DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Status of the Claims
Claims 3, 6, and 18 are canceled.
Claims 1-2, 4-5, 7-17, and 19-22 are pending.
Claims 8-17 and 19-22 are withdrawn from consideration as being drawn to a non-elected invention.
Claims 1-2, 4-5, and 7 are examined herein.
Claims 1-2, 4-5, and 7 are rejected.
Priority
Application No. 18/451,658 filed on 08/17/2023 claims benefit of priority to provisional Application No. 63/398,726 filed on 08/17/2022.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Written Description
Claims 1-2, 4-5, and 7 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
This is a new rejection.
Claims 1-2, 4-5, and 7 are broadly drawn to a cannabis seed comprising a Y chromosome, wherein the Y chromosome is modified to comprise: a) a heterologous promoter operably linked to a marker gene, or b) a gene that is altered through gene editing to generate a marker gene; and wherein expression of the marker gene of (a) or (b) imparts a detectable phenotype, wherein the detectable phenotype is chromogenic or fluorogenic and is visible through the seed coat to allow for sorting of male seed from female seed.
Applicant describes in working examples a 2X35S:RUBY cassette that is introduced into and expressed in cannabis seeds (example 1), a proposed experiment of a CsEde1p:RUBY cassette that may be introduced into and expressed in cannabis seeds (example 2), a proposed experiment of a transforming pollen with a RUBY gene cassette (example 3), and proposes using gene editing (e.g. CRISPR, ZFN, TALEN) to impart a phenotype including knocking out a gene, or enhancing gene expression by altering their promoter element or other expression elements (example 4). The list of proposed genes for knockout or enhanced expression in example 4 include WRI1, KNAT7, NAC2, TTG2, STK, C2H2-like, bZIP44, SHP1, GBF6, PPO, CHS, P, Z, or BIP. Thus, in working examples the Applicant has only provided evidence that a 2X35S:RUBY cassette that is introduced into and expressed in cannabis seeds can impart a detectable phenotype that is visible through the seed coat (example 1).
Applicant has not described in the specification any example of altering any gene through gene editing to generate a marker gene wherein expression of the marker gene imparts a chromogenic or fluorogenic phenotype and is visible through the seed coat. For example, Applicant has not described how WRI1 may be altered though gene editing to generate a marker gene that imparts a detectable phenotype that is chromogenic or fluorogenic and is visible through the seed coat. Furthermore, there is a dearth of description in the prior art as to how any gene, including any of the listed genes in the disclosure, may be altered though gene-editing to impart a detectable phenotype that is chromogenic or fluorogenic and is visible through the seed coat.
The specification fails to provide an adequate written description to describe the genus of altering any gene through gene editing to generate a marker gene that imparts a detectable phenotype which is chromogenic or fluorogenic and is visible through the seed coat. The prior art fails to remedy this deficiency. Therefore, one of ordinary skill in the art would not have recognized the Applicant to be in possession of the claimed invention at the time the application was filed.
Enablement
Claims 1-2, 4-5, and 7 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
This is a new rejection.
In re Wands, 858 F.2d 731 (Fed. Cir. 1988) lists the following eight factors for determining whether undue experimentation would be required to practice an invention: (1) quantity of experimentation necessary; (2) amount of direction or guidance supplied; (3) presence or absence of working examples; (4) nature of the invention; (5) state of the prior art; (6) relative skill of those in the art; (7) predictability or unpredictability or the prior art; (8) breadth of the claims
Claim 1 recites:
A cannabis seed comprising a Y chromosome, wherein the Y chromosome is modified to comprise: a) a heterologous promoter operably linked to a marker gene, or b) a gene that is altered through gene editing to generate a marker gene; and wherein expression of the marker gene of (a) or (b) imparts a detectable phenotype, wherein the detectable phenotype is chromogenic or fluorogenic and is visible through the seed coat to allow for sorting of male seed from female seed.
Thus, the claim requires in an embodiment a Y chromosome is modified to comprise a gene that is altered through gene editing to generate a marker gene; and wherein expression of the marker gene of imparts a detectable phenotype, wherein the detectable phenotype is chromogenic or fluorogenic and is visible through the seed coat to allow for sorting of male seed from female seed.
The claimed invention is not enabled because the effects of any alteration via gene-editing to any gene to impart a chromogenic or fluorogenic phenotype in cannabis seeds is unpredictable, and Applicant has failed to provide any direction or guidance as to how the skilled artisan could/ would alter a gene through gene editing to impart a detectable, visible chromogenic or fluorogenic phenotype in the seeds. Applicant provides no working examples in the specification of how to alter a gene through gene editing to impart a detectable chromogenic or fluorogenic phenotype in the seeds because the proposed examples of altering a gene through gene editing are directed to knocking out or overexpressing endogenous genes unrelated to chromogenic or fluorogenic phenotypes (e.g., WRI1). The state of the prior art also fails to provide any guidance as to how the skilled artisan would alter any gene, including e.g. WRI1, through the methods discussed in the specification (knocking out or enhancing gene expression) so that the edited gene would impart a detectable chromogenic or fluorogenic phenotype visible in the seeds. That is, the level of skill in the prior art would not enable the ordinary artisan to practice the claimed invention and, after reading the specification, the skilled artisan would not understand how to generate a marker gene that imparts a chromogenic or fluorogenic phenotype in cannabis seeds by altering a gene. Given the breadth regarding any alteration to any gene, combined with the nature of the invention, unpredictability in the art, and lack of any guidance as to how to practice the claimed invention, undue experimentation is required to practice Applicant’s invention. Applicant’s claimed invention would require undue trial and error experimentation with no reasonable expectation of success.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1-2, 4-5, and 7 are rejected under 35 U.S.C. 103 as being unpatentable over Hildebrand (US-20210025014-A1), Zhao (Zhao, X., Wei, W., Pan, H., Nie, J., Chen, D., Zhang, P., ... & Zhang, M. (2019). Identification of the sex of pre-implantation mouse embryos using a marked Y chromosome and CRISPR/Cas9. Scientific reports, 9(1), 14315), He (He, Y., Zhang, T., Sun, H., Zhan, H., & Zhao, Y. (2020). A reporter for noninvasively monitoring gene expression and plant transformation. Horticulture research, 7), and Fletcher (US-20220152138-A1).
This is a maintained rejection from the previous rejection set forth in the Office Action dated 12/16/2025.
Claim 1 is drawn to a cannabis seed comprising a Y chromosome, wherein the Y chromosome is modified to comprise:
a) a heterologous promoter operably linked to a marker gene, or
b) a gene that is altered through gene editing to generate a marker gene; and wherein expression of the marker gene of (a) or (b) imparts a detectable phenotype wherein the detectable phenotype is chromogenic or fluorogenic and is visible through the seed coat to allow for sorting of male seed from female seed.
Claim 2 is drawn to the cannabis seed of claim 1, wherein the marker gene is expressed in seeds and pollen grains.
Claim 4 ius drawn to the cannabis seed of claim 3 (interpreted to depend from claim 1), wherein the marker gene is RUBY or a gene encoding tdTomato.
Claim 5 is drawn to the cannabis seed of claim 1, wherein the marker gene is selected from RFP, GFP, YFP, CFP, meffRed, eforRed, aasPink, spisPink, scOrange, crtBl, crtI, crtE, CYP76AD1, DODA, or GT.
Claim 7 is drawn to the cannabis seed of claim 1, wherein the promoter is the ubiquitin promoter, 35S promoter, 2x35S promoter, 7S seed-specific soybean promoter, Edel seed promoter, or the CsEdelp seed promoter.
Regarding claim 1, Hildebrand teaches a method for determining a gender of a Cannabis seed, comprising: obtaining a biological sample from a dry Cannabis seed; determining a presence or absence of a Y-chromosome specific marker in the biological sample from the dry Cannabis seed; and identifying the dry Cannabis seed as being a male or female Cannabis seed based on the presence or absence of the Y-chromosome specific marker in the biological sample (abstract, claim 1 of Hildebrand).
However, Hildebrand does not explicitly teach:
wherein the Y chromosome is modified to comprise a heterologous promoter operably linked to a marker gene, or a gene that is altered through gene editing to generate a marker gene, and wherein expression of the marker gene of (a) or (b) imparts a detectable phenotype that is chromogenic or fluorogenic and is visible through the seed coat (remaining limitations of claim 1).
The cannabis seed of claim 1, wherein the marker gene is expressed in seeds and pollen grains (claim 2).
The cannabis seed of claim 3, wherein the marker gene is RUBY or a gene encoding tdTomato (claim 4).
The cannabis seed of claim 1, wherein the marker gene is selected from RFP, GFP, YFP, CFP, meffRed, eforRed, aasPink, spisPink, scOrange, crtB, crtl, crtE, CYP76AD1, DODA, or GT (claim 5).
The cannabis seed of claim 1, wherein the promoter is the ubiquitin promoter, 35S promoter, 2x35S promoter, 7S seed-specific soybean promoter, Edel seed promoter, or the CsEdelp seed promoter (claim 7).
Regarding the remaining limitations of claim 1, in analogous art, Zhao teaches a method of modifying the y-chromosome of a mouse line with a GFP visual reporter under the control of the CAG promoter for the purpose of easily and accurately identifying male embryos (title, abstract). In other analogous art, He teaches a noninvasive reporter gene to screen for transgenic plants and seeds (title, abstract), and specifically teaches using the 35S promoter (i.e. a heterologous promoter) to express the RUBY visual reporter gene (i.e. a marker gene) in plants and progeny seeds that harbor the T-DNA with the transgene (Fig. 2, Supplemental Fig. 3).
Regarding claim 2, He teaches the marker gene is expressed in seeds driven by the strong constitutive 35S promoter (Supplemental Fig. 3) (i.e. meaning the promoter expresses in all cells). He also teaches betalain was synthesized in all floral organs in 35S:RUBY plants (Supplemental Fig. 3 and caption). Because the strong, constitutively expressed 35S promoter (which is expresses in all cells) was used to drive RUBY expression, and expression was reported to be observed in seeds and all floral organs, it is reasonably interpreted that RUBY was expressed in the pollen cells.
Regarding claim 4, He teaches the marker gene is Ruby (Fig 2., Supplemental Fig. 3).
Regarding claim 5, Zhao teaches the marker gene is GFP (title, abstract).
Regarding claim 7, He teaches the promoter is the 35S promoter (Fig. 2, Supplemental Fig. 3).
It would therefore have been obvious to a person of ordinary skill in the art to modify the invention of as taught by Hildebrand to include the limitations of Zhao and He to arrive at the instantly claimed method with a reasonable expectation of success because Hildebrand teaches a method of identifying Cannabis seeds with a y- chromosome, Zhao teaches a y-chromosome can be modified to comprise a visual reporter gene to easily detect cells with the y-chromosome for sex determination, and He teaches a routine method and product in the biotechnology field that is a plants and seeds carrying a heterologous promoter linked to a visual reporter; this combination would allow identification of plants carrying the y-chromosome and could be achieved by one of ordinary skill in the art without encountering any special technical obstacles. One having ordinary skill in the art would have been motivated to combine the teachings of Hildebrand and Zhao because it would be prima facie obvious to modify a y- chromosome with a marker gene as taught by Zhao in plants rather than mice for the same purpose that is sex determination. One having ordinary skill in the art would have also been motivated to combine the teachings with He because He teaches the RUBY reporter is an effective, noninvasive reporter gene (title, abstract), and plants and seeds harboring the RUBY gene can be easily identified without additional equipment such as a microscope that limits identification of traditional fluorescent reporter genes (Introduction, 41, Fig. 2, Supplemental Fig. 3). Additionally, one of ordinary skill would have applied the method and reporter cassette of He (35S:RUBY, which is one of the claimed promoter X marker gene embodiments) to any other plant including Cannabis for the same purpose that is expressing and observing the RUBY reporter gene in the plant, and by doing so one of ordinary skill would have observed the chromogenic phenotype imparted by the RUBY reporter gene in the cannabis seeds. Furthermore, one having ordinary skill in the art would have been motivated to combine these teachings to modify the y- chromosome of a Cannabis plant to impart a detectable phenotype in the male plants, seed, or pollen because Fletcher teaches Cannabis is an important agronomic crop, and much of the production today is done using feminized seed with the end goal of fields containing only female plants since the female reproductive tissue (e.g. the buds) produce the economically-important cannabinoids (i.e. male plants or plants with the y- chromosome are undesirable) (¶0010).
Response to Arguments
Applicant argues beginning on p. 6 of remarks dated 06/16/2026 the
following arguments:
Claim 1 is objected to for the lack of a period at the end of the claim. Claim 1 is amended to end with a period to overcome the objection. Applicant respectfully requests reconsideration of the objection.
Examiner’s response:
In view of Applicant’s amendments, the previous objection has been withdrawn.
Applicant argues beginning on p. 6 of remarks dated 06/16/2026 the
following arguments:
Claims 4 and 7 are rejected under 35 U.S.C. §112(b) as being indefinite.
Claim 4 recites dependency upon claim 3, which was cancelled in response to the last Office Action wherein the limitations of claim 3 were incorporated into claim 1. Accordingly, claim 4 has been amended to depend upon claim 1.
Claim 7 is rejected for lack of sufficient antecedent basis. Claim 7 has been amended to contain proper antecedent basis.
Applicant respectfully requests entry of these amendments, reconsideration of the claims and withdrawal of the rejections.
Examiner’s response:
In view of Applicant’s amendments, the previous rejections under 35 USC 112(b) have been withdrawn.
Applicant argues beginning on p. 7 of remarks dated 06/16/2026 the
following arguments:
Claims 1, 2, 4, 5, and 7 remain rejected under 35 U.S.C. §103 as being unpatentable over Hildebrand et al. (US 2021/0025014), Zhao et al. (Scientific Reports 9:14315, 2019) He et al. (Horticulture Research 7(1):152, 2020), and Fletcher (US 2022/0152138). The Examiner cites Hildebrand for teaching a method of determining the gender of a Cannabis seed with a Y- chromosome-specific marker but does not teach modifying the Y chromosome with a heterologous promoter linked to a visual marker gene. The Examiner cites He as teaching the use of a strong promoter, such as the 35S promoter, to drive RUBY expression in transgenic plants and to produce a detectable signal in Arabidopsis seeds. Zhao is cited for teaching a method of modifying the Y- chromosome of a mouse line with a GFP reporter to identify male embryos. Fletcher is cited as providing the motivation by noting that male plants are not as economically important as the female plants. The Examiner rejected our previous argument noting that Cannabis seeds have varied coloration and that we provided no reason "why the density of the seed would inhibit RUBY expression in the seed coat when driven by a constitutive promoter." See Office action at page 11.
As set forth in MPEP 2142, in order to establish a prima facie case of obviousness, the prior art references must teach or suggest all of the claim limitations and there must be a reasonable expectation of success in making the claimed combination. Claim 1 as amended now expressly requires that the detectable phenotype allows the cannabis seeds to be sorted from cannabis seeds that do not comprise the Y chromosome comprising the marker gene. This amendment is supported throughout the specification, which teaches that the marker gene must be expressed in seeds in sufficient amount to allow for seed sorting via visual detection of the marker. As explained in more detail below, this is not taught or suggested in the combination of cited references and one of skill in the art would not have a reasonable expectation of success based on the references provided.
The Examiner relies on He to teach the use of the RUBY marker system to produce a detectable signal in seeds using the 35S promoter or a seed specific promoter in Arabidopsis. However, the teaching of the ability to detect the RUBY marker in seeds of Arabidopsis demonstrates the ability to express and detect RUBY in seeds in a distinct plant with distinct seed morphology. He does not demonstrate that the signal would be of sufficient intensity to allow sorting of intact Cannabis seeds and none of the other cited references supplement He to cure this deficiency.
Unlike Arabidopsis seeds, which have an outer layer consisting only of the maternal integuments to form a seed coat, Cannabis seeds have an outer pericarp protective layer that is thick and hard. Arabidopsis seeds lack a pericarp. As shown in Figure 2c of He, the siliques of At2S3:RUBY in Arabidopsis were similar to those of WT, (i.e., the seed pods could not be visually inspected to differentiate between the two plants); however, when the silique was opened, the seeds of At2S3:RUBY were red. Because cannabis seeds lack a siliques and instead have a pericarp, which is absent from Arabidopsis seeds, the teachings of He do not apply to the cannabis seeds of the present invention or provide one of skill in the art with any reasonable expectation of success in Cannabis based on the results in Arabidopsis. Those of skill in the art with the understanding that Cannabis seeds are not found in a silique and instead have a thick pericarp covering the seed, would not reasonably expect the teachings of He in Arabidopsis to apply to Cannabis. One of skill in the art would expect the thicker pericarp to attenuate or prevent detection of a marker. Thus, one skilled in the art would not have a reasonable expectation of success in seeing the marker through the pericarp at an intensity sufficient to allow sorting of cannabis seeds.
Sorting the seeds expressing the marker form those that do not requires that the detectable phenotype be reliably distinguishable on each individual intact seed. None of the cited prior art references, alone or in combination, teach or suggest that a chromogenic or fluorogenic marker expressed in a Cannabis seed would produce a signal detectable through the intact cannabis pericarp at an intensity sufficient to allow reliable sorting. The present inventors demonstrated for the first time in the current specification that RUBY expression is visible through the seed coat of intact cannabis seeds (Fig. 1B).
The amended claims now expressly require that the detectable phenotype allows the cannabis seed to be sorted from seeds lacking the marker gene, and because neither He, Zhao, Hildebrand, Fletcher, nor any combination thereof provides a reasonable expectation of success that such sorting-level detection would be achievable through the intact pericarp of a cannabis seed, Applicant respectfully requests reconsideration of the rejection after entry of the amendment and withdrawal of the § 103 rejection.
Examiner’s response:
Regarding Applicant’s argument that one of ordinary skill in the art would not have a reasonable expectation of success in seeing the marker through the pericarp at an intensity sufficient to allow sorting of cannabis seeds, this argument has been fully considered and is not found persuasive. There are many examples in the prior art of other seeds that have pericarps, and expression of visual markers have successfully been used in these other seeds with pericarps to sort transgenic and non-transgenic seeds. For example, rice also has a pericarp and the expression of RFP under control of the seed-specific LTP2 promoter can be easily visualized through the pericarp to determine transgenic and non-transgenic seed (see, for example, Wei, US-20160326536-A1, Fig. 16 and ¶0102). Additionally, the 35S promoter which would be obvious to use in view of He is a strong constitutive promoter (see Fig. 2a and supplementary Fig. 3 b-d) and would reasonably be expected to express in all tissue including the seed (as observed in He) and also the pericarp tissue. Additionally, techniques are known and routine in the art to increase expression strength, e.g. enhancers and choice of strong promoters. Furthermore, one of ordinary skill would have applied the method and reporter cassette of He (35S:RUBY, which is one of the claimed promoter X marker gene embodiments) to any other plant including Cannabis for the same purpose that is expressing and observing the RUBY reporter gene in the plant, and by doing so one of ordinary skill would have observed the chromogenic phenotype imparted by the RUBY reporter gene in the cannabis seeds. Given what is known in the art, the skilled artisan would have a reasonable expectation of success of observing RUBY expression in the intact cannabis seeds (in and/or through the pericarp) using the strong constitutive 35S promoter, with or without any additional elements to enhance RUBY expression in the seed tissue. Because the RUBY expression would reasonably be expected to be observed in seed and pericarp (or in seed and through pericarp) of the cannabis seeds, one of ordinary skill in the art would also have a reasonable expectation of success of being able to sort the transgenic from non-transgenic seeds. For these reasons, Applicant’s argument has not been found persuasive.
Conclusion and Inquiries
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JESSICA N STOCKDALE whose telephone number is (703)756-5395. The examiner can normally be reached M-F 8:30-5:00 CT.
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JESSICA N. STOCKDALE
Examiner
Art Unit 1663
/JESSICA NICOLE STOCKDALE/Examiner, Art Unit 1663
/CHARLES LOGSDON/Primary Examiner, Art Unit 1662
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