hastens the prospects of achieving routine targeted genome engineering for basic and applied science. Materials and Methods

Construction of Cas9 and sgRNA expressing plasmids

Cas9 was PCR-amplified with primers Cas9GWF and Cas9STR using the clone described in Mali et al.⁵ as a template. The resulting PCR product was cloned into the pENTR/D-TOPO vector (Life Technologies). The entry clone was subsequently recombined into the GW-compatible destination vector pK7WGF2⁹ using the LR clonase (Life Technologies) to produce the 35Sp::GFPCas9 construct.

The Arabidopsis U6 promoter used in this study is the consensus sequence of the 3 U6 promoter variants present in the Arabidopsis genome¹⁰. The AtU6p clone was synthesised with Genescript (FIG. 4). The promoter was PCR-amplified with primers U6p_GGAG_f and U6p_CAAT_r and cloned into a level 0 vector¹¹ via the Bbsl cut- ligation using the Golden Gate (GG) cloning method. The sgRNA was PCR-amplified using primers PDS gRNAl Bsalf and gRNA AGCG Bsalr (PDS locus) or primers gRNA T l GFP Bsalf and gRNA AGCG Bsalr (GFP) using the plasmid

gRNA GFP Tl described in Mali et al.¹ as a template. The resulting PCR product was cloned into the pICH86966 vector (kindly provided by S. Marillonnet, based on the modular cloning system described in Weber et al.ⁿ under the AtU6p via the Bsal cut- ligation using the GG method. Both Cas9 and gRNA GFP Tl plasmids⁵ were obtained from Addgene.

Transient gene expression in N. benthamiana and Cas9 intracellular localisation

Transient expression was performed using the AGL1 strain of Agrobacterium

12

tumefaciens as described in Bos et al. . GFP-Cas9 was localised in the leaf tissue of N. benthamiana 2 days post infiltration using the Leica SP5 confocal microscope in accordance with manufacturer's instructions.

Detection of Cas9-induced indels in plant genomic DNA

GFP-Cas9 and sgRNA carrying the guide sequence matching a site within the PDS gene of N. benthamiana were transiently co-expressed in the N. benthamiana leaf tissue as follows. An Agrobacterium strain carrying a nucleic acid construct of the expression of GFP-Cas9 in plant cells and an Agrobacterium strain carrying a nucleic acid construct of the expression of the sgRNA in plant cells were separately inoculated into L medium containing respective antibiotics for plasmid selection and grown overnight at 28°C. The next day, each of the resulting cultures was centrifuged at 3500 rpm for 10 min, and the resulting pellets of Agrobacteria were separately resuspended in 10 mM MgCl₂. The resuspended Agrobacteria were diluted with the 10 mM MgCl₂ to OD600=0.5 and then the resuspended Agrobacteria carrying the GFP-Cas9 construct were mixed with the resuspended Agrobacteria carrying the sgR A construct were mixed at a ratio 1 : 1 (v/v). Leaves of 4-week-old N. benthamiana plants were infiltrated with pre-mixed Agrobacteria, and after 48 hours, the leaf tissue was harvested.

The tissue was harvested at 2 days post infiltration and the genomic DNA extracted using the DNeasy Plant Mini kit (Qiagen). 100 ng of the genomic DNA was then digested overnight with Mlyl restriction enzyme to reduce greatly the background of the non-modified (wild-type) DNA. Upon digesting the reaction mix was desalted using Sepharose CL-6B (Sigma) and then added as a template into a PCR reaction performed with primers PDS MlylF and PDS MlylR with flank the sgRNA target nucleic acid sequence and Phusion DNA polymerase (New England Biolabs). The resulting PCR band was cloned a high copy vector (pCRTM-Blunt II-TOPO vector, Life Technologies, Inc.). Plasmids from individual Escherichia coli colonies were sequenced using standard primers Ml 3 forward and Ml 3 reverse. Amplicons with mutated Mlyl sites were recovered only when the Cas9 and sgRNA constructs were co-expressed (FIG. 1 and FIG. 5) but weak bands can be occasionally observed in the negative controls probably due to incomplete digestion. To ensure that the amplicons indeed contain DNA fragments resistant to Mlyl digestion, we performed a partial digestion of the genomic DNA with Mlyl followed by a second Mlyl digestion of the amplicons as shown in FIG. 6.

Measurements of the mutation rate

In order to estimate the rate of Cas9/sgRNA-induced mutations in the PDS locus we used the method described by Qi et al. . The PCR product amplified on the non- digested genomic DNA as a template was digested with Mlyl and run on a gel. The intensity of non-saturated bands was quantified using the ImageJ software

(h tt ://rsb . info .nih. gov/ij) . The mutation rate was calculated by dividing the intensity of the uncut band by the total intensity of all bands in a given lane. Analysis of potential off-targets

In order to select potential off-targets, we searched the N. benthamiana genome database using the NCBI BLASTN online tool against the 19 bp sgRNA target site within the PDS gene (CCGTTAATTTGAGAGTCCA). We then picked 44 potential off- targets with varying numbers of bases (14-16) matching the 19 bp PDS target sequence. None of the matching N. benthamiana sequences had more than 16 out of 19 bp matches to the PDS target sequence. All of the off-targets selected had the Mlyl site present in them. Primers were designed to amplify the potential off-target loci across the off-target site. Out of the 44 loci amplified, 18 gave a PCR band of expected size without nonspecific bands. We then proceeded further with these 18 loci (Table 1). First, we tested if we could amplify the loci using Mlyl-digested genomic DNA extracted from the leaf tissue expressing Cas9+sgRNA(PDS) or Cas9 alone. For 12 out of 18 loci it was possible to amplify a PCR product using the Mlyl-digested genomic DNA and for the 1 out of 12 loci a weak band was observed only in the case of the Cas9+sgRNA(PDS) sample indicating potential off-target activity (FIG. 9c). Nevertheless, in all 18 cases the PCR products could be digested by Mlyl (FIG. 9a-c), while the PCR product from the positive control (PDS) showed clear resistance to Mlyl digestion (FIG. 9d).

Table 1. Summary of the off-target analyses. The Mlyl restriction site is shown in blue.

Table 2. Primers used in the Examples.

Table 2. (continued).

Table 2. (continued).

EXAMPLE 2

Targeted, Large Chromosomal Deletions in Nicotiana benthamiana and Tomato

Targeted, large chromosomal deletions were generated by targeting two adjacent sequences within the PDS locus of Nicotiana benthamiana using transient expression. To make the large deletions in PDS locus, two sgRNAs were transiently expressed resulted in chromosomal deletion events. The sgRNAs were designed for target sequences on each side of the region in the PDS locus that was intended to be deleted. A schematic representation of the PDS locus and targets is shown in FIG. 10A. FIG. 10B shows the detection of deletion mutations using the AFLP analysis. The deletions obtained are shown in FIG. IOC. The nucleic acid sequence of the sgRNA constructs are shown in FIG. 12.

Targeted, large chromosomal deletions were generated by targeting two adjacent sequences within the Mlol gene of tomato using transient expression. To make the large deletions in Mlol gene, sgRNAs were transiently expressed resulted in chromosomal deletion events. The sgRNAs were designed for target sequences on each side of the regions in the Mlol gene that was intended to be deleted as shown (FIG. 11 A). FIG. 1 IB shows the detection of deletion mutations by the AFLP analysis and FIG. 1 1C shows the results from the genotyping of TO transgenic tomato plants. The plants were genotyped for the presence of CRISPR/Cas-induced deletions.

The results demonstrate applicability of the CRISPR/Cas system for purposes of creating large chromosomal deletions in plants. Creating a large deletion by targeting with 2 sgRNAs provides a more convenient way to knock-out a gene as such deletion event can be easily detected by the AFLP (PCR band shift) assay. Constructs can be quickly tested using the transient expression systems in crops amenable to transient expression (e.g. tomato) before proceeding with generation of stable transgenic lines. In addition to generating single gene-knockouts, ability to create large chromosomal deletions provides an opportunity for deleting whole gene clusters as well as deleting certain promoter elements or protein domains.

Materials and Methods

Plasmid construction

In the case of the lane 2 (Figure IB), pK7WGF2::Cas9 and

pICH86966::AtU6p::sgRNAl_ D5^* [1] were co-expressed with

pICH86966::AtU6p::sgRNA2_ NbPDS in N. benthamiana using the standard agroinfiltration protocol. In the case of the lane 4 (Figure IB), Cas9 and both sgRNAl_A/¾ ZXS' and sgRNA2_A/¾ ZXS' were expressed in N. benthamiana from the single construct.

In order to use the human codon optimised Cas9⁵ in the Golden Gate cloning system¹¹, all Bsal and Bbsl sites had to be removed from its sequence, while preserving the amino acid composition of the protein, in a process called "domestication. The "domesticated" Cas9 was cloned into a level 1 vector pICH47742ⁿ.

N. benthamiana PDS locus

NbPDS sgRNAs 1 and 2:

pICH47732 : : AtU6p : :sgKNA2_NbPDS, pICH47742 : :2x35 S- 5'UTR::Cas9::NOST, pICH47751 ::AtU6p::sgR Al_M> D5 level 1 constructs plus the pELE-3 linker [2] were cut-ligated into the pAGM4723 level 2 vector. Tomato SlMlol locus

SlMlol sgR As 1 and 3:

pICH47732::NOSp::NPTII::OCST, pICH47742::2x35S-5'UTR::Cas9::NOST, pICH47751 ::AtU6p::sgRNAl_S/ oi, pICH47761 ::AtU6p::sgRNA3_S/ oi level 1 constructs plus the pELE-4 linker [2] were cut-ligated into the pAGM4723 level 2 vector.

SlMlol sgRNAs 2 and 3:

pICH47732::NOSp::NPTII::OCST, pICH47742::2x35S-5'UTR::Cas9::NOST, pICH47751 ::AtU6p::sgRNA2_S/ oi, pICH47761 ::AtU6p::sgRNA3_S/ oi level 1 constructs plus the pELE-4 linker were cut-ligated into the pAGM4723 level 2 vector.

SlMlol sgRNAs 3 and 4:

pICH47732::NOSp::NPTII::OCST, pICH47742::2x35S-5'UTR::Cas9::NOST, pICH47751 ::AtU6p::sgRNA3_S/ oi, pICH47761 ::AtU6p::sgRNA4_S/ oi level 1 constructs plus the pELE-4 linker were cut-ligated into the pAGM4723 level 2 vector.

The resulting level 2 constructs were transformed into the AGL1 strain of A. tumifaciens.

Transient gene expression in N. benthamiana and tomato

Transient expression in N. benthamiana and tomato was performed as the AGL1

12

strain of A. tumifaciens as described in Bos et al. .

Generation of stable transgenic tomato lines

Tomato transgenic lines were generated with same constructs as the ones used for the transient assay by agrotransformation of the tomato Moneymaker variety using the AGL1 A. tumifaciens strain. Transformants were selected on kanamycin.

Detection of Cas9-induced deletions in plant genomic DNA

In the case of the transient expression, Cas9 and sgRNAs were expressed in the N. benthamiana or tomato Moneymaker leaf tissue. The tissue was harvested at 2 days post infiltration and the genomic DNA extracted using the DNeasy Plant Mini kit (Qiagen). The genomic DNA extracted from the transiently expressing or stable transgenic tissue was digested with Sacl (New England Biolabs) in the case of N.

benthamiana PDS targets 1 and 2; with Bsrl (New England Biolabs) in the case of tomato Mlol targets 1 and 3, and 2 and 3; with Ncol (New England Biolabs) in the case of tomato Mlol targets 3 and 4.

50 ng of pre-digested DNA was added in a PCR reaction and amplified with respective primers using Phusion DNA polymerase (New England Biolabs).

PCR products were run on an agarose gel. The DNA from bottom bands in lanes

2 and 4 (Figure 10B) was extracted and cloned into pCR-Blunt II-TOPO vector (Life Technologies). 15 individual clones were sequenced using standard Ml 3 forward and Ml 3 reverse primers. REFERENCES

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The article "a" and "an" are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one or more element. As used herein, the word "comprising," or variations such as "comprises" or "comprising," will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

THAT WHICH IS CLAIMED:

1. A method for modifying the genome of a plant, plant part, or plant cell, the method comprising producing at least one plant cell comprising a Cas protein and at least one engineered, single guide RNA (sgRNA) that is capable of recognizing a target nucleic acid sequence, wherein the plant, plant part, plant cell, or descent thereof, comprises in its genome the target nucleic acid sequence.

2. The method of claim 1 , wherein the Cas protein is Cas9 or a mutant or derivative of Cas9.

3. The method of claim 2, wherein the mutant or derivative is naturally occurring or engineered.

4. The method of any one of claims 1-3, wherein the Cas protein is capable of causing a double-strand break in DNA.

5. The method of any one of claims 1-3, wherein the Cas protein is capable of causing a single-strand break in DNA.

6. The method of claim 2 or 3, wherein the mutant or derivative comprises the DIOA amino acid substitution or the H841A amino acid substitution, and wherein the mutant or derivative is capable of causing a single-strand break in DNA.

7. The method of any one of claims 1-6, wherein producing at least one plant cell comprising a Cas protein and an engineered sgRNA, comprises:

(b) introducing into the at least one a plant cell or a progenitor cell thereof a nucleic acid construct for expression of the Cas protein in the plant cell, whereby the Cas protein is expressed in the at least one plant cell; or

(c) both (a) and (b).

8. The method of any one of claims 1-7, wherein the sgRNA, the Cas protein, or both are transiently expressed in the plant cell.

9. The method of claim 7 or 8, wherein the nucleic acid construct for expression of the sgRNA, the nucleic acid construct for expression of the Cas protein, or both are stably incorporated in the genome of the plant cell.

10. The method of any one of claims 1-9, wherein producing at least one plant cell comprising a Cas protein and an engineered sgRNA, comprises:

(ii) directly introducing the Cas protein into the plant cell; or

(iii) both (i) and (ii).

11. The method of any one of claims 1-10, wherein the Cas protein or the nucleic acid construct for expression of the Cas protein is introduced into the plant cell or the progenitor, before, concurrently with, or after introducing the sgRNA or the nucleic acid construct for expression of the sgRNA.

12. The method of any one of claims 1-11, wherein at least one modification is introduced into the genome of the plant, plant part, or plant cell, or descendant thereof.

13. The method of claim 12, wherein the modification occurs within the target nucleic acid sequence.

14. The method of claim 12 or 13, wherein the modification comprises the insertion of at least one base pair in the genome of the plant, plant part, plant cell, or descendant thereof.

15. The method of claim 12 or 13, wherein the modification comprises the deletion of at least one base pair in the genome of the plant, plant part, plant cell, or descendant thereof.

16. The method of claim 12 or 13, wherein the modification comprises the insertion of at least one base pair in the genome of the plant, plant part, plant cell, or descendant thereof and the deletion of at least one base pair in the genome of the plant, plant part, plant cell, or descendant thereof.

17. The method of any one of claims 1-16, wherein the target nucleic acid sequence is within a gene or wherein the target nucleic acid sequence affects the level of expression of a gene.

18. The method of claim 17, wherein the gene is selected from the group consisting of a disease resistance gene, a disease susceptibility gene, a gene encoding an enzyme, a gene encoding a structural protein, a gene encoding a receptor, a gene encoding a membrane channel protein, a gene encoding a transcription factor, a gene that is associated with herbicide tolerance, a gene that affects development of male or female reproductive structures, and a gene that affects self-incompatibility.

19. The method of claim 17 or 18, wherein modification is a mutation in the gene that results in an alteration or loss of the wild-type function of the gene and/or the protein encoded thereby.

20. The method of claim 19, wherein the mutation is a loss-of- function mutation.

21. The method of any one of claims 1-20, further comprising introducing into the plant, plant part, plant cell, or descendant thereof a donor nucleic acid .

22. The method of claim 21 , wherein the donor nucleic acid molecule or part thereof is incorporated into the genome of the plant, plant part, or plant cell or descendant thereof.

23. The method of claim 21 or 22, wherein the donor nucleic acid molecule or part thereof is incorporated into the genome of the plant, plant part, plant cell, or descendant thereof within the target nucleic acid sequence.

24. The method of any one of claims 21-23, wherein the donor nucleic acid molecule comprises a nucleic acid sequence which comprises in a 5' to 3' order: a first homologous region, a donor region, and a second homologous region.

25. The method of claim 25, wherein the first homologous region comprises nucleic acid sequence homology to a first genomic region and the second homologous region comprises nucleic acid sequence homology to a second genomic region.

26. The method of claim 26, wherein the first genomic region and the second genomic region are on opposite sides of the site of incorporation of the donor nucleic acid molecule or part thereof into the genome of the plant, plant part, plant cell, or descendant thereof.

27. The method of any one of claims 21-26, wherein the donor region comprises a coding and/or a non-coding nucleic acid sequence.

28. The method of any one of claims 21-27, wherein the donor region comprises at least one transgene.

29. The method of claim 28, wherein the transgene comprises a promoter operably linked to a nucleic acid sequence, and wherein the promoter is capable of driving expression of the operably linked nucleic acid in a plant cell.

30. The method of claim 28 or 29, where the transgene encodes a protein.

31. The method of claim 30, wherein the protein is native to the plant, plant part, plant cell.

32. The method of claim 30, wherein the protein is a foreign protein that is not native the plant, plant part, or plant cell.

33. The method of any one of claims 21-32, wherein the donor nucleic acid comprises or further comprises at least one selectable marker gene.

34. The method of any one of claims 22-33, wherein incorporation of the donor nucleic acid into the genome of the plant, plant part, or plant cell or descendant thereof produces a selectable marker gene in the genome of the plant, plant part, or plant cell or descendant thereof.

35. The method of claim 34, wherein the donor nucleic acid comprises the entire, at least a portion of, or none of, the coding region of a protein encoded by the selectable marker gene.

36. The method of any one of claims 21-35, wherein the donor nucleic acid molecule is single stranded or double stranded.

37. The method of any one of claims 21-36, wherein the donor nucleic acid molecule is a linear nucleic acid molecular or a circular nucleic acid molecule.

38. The method of any one of claims 7-37, wherein the nucleic acid construct for expression of the sgR A comprises a promoter operably linked to a nucleic acid sequence encoding the sgRNA and wherein the promoter is capable of driving expression of a nucleic acid sequence in a plant cell.

39. The method of claim 38, wherein the promoter is an RNA polymerase III promoter.

40. The method of claim 38 or 39, wherein the promoter is the Arabidopsis U6 promoter.

41. The method of any one of claims 38-40, wherein the promoter comprises the Arabidopsis U6 promoter-derived synthetic nucleic acid sequence set forth in FIG. 4B.

42. The method of any one of claims 7-41, wherein nucleic acid construct for expression of the sgR A comprises the nucleic acid sequence of the U6::sgRNA construct set forth in FIG. 4B.

43. The method of any one of claims 7-42, wherein the nucleic acid construct for expression of the Cas protein comprises a promoter operably linked to a nucleic acid sequence encoding the Cas protein and optionally an operably linked eukaryotic nuclear localization signal sequence, wherein the promoter is capable of driving expression of a nucleic acid sequence in a plant cell.

44. The method of claim 43, wherein the promoter is an RNA polymerase II promoter.

45. The method of claims 43 or 44, wherein the promoter is a constitutive promoter, a tissue-specific promoter or an inducible promoter.

46. The method of any one of claims 43-45, wherein the promoter is selected from the group consisting of the CaMV 35S promoter, a ubiquitin promoter, an estradiol- inducible promoter, a dexamethasone-inducible promoter, an alcohol-inducible promoter, a heat shock promoter, an inflorescence-specific promoter, the Arabidopsis Apetala2 promoter, and the Arabidopsis Agamous promoter.

47. The method of any one of claims 1-46, wherein the target nucleic acid sequence comprises or consists of 19 bases.

48. The method of any one of claims 1-47, wherein immediately following the 3' end of the target nucleic acid sequence in the genome of the plant, plant part, or plant cells is a protospacer-adjacent motif (PAM).

49. The method of claim 48, wherein the PAM is NGG, wherein N can be A, C, G, or T.

50. The method of claim 47, wherein the Cas protein recognizes a PAM that is not NGG, wherein N can be A, C, G, or T.

51. The method of any one of claims 1-50, wherein the sgRNA comprises a guide nucleic acid sequence that is capable of binding to or recognizing the target nucleic acid or its complement.

52. The method of claim 51 , wherein the guide nucleic acid sequence is identical to the target nucleic acid sequence or its complement, when a U in the guide nucleic acid sequence is equivalent to a T in the target nucleic acid sequence or its complement.

53. The method of claim 51 , wherein the guide nucleic acid sequence is not identical to the target nucleic acid sequence or its complement, when a U in the guide nucleic acid sequence is equivalent to a T in the target nucleic acid sequence or its complement.

54. The method of claim 51 or 53, wherein there are one or more mismatched bases between the guide nucleic acid sequence and the target nucleic acid sequence or its complement.

55. The method of claim 54, wherein the one or more mismatches occur at a position or positions in the target nucleic acid sequence that are located within the first 8, 9, 10, 11, or 12 bases beginning from the 5' end of the target nucleic acid sequence.

56. The method of any of claims 1-55, further comprising regenerating the plant cell or descendent thereof into a plant comprising at least one modification in its genome or in the genome of a progeny plant or descendent of the regenerated plant, wherein at least one modification in the genome of a progeny plant or descendent is due to the presence of the sgRNA and the Cas protein in the progeny plant or descendent.

57. A plant, plant part, seed, or plant cell produced by the method of any one of claims 1-56, wherein the genome of the plant, plant part, seed, or plant cell is modified when compared to the genome of a control plant cell, and wherein said control plant cell is plant cell before its genome has been modified by the method.

58. A plant, plant part, seed, or plant cell comprising a modification in its genome that was introduced into the genome according to the method of any one of claims 1-56.

59. Use of the plant, plant part, or seed of claim 57 or 58 in agriculture and/or in the production of a human and/or animal food product.

60. Use of the plant, plant part, or seed of claim 57 or 58 in a plant breeding method and/or in the production of hybrid seed.

61. A method for targeting a fusion protein to a target nucleic acid sequence in the genome of a plant, plant part, or plant cell, producing at least one plant cell comprising a fusion protein and an engineered, single guide R A (sgRNA), wherein the fusion protein comprises a Cas protein domain that is completely or partially defective in nuclease activity and an additional domain, wherein the sgRNA is capable of recognizing the target nucleic acid sequence, whereby the fusion protein is capable of binding to or recognizing the target sequence when in the presence of the sgRNA.

62. The method of claim 61, wherein the Cas protein domain is a Cas9 protein domain or mutant or dervitive of Cas9 protein domain.

63. The method of claim 62, wherein Cas9 protein domain comprises the DIOA amino acid substitution, the H841A amino acid substitution, or both the DIOA and H841A amino acid substitutions.

64. The method of any one of claims 61-63, wherein producing the at least one plant cell comprising the fusion protein and the engineered, single guide RNA, comprises:

(c) both (a) and (b).

65. The method of claim of claim 64, wherein the sgRNA, the fusion protein, or both are transiently expressed in the plant cell.

66. The method of claim 64 or 65, wherein the nucleic acid construct for expression of the sgRNA, the nucleic acid construct for expression of the fusion protein, or both are stably incorporated in the genome of the plant cell.

67. The method of any one of claims 61-66, wherein producing the at least one plant cell comprising the fusion protein and the sgRNA, comprises:

(ii) directly introducing the fusion protein into the plant cell; or

(iii) both (i) and (ii).

68. The method of any one of claims 61-67, wherein the fusion protein or the nucleic acid construct for expression of the fusion protein is introduced into the plant cell or the progenitor, before, concurrently with, or after introducing the sgRNA or the nucleic acid construct for expression of the sgRNA.

69. The method of any one of claims 61-68, wherein the additional domain is selected from the group consisting of a transcriptional activator domain, a transcriptional repressor or co-repressor domain, a protein domain known to elevate the local mutagenesis rate, and a protein that can elevate the local recombination rate.

70. The method of any one of claims 61-69, wherein the additional domain is selected from the group consisting of an EAR domain, a SRDX domain, a TOPLESS domain, a cytidine deaminase or other DNA or histone modifying enzyme domain, a MAGI domain, and a RAD54 domain.

71. The method of any one of claims 61 -70, wherein the target nucleic acid sequence is within a gene.

72. The method of any one of claims 61-71, wherein the target nucleic acid sequence is within a promoter.

73. The method of any one of claims 64-72, wherein the nucleic acid construct for expression of the fusion protein comprises a promoter operably linked to a nucleic acid sequence encoding the fusion protein and optionally an operably linked eukaryotic nuclear localization signal sequence, wherein the promoter is capable of driving expression of a nucleic acid sequence in a plant cell.

74. The method of claim 71, wherein the promoter is an RNA polymerase II promoter.

75. The method of claim 73 or 74, wherein the promoter is a constitutive promoter, an inducible promoter, a developmentally regulated promoter, a tissue-specific promoter, and a seed-specific promoter.

76. The method of any one of claims 73-75, wherein the promoter is selected from the group consisting of the CaMV 35S promoter, a ubiquitin promoter, an estradiol- inducible promoter, a dexamethasone-inducible promoter, an alcohol-inducible promoter, a heat shock promoter, an inflorescence-specific promoter, the Arabidopsis Apetala2 promoter, and the Arabidopsis Agamous promoter.

77. The method of any one of claims 64-76, wherein the nucleic acid construct for expression of the sgRNA comprises a promoter operably linked to a nucleic acid sequence encoding the sgRNA, wherein the promoter is capable of driving expression of a nucleic acid sequence in a plant cell.

78. The method of claim 77, wherein the promoter is an RNA polymerase III promoter.

79. The method of claim 77 or 78, wherein the promoter is the Arabidopsis U6 promoter.

80. The method of any one of claims 77-79, wherein the promoter comprises the Arabidopsis U6 promoter-derived synthetic nucleic acid sequence set forth in FIG. 4B.

81. The method of any one of claims 77-80, wherein nucleic acid construct for expression of the sgRNA comprises the nucleic acid sequence of the U6::sgRNA construct set forth in FIG. 4B.

82. The method of any one of claims 61-81, wherein the target nucleic acid sequence comprises or consists of 19 bases.

83. The method of any one of claims 61-82, wherein immediately following the 3' end of the target nucleic acid sequence in the genome of the plant, plant part, or plant cells is a protospacer-adjacent motif (PAM).

84. The method of claim 83, wherein the PAM is NGG, wherein N can be A, C, G, or T.

85. The method of claim 83, wherein the Cas protein recognizes a PAM that is not NGG, wherein N can be A, C, G, or T.

86. The method of any one of claims 61-85, wherein the sgRNA comprises a guide nucleic acid sequence that is capable of binding to or recognizing the target nucleic acid or its complement.

87. The method of claim 86, wherein the guide nucleic acid sequence is identical to the target nucleic acid sequence or its complement, when a U in the guide nucleic acid sequence is equivalent to a T in the target nucleic acid sequence or its complement.

88. The method of claim 86, wherein the guide nucleic acid sequence is not identical to the target nucleic acid sequence or its complement, when a U in the guide nucleic acid sequence is equivalent to a T in the target nucleic acid sequence or its complement.

89. The method of claim 86 or 88, wherein there are one or more mismatched bases between the guide nucleic acid sequence and the target nucleic acid sequence or its complement.

90. The method of claim 89, wherein the one or more mismatches occur at a position or positions in the target nucleic acid sequence that are located within the first 8, 9, 10, 11, or 12 bases beginning from the 5' end of the target nucleic acid sequence.

91. The method of any one of claims 61-90, further comprising introducing at least one additional engineered sgRNA, wherein the sgRNA is capable of recognizing an additional target nucleic acid sequence.

92. The method of claim 91, wherein the fusion protein is also capable of binding to or recognizing the additional target sequence when in the presence of the additional sgRNA.

93. The method of any of claims 61-92, further comprising regenerating the plant cell or descendent thereof into a plant comprising the fusion protein and/or the sgRNA and/or at least one modification in the genome of the plant cell or descendent that is due the fusion protein and the sgRNA.

94. A plant, plant part, seed, or plant cell produced by the method of any one of claims 61-93, wherein the plant, plant part, seed, or plant cell comprises the fusion protein and the engineered sgRNA.

95. A plant, plant part, seed, or plant cell comprising the fusion protein and/or sgRNA introduced into the genome according to the method of any one of claims 61-93 or comprising at least one modification in its genome that is due to the fusion protein and the sgRNA according to the method of any one of claims 61-93.

96. Use of the plant, plant part, or seed of claim 94 or 95 in agriculture and/or in the production of a human and/or animal food product.

97. Use of the plant, plant part, or seed of claim 94 or 95 in a plant breeding method and/or in the production of hybrid seed.

98. A fusion protein according to any of claims 61-93 or a nucleic acid molecule encoding the fusion protein.

(b) contacting the plant tissue with one or more cells of a second bacterial strain comprising a second nucleic acid construct, wherein the second nucleic acid construct comprises an RNA polymerase III promoter that is expressible in a plant cell operably linked to nucleic acid sequence encoding an engineered, single guide RNA (sgRNA) that is capable of recognizing a target nucleic acid sequence in the genome of plant tissue, and wherein a cell of the second bacterial strain is capable of transferring a nucleic acid molecule to a plant cell; and

monitoring the plant tissue for a modification of the genome

100. The method of claim 99, wherein the Cas protein is Cas9 or a mutant or derivative of Cas9.

101. The method of claim 100, wherein the mutant or derivative is naturally ocurring or engineered.

102. The method of any one of claims 99-101, wherein the Cas protein is capable of causing a double-strand break in DNA.

103. The method of any one of claims 99- 101, wherein the Cas protein is capable of causing a single-strand break in DNA.

104. The method of any one of claims 100, 101, and 103, wherein the mutant or derivative comprises the D10A amino acid substitution or the H841A amino acid substitution, and wherein the mutant or derivative is capable of causing a single-strand break in DNA.

105. The method of any one of claims 99-104, wherein at least one of the first and the second bacterial strains is a strain from a bacterial species that is selected from the group consisting of Agrobacterium tumefaciens, Agrobacterium rhizogenes, Ensifer adhaerens and other Ensifer spp., Sinorhizobium meliloti, and Mesorhizobium loti.

106. The method of any one of claims 99-105, wherein at least one of the first and the second bacterial strains is selected from the group consisting of Agrobacterium tumefaciens strain AGL1 and Ensifer adhaerens strain OV14.

107. The method of any one of claims 99-106, wherein at least one of the first and the second nucleic acid constructs is contained in a Ti plasmid or an Agrobacterium T-DNA border-containing broad host range binary vector..

108. The method of any one of claims 99-107, wherein the contacting of step

(a) comprises infiltration of the plant tissue with one or more cells of the first bacterial strain and/or the contacting of step (b) comprises infiltration of the plant tissue with one or more cells of the second bacterial strain.

109. The method of claim 108, wherein infiltration comprises use of a syringe to deliver the cells to the plant tissue.

110. The method of any one of claims 99-109, wherein steps (a) and (b) are performed at the same time.

111. The method of claim 110, wherein the plant tissue is infiltrated with a mixture comprising cells of the first bacterial strain and the second bacterial strain.

112. The method of any one of claims 99-111, wherein the plant tissue is from a plant selected from the group consisting of Nicotiana benthamiana, Nicotiana tabacum, Lactuca sativa, Brassica rapa, and any other plant species in which transient expression of genes after delivery of DNA either from bacteria or by other means can be achieved.

113. The method of any one of claims 99-112, wherein the plant tissue is leaf tissue.

114. The method of claim 112, wherein the leaf tissue is, or is part of, a whole leaf.

115. The method of any one of claims 99-114, wherein the plant tissue is attached to a plant or is detached from a plant.

116. The method of any one of claims 99-115, wherein the target nucleic acid sequence comprises or consists of 19 bases.

117. The method of any one of claims 99-116, wherein immediately following the 3' end of the target nucleic acid sequence in the genome of the plant, plant part, or plant cells is the protospacer-adjacent motif (PAM).

118. The method of claim 117, wherein the PAM is NGG, wherein N can be A, C, G, or T.

119. The method of claim 117, wherein the Cas protein recognizes a PAM that is not NGG, wherein N can be A, C, G, or T.

120. The method of any one of claims 99-119, wherein the sgRNA comprises a guide nucleic acid sequence that is capable of binding to or recognizing the target nucleic acid or its complement.

121. The method of claim 120, wherein the guide nucleic acid sequence is identical to the target nucleic acid sequence or its complement, when a U in the guide nucleic acid sequence is equivalent to a T in the target nucleic acid sequence or its complement.

122. The method of claim 120, wherein the guide nucleic acid sequence is not identical to the target nucleic acid sequence or its complement, when a U in the guide nucleic acid sequence is equivalent to a T in the target nucleic acid sequence or its complement.

123. The method of claim 120 or 123, wherein there are one or more mismatched bases between the guide nucleic acid sequence and the target nucleic acid sequence or its complement.

124. The method of claim 123, wherein the one or more mismatches occur at a position or positions in the target nucleic acid sequence that are located within the first 8, 9, 10, 11, or 12 bases beginning from the 5' end of the target nucleic acid sequence.

125. The method of any one of claims 99-124, wherein the target nucleic acid sequence is:

(i) native to the genome,

(iv) co-delivered with the Cas gene and the sgR A.

126. The method of any one of claims 99-125, wherein monitoring the plant tissue for a modification of the genome within the target site comprises determining the nucleotide sequence of all or a part of the target nucleic acid sequence.

127. The method of any one of claims 99-126, wherein the target nucleic acid sequence comprises a restriction enzyme recognition sequence.

128. The method of claim 127, wherein step (c) comprises:

digesting the genomic DNA with a restriction enzyme that recognizes the restriction enzyme recognition sequence;

amplifying the digested DNA by polymerase chain reaction using primers designed to amplify across the target nucleic acid sequence so as to produce PCR products comprising one or more modifications within target nucleic sequence;

cloning the PCR products; and

determining the nucleic acid sequence of the cloned PCR products.

129. The method of claim 127 or 128, wherein the restriction enzyme recognition sequence is a Mlyl restriction enzyme recognition sequence.

130. The method of claim 128 or 129, wherein the DNA is extracted from plant tissue harvested at about 1-48 hours after the later of the contacting steps.

131. The method of claim 128 or 129, wherein the DNA is extracted from leaves harvested at about 6, 12, 18, 24, 30, 36, 42, or 48 hours after the later of the contacting steps.

132. A method for modifying the genome of a plant, plant part, or plant cell, the method comprising producing at least one plant cell comprising a Cas protein and at least two engineered, sgRNAs that are each capable of recognizing a different target nucleic acid sequence, wherein the plant, plant part, plant cell, or descent thereof, comprises in its genome the target nucleic acid sequences.

133. The method of claim 132, wherein a first sgRNA is capable of recognizing a first target sequence and a second sgRNA is capable of recognizing a second target sequence.

134. The method of claim 133, wherein the first target sequence and the second target sequence are on the same chromosome.

135. The method of claim 133 or 134, wherein the first target sequence and the second target sequence are within the same gene.

136. The method of any one of claims 133-135, wherein the first target sequence and the second target sequence are separated by at least 10, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500, 1000, 1500, 2000, 3000 or more nucleotides.

137. The method of any one of claims 133-136, wherein the modification comprises the deletion of at least one base pair in the genome of the plant, plant part, plant cell, or descendant thereof.

138. The method claim 137, wherein the deletion occurs between the first target sequence and the second target sequence.

139. The method of any one of claims 133-138, wherein at least one of the Cas protein, the first sgRNA, and the second sgRNA are transiently expressed in the plant cell.