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 .
This is a Final Office Action in response to amendment filed on 04/06/2026.
Claim Status
Claims 1, 3-7, 11-15, 17-20, 24-27 and 29-36 are pending. Claims 2, 8-10, 16, 21-23, 28, and 37 are canceled. Claims 1, 3-7, 11-15, 17-19, 24-27, 29, 30, 32, 34 and 36 are currently amended. Claims 1, 3-7, 11-15, 17-20, 24-27 and 29-36 are examined on the merits.
Response to Amendment
The rejection of Claim 28 rejected under 35 U.S.C. 112(b) is withdrawn because claim 28 has been canceled.
The rejection of Claims 1, 3, 4, 15, 18 and 19 over González and Claims 1-3, 5, 7-10 over Beecher rejected under 35 USC § 102(a)(1) is withdrawn in view of amendment to the claims. However, claims 1, 3-5, 7, 15 and 18-19 are now rejected under 35 U.S.C. §103 for the reasons set forth below.
The rejection of claims 2, 8-10, 16, 21-23, 28, and 37 rejected under 35 U.S.C. §103 is withdrawn in view of the cancellation of claims 2, 8-10, 16, 21-23, 28, and 37.
Claim Rejections - 35 USC § 103
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.
The rejection under 35 U.S.C. 103 is modified to address Applicant’s claim amendments.
Claims 1, 3-7, 11-15, 17-20, 24-27 and 29-36 are rejected under 35 U.S.C. §103 as being unpatentable over Liu (Congcong Liu et. al., Genomics 112 (2020) 4690–4700) in view of Beecher (Brian S. Beecher et.al., Theoretical and Applied Genetics (2012) 124:1463–1473) and Zhang (Shujuan Zhang et. al., Plant Biotechnology Journal (2021) 19, pp. 1684–1686).
Claim 1 recites a modified wheat plant having reduced polyphenol oxidase (PPO) activity, the wheat plant comprising loss-of-function mutations in each of the endogenous PPO1 and PPO2 genes of the wheat plant, wherein the loss-of-function mutations are in a region of each of the endogenous PPO 1 and PPO2 genes that encodes a copper binding domain.
Under the broadest reasonable interpretation, “PPO1” and “PPO2” refer to endogenous wheat PPO gene family/locus members recognized in the art, including PPO1-type loci such as PPO-A1/PPO-D1 and PPO2-type loci such as PPO2A-1, PPO2a-2, PPO2B-1, PPO2B-2, PPO2B-3, PPO2D-1 and PPO2D-2.
Liu teaches that wheat grain PPO activity is regulated by multiple PPO genes, including PPO genes on chromosomes 2A, 2B, and 2D (p4694, 3.6. Expression profiling of PPO genes). Liu further identifies PPO genes including TaPPO2A-1, TaPPO2A-2, TaPPO2B-1, TaPPO2B-2, TaPPO2B-3, TaPPO2D-1 and TaPPO2D-2 as PPO genes associated with grain PPO activity (p4698, left and right column), and teaches that pyramiding favorable PPO alleles reduce PPO activity (p4694, left column, paragraph 1).
Beecher teaches that PPO activity causes time-dependent discoloration/browning in wheat products (p1463, introduction), that PPO exists as a multigene family in wheat (p1470, right column, paragraph 2), and that PPO proteins contain catalytic copper-binding regions (p1467, left column). The copper-binding sites are necessary for PPO enzymatic catalysis and are conserved functional regions of the enzyme (p1468, Fig. 2). Beecher further teaches wheat PPO1/PPO loci, including PPO-A1/PPO-D1 type loci, as PPO genes associated with kernel PPO activity (p1470, right column, paragraph 3).
Liu and Beecher do not expressly teach a single modified wheat plant comprising loss-of-function mutations in each endogenous PPO1 and PPO2 gene.
Zhang teaches using genome editing in wheat to introduce targeted mutations in grain-quality genes, including multiple PPO genes, to obtain plants with reduced PPO function /activity (p1684-1685).
It would have been obvious to a POSITA, motivated by Liu’s and Beecher’s teaching that multiple endogenous wheat PPO genes contribute to grain/kernel PPO activity and that reduced PPO activity improves wheat product quality, to apply Zhang’s CRISPR/Cas9 editing method to introduce loss-of-function mutations into each endogenous PPO1 and PPO2 gene of wheat. A POSITA would have been further motivated to target regions encoding copper-binding domains because Beecher teaches that copper-binding regions are catalytic/functional PPO domains, and disrupting such domains would predictably reduce PPO enzymatic function. Because Liu teaches that pyramiding favorable PPO alleles reduces PPO activity, and Zhang teaches that higher PPO editing ratios produce lower PPO activity, a POSITA would have had a reasonable expectation that editing each endogenous PPO1/PPO2 gene would maximize reduction of PPO activity.
Accordingly, claim 1 is obvious over Liu in view of Beecher and Zhang.
Claim 3 recites the modified wheat plant of claim 1, wherein the loss-of- function mutations are an insertion, a deletion, or a substitution of one or more nucleotides. Claim 4 recites the modified wheat plant of claim 1, wherein the loss-of- function mutations introduce a frameshift mutation, a pre-mature stop codon, or an in-frame deletion.
Zhang teaches CRISPR/Case-induced mutations in wheat PPO genes, including deletions and insertions ( p1685-1686, fig. 1(a)and 1(b)), and deletion and insertion mutation causing frameshift/premature termination in edited wheat genes. Accordingly, the mutation types recited in claims 3 and 4 would have been obvious for the same reason set forth for claim 1.
Claim 5 recites the modified wheat plant of claim 1, wherein the PPO activity in the grain of the wheat plant is reduced relative to the PPO activity in grain of a wheat plant without the loss-of-function mutations.
Liu (p4694, left column, paragraph 2) and Zhang (p1686 fig.1(i)) teach reduced PPO activity in wheat grain/kernel; therefore, claim 5 is obvious for the same reasons set forth for claim 1.
Claim 6 recites the modified wheat plant of claim 5, wherein the PPO activity is reduced by at least 70%, 80%, or 90%. Claim 7 recites the modified wheat plant of claim 1, wherein browning of processed grain of the modified plant is reduced.
Zhang teaches WT PPO activity of 9.0 Ug-1 min-1 and T3 PPO mutants with PPO activity of 1.24Ug-1min-1, which is an about 86% reduction (p1686, left column, paragraph 1).
Beecher teaches PPO activity causes wheat product discoloration/browning (Abstract), and Zhang teaches reducing PPO activity by editing wheat PPO gene (p1686, left column, paragraph 1). Therefore, reduced browning would have been the expected results of reduced PPO activity.
Claims 6 and 7 are prima facie obvious for the same reasons set forth for claim 1.
Claims 11 and 12 recites the modified wheat plant of claim 1, wherein the loss-of- function mutations are in endogenous PPO1 and PPO2 genes having at least 95% sequence identity to SEQ ID NO: 1-7.
Under the broadest reasonable interpretation consistent with the specification, the recited SEQ ID NOs correspond to the endogenous wheat PPO genes identified in Table 1 (paragraph 0036) of the instant specification. Specifically:
SEQ ID NO:1 = TaPPO2A-1 (TraesCS2A02G468200);
SEQ ID NO: 2 = TaPPO2A-2 (TraesCS2A02G468500);
SEQ ID NO: 3 = TaPPO2B-1 (TraesCS2B02G491000);
SEQ ID NO:4 = TaPPO2B-2 (TraesCS2B02G491100);
SEQ ID NO: 5 = TaPPO2B-3 (TraesCS2B02G491400);
SEQ ID NO: 6 = TaPPO2D-1 (TraesCS2D02G468600);
SEQ ID NO:7 = TaPPO2D-2 (TraesCS2D02G468600).
Therefore claims 11 and 12 recite endogenous wheat PPO2 gene targets identified by sequence designation.
Liu teaches the same seven endogenous wheat PPO2 genes by gene name and Chinese Spring locus identifiers, including (p4698, left and right column, p4694, right column, paragraph 1, Table 1):
TaPPO2A-1 (TraesCS2A01G468200),
TaPPO2A-2 (TraesCS2A01G468500),
TaPPO2B-1 (TraesCS2B01G491000),
TaPPO2B-2 (TraesCS2B01G491100),
TaPPO2B-3 (TraesCS2B01G491400),
TaPPO2D-1 (TraesCS2D01G468200),
TaPPO2D-2 (TraesCS2D01G468600),
Although the instant specification uses updated locus identifiers with “02G” numbering, while Liu uses “01G” numbering, it only reflects updated annotations in different versions of the wheat reference genome database, and represent revised gene modeling numbering of the same underlying genomic loci rather than the gene names correspond to the same endogenous wheat PPO2 genes. Thus, Liu teaches the same PPO2 gene targets corresponding to SEQ ID NO: 1-7.
Accordingly, claims 11 and 12 are prima facie obvious for the same reasons set forth for claim 1.
Claim 13 recites modified wheat plant of claim 1, wherein the wheat plant is of the variety Fielder, Guardian, or Steamboat.
Zhang expressly teaches CRISPR/Cas9 editing in wheat cultivar Fielder (p1686, left column, paragraph 3). Therefore, at least the Fielder embodiment is obvious.
Fielder, Guardian, and Steamboat varieties and information can be found at the Agricultural Experiment Station of Colorado State University (https://webdoc.agsci.colostate.edu/csucrops/reports/winterwheat/wheatreport_2022.pdf, accessed 12/29/2025); Fielder variety and information can be found from Agricultural Extension Service University of Idaho (https://washingtoncrop.com/documents/Wheat/Spring/Soft%20White/Fielder.pdf, accessed 12/29/2025). It would have been obvious to implement the modified PPO genotype taught by Beecher and Zhang in any known wheat variety, including Fielder, Guardian, or Steamboat, because selecting among known wheat varieties for introducing a known genetic modification is a predictable substitution of one known cultivar for another. And the teaching of reducing PPO activity would reasonably be expected to apply across wheat genetic backgrounds.
Claim 14 recites a wheat plant part, plant cell, or seed of the modified plant of claim 1.
Beecher further teaches analysis of Kernel/seed in connection with PPO activity (e.g., “Kernel PPO analysis” performed on whole kernels/seeds; and PPO gene described as “seed-expressed” and “expressed in developing wheat kernels” mentioned in abstract and introduction), and thus discloses seed of the plant meeting claim 1. Accordingly, claim 14 is obvious.
Claim 15 recites a method of reducing PPO activity in a wheat plant by introducing loss-of-function mutations in each of the endogenous PPO 1 and PPO2 genes, wherein the mutations are in copper binding domains.
For the same reasons set forth for claim 1, Zhang teaches introducing PPO mutations by CRISPR/Cas9 in wheat and obtaining reduced PPO activity (p1684-1685). Liu and Beecher provide the PPO target rationale. Claim 6 is obvious over Liu, Beecher and Zhang.
Claim 17 recites the method of claim 15, wherein the PPO activity in the grain of the wheat plant is reduced relative to the PPO activity in grain.
Claim 17 is obvious for the same reasons set forth for claim 5.
Claim 18 recites the method of claim 15, wherein the loss-of-function mutations are introduced by genome editing. Claim 19 recites the method of claim 15, wherein the method comprises introducing a Cas nuclease and a guide RNA targeting each of the endogenous PPO1 and PPO2 genes. Claim 20 recites method of claim 19, wherein the guide RNA comprises the nucleotide sequence of SEQ ID NO: 34.
For the same reasons set forth for claim 15, Zhang teaches introducing PPO mutations by CRISPR/Cas9 in wheat and obtaining reduced PPO activity (p1684-1685). Zhang teaches CRISPR/Cas9 editing using sgRNA targeting conserved PPO sites (p1685-1686, Table 1). A POSITA would have been motivated to design guide RNA(s) targeting each endogenous PPO1/PPO2 gene to maximize PPO reduction. Selecting a guide RNA sequence targeting conserved PPO1/PPO2 regions, including SEQ ID NO:34, would have been a routine guide-design choice to carry out the PPO-reducing edits. Accordingly, claims 18-20 are prima facie obvious for the same reasons set forth for claim 15.
Claims 24 and 25 recites the method of claim 15, wherein the loss-of-function mutations are introduced in endogenous PPO1 and PPO2 genes having at least 95% sequence identity to SEQ ID NO: 1- 7.
Claims 24 and 25 are obvious for the same reasons set forth for claims 11 and 12.
Claim 26 recites the method of claim 15, wherein the wheat plant is of the variety Fielder, Steamboat, or Guardian.
Claim 26 is obvious for the same reasons set forth for claim 13.
Claim 27 recites a method of producing a wheat plant having reduced PPO activity by (a) crossing the wheat plant of claim 1; (b) growing wheat plants from the seed; and (c) selecting a progeny to produce a wheat plant having reduced PPO activity. Claim 29 recites a crop comprising a plurality of the wheat plants of claim 1 planted together in an agricultural field.
Beecher teaches wheat plants with reduced PPO activity and recognizes PPO traits are inherited (p1463, abstract and introduction). Beecher does not teach the method of producing a plant having reduced PPO activity. Zhang teaches generating wheat plants with PPO gene mutation and evaluating plants with reduce PPO activity. It would have been obvious to one of ordinary skill to cross such a reduced-PPO plant (mutant plant) with itself or another wheat plant, grown progeny, and select progeny carrying the PPO mutation, because these are routine breeding steps used to propagate and fix a desired, heritable mutation/trait and to obtain plants having reduced PPO activity. Also, planting a plurality of the wheat plants of claim 1 together in a field is the routine agricultural use of wheat plants.
Accordingly, claims 27 and 29 are prima facie obvious for the same reasons set forth for claim 1.
Claim 30 recites a commodity plant product prepared from the wheat plant of claim 1, wherein the product has reduced browning.
Beecher teaches that PPO activity is responsible for enzymatic browning (Abstract); Zhang teaches plant with PPO mutations and reduced PPO activity (p1686, left column, paragraph 1); therefore, it would have been obvious that a commodity product prepared from such plants would exhibit reduced browning. Claim 30 is obvious.
Claim 31 recites the commodity plant product of claim 30, wherein the product is grain, flour, a baked good, cereal, pasta, a beverage, livestock feed, biofuel, straw, a construction material, or starch. Claim 32 recites a method for producing a commodity plant product, the method comprising processing the wheat plant of claim 1, or a part thereof, to obtain the product. Claim 33 recites the method of claim 32, wherein the commodity plant product is grain, flour, a baked good, cereal, pasta, a beverage, livestock feed, biofuel, straw, a construction material, or starch.
The limitation of claim 31 is the listed items (grain, flour, a baked good, cereal, pasta, a beverage, livestock feed, biofuel, straw, a construction material, or starch) are conventional commodity products routinely prepared from wheat, and specifying them does not add a patentable distinction. Also, processing wheat plants or gain into commodity products is routine.
Claims 31-33 are obvious.
Claim 34 recites a guide RNA for editing PPO1 and PPO2 genes comprising the nucleotide sequence of SEQ ID NO: 34. Claim 35 recites an expression cassette or vector encoding the guide RNA of claim 34. Claim 36 recites a wheat plant cell comprising a Cas9 nuclease and the guide RNA of claim 34.
Zhang teaches designing sgRNA to target conserved PPO site in wheat, selecting a guide RNA sequence targeting conserved PPO1/PPO2 regions (p1684-1686, Table 1). Selecting SEQ ID NO: 34, would have been an obvious matter of routine CRISPR guide design. Zhang teaches CRISPR/Cas9 editing using constructed vectors for wheat transformation (p1684, left column, paragraph 1). Encoding the guide RNA in an expression cassette or vector would have been an obvious implementation of CRISPR editing. Zhang teaches CRISPR/Cas9-mediated editing in wheat cells/plant using Cas 9 and sgRNA. A wheat plant cell comprising Cas9 and the PPO-targeting guide RNA would have been an obvious intermediate int eh editing method.
Claims 34-36 are obvious.
Response to Applicant’s Remarks:
Applicant’s Main 103 Arguments:
Amended claims now require loss-of -function mutations in each endogenous PPO1 and PPO2 gene.
Applicant argues that Liu, Beecher, and Zhang, alone or combined, do not teach or suggest editing each endogenous PPO1 and PPO2 gene in wheat.
Zhang allegedly edits only a limited subset of PPO genes.
Applicant argues Zhang’s sgRNA targets a region between the conserved CuA and CuB domain, and due to sequence mismatches in Fielder PPO genes, Zhang would at most target only three PPO1/PPO2 genes, not all seven.
The present application allegedly targets a different region.
Applicant emphasizes that the claimed invention targets the CuA and CuB directly, while Zhang targets a region between CuA and CuB.
Applicant relies on specification data as a distinction.
Applicant points to the present specification showing editing of all seven PPO1/PPO2 genes in Fielder and all five target PPO genes in Steamboat and Guardian, with reduced PPO activity.
For claim 34-37, Applicant argues SEQ ID NO:34 is not routine guide design.
Applicant asserts Zhang’s guide RBA is different because it targets a different genomic region and would not target the same set of PPO1/PPO2 genes. therefore, selecting SEQ ID NO :34 was allegedly not an obvious routine design choice.
Applicant’s arguments have been considered but not persuasive.
Applicant argues that the cited references do not teach or suggest introducing loss-of-function mutations in each endogenous PPO1 and PPO2 gene of wheat. However, Beecher expressly teaches that the wheat PPO gene family includes multiple PPO1/PPO2 genes, including PPO-A1, PPO-D1, PPO-A2, PPO-D2, and PPO-B2, and further teaches that PPO-A2, PPO-B2, and PPO-D2 together accounted for over 72% of PPO transcripts in developing wheat kernels (p1464, left column). Beecher therefore identifies the PPO2 gene family as a substantial contributor to kernel PPO activity and teaches these PPO genes are relevant to wheat Kernel browning.
Liu further supports this same understanding by providing an in-depth genetic analysis of the PPO gene family and identifying 20 PPO genes in wheat (p4692, left column, paragraph 3, and Table 1). Liu teaches that PPO activity in wheat grain is controlled by expression of PPO-family genes, including TaPPO2A-1, and connects PPO express with grain PPO activity and browning (p4694, left and right column; p4690, Introduction). Liu further teaches, for the 9 PPO genes in the GWAS peak of PPO activity, 7 were expressed in wheat grains (p4694, right column, paragraph 2). Thus, Liu does not merely identify a single gene; Liu places TaPO2A-1 within a lager wheat PPO gene-family context and confirms that PPO-family expression is directly relevant to Kernel PPO activity.
Beecher also taches that PPO enzymes contain conserved copper-binding regions and that allelic variation associated with PPO activity occurs in or near the conserved CuA/CuB catalytic regions (p1647, left column; p1648, fig. 2; p1471, right column, paragraph 1). Because the copper-binding regions are essential to PPO enzymatic function, a POSITA seeking to reduce or eliminate PPO activity would have been motivated to target conserved copper-binding /catalytic regions of PPO genes, including PPO1 and PPO2 family members, to generate loss-of-function mutation.
Accordingly, the references do more than generally suggest “PPO editing”. Beecher identifies PPO1/PPO2 kernel-expressed gene family members and teaches that PPO2 members account for a major portion of Kernel PPO transcripts; Liu confirms the broader PPO gene family and its relationship to grain PPO activity and browning; and Zhang teaches the CRISPR/Cas9 editing of wheat PPO genes is an available and predictable approach for reducing PPO function. In view of these combined teaching, it would have been obvious to a POSITA to target multiple, and preferably all, endogenous PPO1/PPO2 genes contributing to wheat kernel PPO activity, including by targeting conserved copper-binding regions, with a reasonable expectation of reducing PPO activity and browning.
Applicant’s distinction that Zhang targeted a region between CuA and CuB, whereas the present application targets the CuB domain directly, is not persuasive. Th rejection is not limited to bodily incorporating Zhang’s exact sgRNA sequence. Rather, Zhang is relied upon for teaching the use of CRISPR/Cas9 to introduce targeted mutations into wheat PPO genes to reduce PPO activity. Beecher supplies the motivation to target conserved copper-binding /catalytic regions because those regions are critical for PPO enzyme activity. Therefore, selecting a guide RNA that gargets the conserved CuB-encoding region would have been a predictable design choice to disrupt PPO function, particularly where the goal was to create loss-of-function PPO mutations.
Thus, the recited art provides a specific reason to edit the PPO genes responsible for kernel PPO activity, including the PPO2 genes, and provides a specific reason to target conserved copper-binding regions required for PPO function. Applicant’s argument that the art teaches only editing three PPO genes is not persuasive because a POSITA would have understood from Beecher and Liu that additional PPO1/PPO2 family members contribute to kernel PPO activity and would have been motivated to target those genes as well to further reduce PPO activity.
Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to YANXIN SHEN whose telephone number is (571)272-7538. The examiner can normally be reached Monday-Friday.
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/YANXIN SHEN/Examiner, Art Unit 1663
/WEIHUA FAN/Primary Examiner, Art Unit 1663
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