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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 3/24/2026 has been entered.
Status of Claims
Claims 1, 3-6, 8-9, 13-15, 27-33 & 35-36 are under examination on the merits.
The rejection of claims 1, 3-6, 9, 13-15 & 27-30 under 35 U.S.C. 103 as being unpatentable over Lam et al (2019). New Phytologist. 223: 204–219, in view of Spini et al (2016) Plant Soil. 399: 159–178, taken with the evidence of NCBI GenBank accession AK064736 is withdrawn in light of Applicant’s amendments.
The rejection of claims 1, 3-6, 9, 13-15, 27-31 & 35-36 under 35 U.S.C. 103 as being unpatentable over Lam 2019 and Spini, taken with the evidence of GenBank accession AK064736, and further in view of Zhang et al (2017) Plant Physiology and Biochemistry. 111: 30-38 with withdrawn in light of Applicant’s amendments.
The rejection of claims 1, 3-6, 9, 13-15, 27-33 & 35-36 under 35 U.S.C. 103 as being unpatentable over Lam 2019, Spini, and Zhang taken with the evidence of GenBank accession AK064736 and further in view of Shao et al (2020). PLoS ONE. 15(7): e0235975 is withdrawn in light of Applicant’s amendments.
The rejection of claims 1, 3-6, 8-9, 13-15 & 27-30 under 35 U.S.C. 103 as being unpatentable over Lam 2019 and Spini with the evidence of GenBank accession AK064736 and further in view of Li et al (2016) Nature Plants. 2(10): 1-6 (published 9/12/2016, hereafter Li) is withdrawn in light of Applicant’s amendments.
Nucleotide and/or Amino Acid Sequence Disclosures
This application contains sequence disclosures that are encompassed by the definitions for nucleotide and/or amino acid sequences set forth in 37 CFR 1.821(a)(1) and (a)(2). However, this application fails to comply with the requirements of 37 CFR 1.821 through 1.825.
Figure 18 depicts 9 sequences. The brief description in paragraph [0040] provides that these are SEQ ID NOs: 146-151, 11, 13 & 12. However, the 6th sequence in figure 18 is 30 nucleotides long and has an additional C relative to the 29 nucleotide-long SEQ ID NO: 151. The sequence in the sequence listing file should match the sequence in the figure.
Full compliance with the sequence rules is required in response to this Office action. A complete response to this Office action must include both compliance with the sequence rules and a response to the issues set forth herein. Failure to fully comply with both of these requirements in the time period set forth in this Office action will be held to be non-responsive.
Claim Rejections - 35 USC § 112
Written Description
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.
Claims 1, 3-6, 8-9, 14-15, 27-33 & 35-36 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.
Due to Applicant' s amendment of the claims, the rejection is modified from the rejection as set forth in the Office action mailed 11/25/2025, as applied to claims 1,3-6,8-9,13-15,27-33 and 35-36. Applicant' s arguments filed 3/24/2026 have been fully considered but they are not persuasive.
Claims 1, 3-6, 8-9, 14-15, 27-33 & 35-36 are drawn to or require a crop plant in which expression of a CYP75B3 gene encoding a polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 31 has been modified. Claims 27, 30 & 36 require that the plant is selected from the group consisting of corn, wheat, rice, soy, cotton, canola, and sugarcane. Claim 33 requires that the crop plant is rice. Claims 6, 8, 9, 14 & 15 further require that an endogenous copy of the gene is modified, while claims 29-30 & 35 require a mutation or deletion in a CYP75B3 gene.
Claim 13, which further requires that the gene encodes SEQ ID NO: 31, is interpreted to be limited to only maize crop plants. Maize plants comprising a gene encoding SEQ ID NO: 31 are supported by Written Description, and so claim 13 is not included in this rejection.
Genes encoding CYP75B3 polypeptides with 95% identity to 515 amino acid-long SEQ ID NO: 31 encompass those encoding polypeptides with 25 amino acid substitutions relative to SEQ ID NO: 31.
The specification provides NCBI accession number AK064736 and UniProt Q7G602 as examples of CYP75B3, as well as 107 orthologs in table 1 (paragraph [0064]). The specification describes 111 amino acid sequences of CYP75B3 or CYP75B4 proteins in 16 different species, SEQ ID NOs: 1, 3, 5, 7 & 14-120. Of these, SEQ ID NO: 35 is identical to SEQ ID NO: 31, and the next nearest sequence, SEQ ID NO: 38, has only 93.9% sequence identity to SEQ ID NO: 31 and is also a sequence from maize. See alignment below. Thus, the specification does not describe species over the full scope of polypeptides comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 31.
US-18-015-229-38
Sequence 38, US/18015229
GENERAL INFORMATION
APPLICANT: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
TITLE OF INVENTION: PLANT METABOLITE-MEDIATED INDUCTION OF BIOFILM FORMATION IN SOIL
TITLE OF INVENTION: BACTERIA TO INCREASE BIOLOGICAL NITROGEN FIXATION AND PLANT
TITLE OF INVENTION: NITROGEN ASSIMILATION
FILE REFERENCE: 081906-1252964(GENERIC)
CURRENT APPLICATION NUMBER: US/18/015,229
CURRENT FILING DATE: 2023-01-09
PRIOR APPLICATION NUMBER: 63/051,267
PRIOR FILING DATE: 2020-07-13
NUMBER OF SEQ ID NOS: 151
SEQ ID NO 38
LENGTH: 491
TYPE: PRT
ORGANISM: Zea mays
Query Match 93.9%; Score 2496; Length 491;
Best Local Similarity 94.8%;
Matches 488; Conservative 1; Mismatches 2; Indels 24; Gaps 1;
Qy 1 MDVPLPLLLGSVAVSLVVWCLLLRRGGAGKGKRPLPPGPRGWPVLGNLPQVGAKPHHTMC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MDVPLPLLLGSVAVSLVVWCLLLRRGGAGKGKRPLPPGPRGWPVLGNLPQVGAKPHHTMC 60
Qy 61 AMAREYGPLFRLRFGSAEVVVAASARVAAQFLRAHDANFSNRPPNSGAEHVAYNYQDLVF 120
|:||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 ALAREYGPLFRLRFGSAEVVVAASARVAAQFLRAHDANFSNRPPNSGAEHVAYNYQDLVF 120
Qy 121 APYGSRWRALRKLCALHLFSAKALDDLRGVREGEVALMVRELARQGERGRAAVALGQVAN 180
|||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||
Db 121 APYGSRWRALRKLCALHLFSAKALDDLRGVREGEVALMVRELARQGERERAAVALGQVAN 180
Qy 181 VCATNTLARATVGRRVFAVDGGEGAREFKEMVVELMQLAGVFNVGDFVPALAWLDPQGVV 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 VCATNTLARATVGRRVFAVDGGEGAREFKEMVVELMQLAGVFNVGDFVPALAWLDPQGVV 240
Qy 241 GRMKRLHRRYDDMMNGIIRERKAAEEGKDLLSVLLARMREQQPLAEGDDTRFNETDIKAL 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 GRMKRLHRRYDDMMNGIIRERKAAEEGKDLLSVLLARMREQQPLAEGDDTRFNETDIKAL 300
Qy 301 LLNLFTAGTDTTSSTVEWALAELIRHPDVLRKAQQELDAVVGRDRLVSESDLPRLTYLTA 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 LLNLFTAGTDTTSSTVEWALAELIRHPDVLRKAQQELDAVVGRDRLVSESDLPRLTYLTA 360
Qy 361 VIKETFRLHPSTPLSLPRVAAEECEVDGFRIPAGTTLLVNVWAIA RDPEAWPEPLEFRPA 420
|||||||||||||||||||||||| |||||||||||
Db 361 VIKETFRLHPSTPLSLPRVAAEEC------------------------EAWPEPLEFRPG 396
Qy 421 RFLPGGSHAGVDVKGSDFELIPFGAGRRICAGLSWGLRMVTLMTATLVHALDWDLADGMT 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 397 RFLPGGSHAGVDVKGSDFELIPFGAGRRICAGLSWGLRMVTLMTATLVHALDWDLADGMT 456
Qy 481 ADKLDMEEAYGLTLQRAVPLMVRPAPRLLPSAYAE 515
|||||||||||||||||||||||||||||||||||
Db 457 ADKLDMEEAYGLTLQRAVPLMVRPAPRLLPSAYAE 491
The instant specification teaches that CYP75B3 is a hydroxylase involved in the conversion of apigenin to luteolin (paragraph [0063]).
The art describes CYP75B3 genes with at least 95% sequence identity to SEQ ID NO: 31, but not in species other than maize. UniProt record A0A811QCC1_9POAL (available 9/29/2021) describes a Miscanthus lutarioriparius flavonoid 3'-monooxygenase gene with 93.9% sequence identity to SEQ ID NO: 31. See alignment below.
A0A811QCC1_9POAL
ID A0A811QCC1_9POAL Unreviewed; 514 AA.
AC A0A811QCC1;
DT 29-SEP-2021, integrated into UniProtKB/TrEMBL.
DT 29-SEP-2021, sequence version 1.
DT 18-JUN-2025, entry version 16.
DE RecName: Full=flavonoid 3'-monooxygenase {ECO:0000256|ARBA:ARBA00066562};
DE EC=1.14.14.82 {ECO:0000256|ARBA:ARBA00066562};
GN ORFNames=NCGR_LOCUS37378 {ECO:0000313|EMBL:CAD6253756.1};
OS Miscanthus lutarioriparius.
OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
OC Spermatophyta; Magnoliopsida; Liliopsida; Poales; Poaceae; PACMAD clade;
OC Panicoideae; Andropogonodae; Andropogoneae; Saccharinae; Miscanthus.
OX NCBI_TaxID=422564 {ECO:0000313|EMBL:CAD6253756.1, ECO:0000313|Proteomes:UP000604825};
RN [1] {ECO:0000313|EMBL:CAD6253756.1}
RP NUCLEOTIDE SEQUENCE.
RA Han B., Lu T., Zhao Q., Huang X., Zhao Y.;
RL Submitted (OCT-2020) to the EMBL/GenBank/DDBJ databases.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=a 3'-unsubstituted flavone + reduced [NADPH--hemoprotein
CC reductase] + O2 = a 3'-hydroxyflavone + oxidized [NADPH--hemoprotein
CC reductase] + H2O + H(+); Xref=Rhea:RHEA:16337, Rhea:RHEA-COMP:11964,
CC Rhea:RHEA-COMP:11965, ChEBI:CHEBI:15377, ChEBI:CHEBI:15378,
CC ChEBI:CHEBI:15379, ChEBI:CHEBI:27741, ChEBI:CHEBI:57618,
CC ChEBI:CHEBI:58210, ChEBI:CHEBI:138726; EC=1.14.14.82;
CC Evidence={ECO:0000256|ARBA:ARBA00052910};
CC -!- COFACTOR:
CC Name=heme; Xref=ChEBI:CHEBI:30413;
CC Evidence={ECO:0000256|ARBA:ARBA00001971,
CC ECO:0000256|PIRSR:PIRSR602401-1};
CC -!- PATHWAY: Secondary metabolite biosynthesis; flavonoid biosynthesis.
CC {ECO:0000256|ARBA:ARBA00004966}.
CC -!- SUBCELLULAR LOCATION: Membrane {ECO:0000256|ARBA:ARBA00004167}; Single-
CC pass membrane protein {ECO:0000256|ARBA:ARBA00004167}.
CC -!- SIMILARITY: Belongs to the cytochrome P450 family.
CC {ECO:0000256|ARBA:ARBA00010617, ECO:0000256|RuleBase:RU000461}.
CC -!- CAUTION: The sequence shown here is derived from an EMBL/GenBank/DDBJ
CC whole genome shotgun (WGS) entry which is preliminary data.
CC {ECO:0000313|EMBL:CAD6253756.1}.
CC ---------------------------------------------------------------------------
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DR EMBL; CAJGYO010000009; CAD6253756.1; -; Genomic_DNA.
DR AlphaFoldDB; A0A811QCC1; -.
DR OrthoDB; 2789670at2759; -.
DR Proteomes; UP000604825; Unassembled WGS sequence.
DR GO; GO:0020037; F:heme binding; IEA:InterPro.
DR GO; GO:0005506; F:iron ion binding; IEA:InterPro.
DR GO; GO:0004497; F:monooxygenase activity; IEA:UniProtKB-KW.
DR GO; GO:0016705; F:oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen; IEA:InterPro.
DR FunFam; 1.10.630.10:FF:000056; Red aleurone1; 1.
DR Gene3D; 1.10.630.10; Cytochrome P450; 1.
DR InterPro; IPR001128; Cyt_P450.
DR InterPro; IPR017972; Cyt_P450_CS.
DR InterPro; IPR002401; Cyt_P450_E_grp-I.
DR InterPro; IPR036396; Cyt_P450_sf.
DR PANTHER; PTHR47944; CYTOCHROME P450 98A9; 1.
DR PANTHER; PTHR47944:SF18; FLAVONOID 3'-MONOOXYGENASE; 1.
DR Pfam; PF00067; p450; 1.
DR PRINTS; PR00463; EP450I.
DR PRINTS; PR00385; P450.
DR SUPFAM; SSF48264; Cytochrome P450; 1.
DR PROSITE; PS00086; CYTOCHROME_P450; 1.
PE 3: Inferred from homology;
KW Flavonoid biosynthesis {ECO:0000256|ARBA:ARBA00023241};
KW Heme {ECO:0000256|ARBA:ARBA00022617, ECO:0000256|PIRSR:PIRSR602401-1};
KW Iron {ECO:0000256|ARBA:ARBA00023004, ECO:0000256|PIRSR:PIRSR602401-1};
KW Metal-binding {ECO:0000256|ARBA:ARBA00022723,
KW ECO:0000256|PIRSR:PIRSR602401-1};
KW Monooxygenase {ECO:0000256|ARBA:ARBA00023033,
KW ECO:0000256|RuleBase:RU000461}; NADP {ECO:0000256|ARBA:ARBA00022857};
KW Oxidoreductase {ECO:0000256|ARBA:ARBA00023002,
KW ECO:0000256|RuleBase:RU000461};
KW Reference proteome {ECO:0000313|Proteomes:UP000604825}.
FT BINDING 448
FT /ligand="heme"
FT /ligand_id="ChEBI:CHEBI:30413"
FT /ligand_part="Fe"
FT /ligand_part_id="ChEBI:CHEBI:18248"
FT /note="axial binding residue"
FT /evidence="ECO:0000256|PIRSR:PIRSR602401-1"
SQ SEQUENCE 514 AA; 56270 MW; 374BD99C2E288838 CRC64;
Query Match 93.9%; Score 2496; Length 514;
Best Local Similarity 94.2%;
Matches 485; Conservative 13; Mismatches 13; Indels 4; Gaps 2;
Qy 1 MDVPLPLLLGSVAVSLVVWCLLLRRGGAG-KGKRPLPPGPRGWPVLGNLPQVGAKPHHTM 59
|||||||||||:|||:||||||||||| | |||||||||||||||||||||||: |||||
Db 1 MDVPLPLLLGSLAVSVVVWCLLLRRGGDGKKGKRPLPPGPRGWPVLGNLPQVGSHPHHTM 60
Qy 60 CAMAREYGPLFRLRFGSAEVVVAASARVAAQFLRAHDANFSNRPPNSGAEHVAYNYQDLV 119
||:|:|||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 CALAKEYGPLFRLRFGSAEVVVAASARVAAQFLRAHDANFSNRPPNSGAEHVAYNYQDLV 120
Qy 120 FAPYGSRWRALRKLCALHLFSAKALDDLRGVREGEVALMVRELARQGERGRAAVALGQVA 179
||||||||||||||||||||||||||||||||||||||||||||| | | |||||
Db 121 FAPYGSRWRALRKLCALHLFSAKALDDLRGVREGEVALMVRELAR---HQLAPVVLGQVA 177
Qy 180 NVCATNTLARATVGRRVFAVDGGEGAREFKEMVVELMQLAGVFNVGDFVPALAWLDPQGV 239
|||| ||||||||||||||||||| |||||:|||||||||||||||||||||||||||||
Db 178 NVCAINTLARATVGRRVFAVDGGEEAREFKDMVVELMQLAGVFNVGDFVPALAWLDPQGV 237
Qy 240 VGRMKRLHRRYDDMMNGIIRERKAAEEGKDLLSVLLARMREQQPLAEGDDTRFNETDIKA 299
||:|||||||||||||||||||||| ||||||||||||||||||||:|:|:| |||||||
Db 238 VGKMKRLHRRYDDMMNGIIRERKAAGEGKDLLSVLLARMREQQPLADGEDSRINETDIKA 297
Qy 300 LLLNLFTAGTDTTSSTVEWALAELIRHPDVLRKAQQELDAVVGRDRLVSESDLPRLTYLT 359
|||||||||||||||||||||||||||||||:||||||||||||||||||:|||||||||
Db 298 LLLNLFTAGTDTTSSTVEWALAELIRHPDVLKKAQQELDAVVGRDRLVSETDLPRLTYLT 357
Qy 360 AVIKETFRLHPSTPLSLPRVAAEECEVDGFRIPAGTTLLVNVWAIA RDPEAWPEPLEFRP 419
||||||||||||||||||||||||||||||||||||||||||||||||||||||||:|||
Db 358 AVIKETFRLHPSTPLSLPRVAAEECEVDGFRIPAGTTLLVNVWAIA RDPEAWPEPLQFRP 417
Qy 420 ARFLPGGSHAGVDVKGSDFELIPFGAGRRICAGLSWGLRMVTLMTATLVHALDWDLADGM 479
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 418 GRFLPGGSHAGVDVKGSDFELIPFGAGRRICAGLSWGLRMVTLMTATLVHALDWDLADGM 477
Qy 480 TADKLDMEEAYGLTLQRAVPLMVRPAPRLLPSAYA 514
|| ||||||||||||||||||||||||||||||||
Db 478 TAGKLDMEEAYGLTLQRAVPLMVRPAPRLLPSAYA 512
Cytochrome P450 (CYP) enzymes form a superfamily of plant genes known in the art, and CYP75B genes perform diverse functions in plant growth and development (Xiao et al (2021). PeerJ. 9:e12174 (published 9/15/2021, after the effective filing date of the instant application; hereafter Xiao), page 2, paragraph 2 & page 11, paragraph 3). CYP450s have a heme-binding region, a PERF motif, a K-helix region, and an I-helix region (Xiao page 5, paragraph 5), but the presence of other motifs in CYP75B homologs varies (Xiao figure 2). Substrate specificities are determined near the N-terminal end (Xiao page 11, paragraph 2).
Flavonoid 3’-hydroxylase (F3'H) is a poorly understood flavonoid biosynthetic gene in monocot plants, and CYP75B3 is classified as a Class 1 F3’H (Jia et al (2019) BMC Plant Biology. 19:347, published 8/8/2019, hereafter Jia; page 12 right column, paragraph 2). Class I and Class II F3'H have divergent expression profiles (page 13 left column, paragraph 2) and different substrate specificity (Jia, page 2, right column, paragraph 2). In addition, different species have different numbers of F3'H genes; Zea mays and sorghum bicolor have 3 and 5 respectively (page 3, left column, paragraph 1).
Plants other than maize comprising a CYP75B3 gene that is at least 95% identical to SEQ ID NO: 31, especially an endogenous gene, are not known in the art or described in the instant specification. One of skill in the art would not recognize that Applicant was in possession of the necessary common attributes or features of the genus in view of the disclosed species.
Because plants comprising CYP75B3 genes that encode a polypeptide with 95% sequence identity to SEQ ID NO: 31 are not described over the full scope of the claims, the method of using the sequences to increase the ability of a crop plant to assimilate nitrogen is likewise not described, and the specification fails to provide an adequate written description of the claimed invention. Therefore, given the lack of written description in the specification, Applicant does not appear to have been in possession of the claimed genus at the time this application was filed.
Applicant urges that a patent specification must describe the claimed invention in sufficient detail such that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention. Applicant urges that the amended claims no longer recite homolog or ortholog, so these limitations are no longer relevant. Applicant urges that 95% sequence identity does reasonably define subject matter a person of skill in the art would believe the inventors had in their possession, because CYP75B3 genes in different species are known in the art. Applicant urges that one of skill in the art would recognize that the inventors could have readily obtained a functional CYP75B3 protein at least 95% identical to SEQ ID NO: 31 by modifying SEQ ID NO: 31 guided by information derived from sequence alignment of known CYP75B3 proteins (Remarks, page 6, paragraph 3-page 7, paragraph 3).
This argument is unpersuasive, because the amended claims require a crop plant comprising a CYP75B3 gene encoding a protein with 95% sequence identity to SEQ ID NO: 31, not merely a homolog or ortholog. However, species other than maize comprising a CYP75B3 gene encoding a protein with at least 95% sequence identity to SEQ ID NO: 31 are not known in the art or described by Applicant. One of skill in the art would not have recognized Applicant to be in possession of a crop plant comprising a CYP75B3 gene over the full scope of the encompassed species. Whether or not Applicant could have readily obtained a functional CYP75B3 protein at least 95% identical to SEQ ID NO: 31 by modifying SEQ ID NO: 31 is a question of Enablement, not Written Description. Regardless, the claims are not drawn to a CYP75B3 gene in isolation, but to a CYP75B3 gene in a crop plant, with reduced or modified expression, amount, or enzymatic activity. The originally filed specification does not demonstrate possession of the invention over the full scope of the claims.
New Matter
Claim 15 is 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 Matter Rejection.
Claim 15 requires the guide RNA comprises a target sequence that is at least 95% identical to a 20-nucleotide sequence within SEQ ID NO: 9.
The instant specification provides support for a guide RNA comprising a target sequence substantially identical to SEQ ID NOs: 11-13 or to a sequence within SEQ ID NO: 9 or 10 (paragraph [0014 & 0064-0065]). Figure 18 describes SEQ ID NOs: 11-13 and 146-151, although SEQ ID NOs: 149-151 are target sites in CYP75B4 rather than CYP75B3. These sequences are of varying lengths, not necessarily 20 nucleotides. The originally filed specification does not provide support for the genus of target sequences at least 95% identical to 20-nucleotide sequences within SEQ ID NO: 9. Because the genus of target sequences at least 95% identical to 20-nucleotide sequences within SEQ ID NO: 9 is not the same as the genus of target sequences found within SEQ ID NO: 9, or the same as the originally required target sequence substantially identical e.g. comprising 0, 1, 2, or 3 mismatches to a sequence within SEQ ID NO: 9 or 10, this limitation constitutes New Matter. In response to this rejection, Applicant is required to point specifically to support for the concept in the specification or to cancel the new matter.
Scope of Enablement
Claims 1, 3-6, 8-9, 14-15, 27-33 & 35-36 rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for maize crop plants comprising an a CYP75B3 gene of SEQ ID NO: 31, does not reasonably provide enablement for any crop plant or any method comprising reducing or modifying expression of a gene with 95% sequence identity to SEQ ID NO: 31. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims.
Claims 1, 3-6, 8-9, 14-15, 27-33 & 35-36 are drawn to or require a crop plant in which expression of a CYP75B3 gene encoding a polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 31 has been modified. Claims 27, 30 & 36 require that the plant is selected from the group consisting of corn, wheat, rice, soy, cotton, canola, and sugarcane. Claim 33 requires that the crop plant is rice. Claims 6, 8, 9, 14 & 15 further require that an endogenous copy of the gene is modified, while claims 29-30 & 35 require a mutation or deletion in a CYP75B3 gene. The claims all require a plant comprising a CYP75B3 gene encoding a polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 31.
Genes encoding CYP75B3 polypeptides with 95% identity to 515 amino acid-long SEQ ID NO: 31 encompass those encoding polypeptides with 25 amino acid substitutions relative to SEQ ID NO: 31.
The instant specification teaches examples of CYP75B3, including NCBI accession number AK064736 and UniProt Q7G602, as well as 107 orthologs in table 1 (paragraph [0064]). The specification teaches 111 amino acid sequences of CYP75B3 or CYP75B4 proteins in 16 different species, SEQ ID NOs: 1, 3, 5, 7 & 14-120. Of these, SEQ ID NO: 35 is identical to SEQ ID NO: 31, and the next nearest sequence, also a sequence from maize, SEQ ID NO: 38, has only 93.9% sequence identity to SEQ ID NO: 31. See alignment above. Thus, the specification does not teach examples of crop plants or even of maize crop plants comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 31 over the full scope of the claims.
CYP75B3 genes with at least 95% sequence identity to SEQ ID NO: 31 are known in the art, but not in species other than maize. UniProt record A0A811QCC1_9POAL (available 9/29/2021) teaches a Miscanthus lutarioriparius flavonoid 3'-monooxygenase gene with 93.9% sequence identity to SEQ ID NO: 31. See alignment above.
Flavonoid 3’-hydroxylase (F3'H) is a poorly understood flavonoid biosynthetic gene in monocot plants, and CYP75B3 is classified as a Class 1 F3’H (Jia et al (2019) BMC Plant Biology. 19:347, published 8/8/2019, hereafter Jia; page 12 right column, paragraph 2). Class I and Class II F3'H have divergent expression profiles (page 13 left column, paragraph 2) and different substrate specificity (Jia, page 2, right column, paragraph 2). In addition, different species have different numbers of F3'H genes; Zea mays and sorghum bicolor have 3 and 5 respectively (page 3, left column, paragraph 1).
Because crop plants other than maize comprising a CYP75B3 gene that is at least 95% identical to SEQ ID NO: 31, especially an endogenous gene, are not known in the art and representative working examples are not taught in the instant specification over the full scope of the genus, one of ordinary skill in the art would be required to screen crop plants other than maize to find any that are encompassed, if such plants even exist. Alternatively, one of ordinary skill in the art would be required to generate such crop plants and to modify, reduce, and/or eliminate expression or reduce the amount or enzymatic activity of the CYP75B3. Given the poor understanding of flavonoid biosynthetic genes in monocots, it would require undue trial and error experimentation to generate such plants. Thus, the claims are not enabled over the full scope of the claims.
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.
Claim(s) 1, 3-4 & 27 are rejected under 35 U.S.C. 103 as being unpatentable over Sharma et al (2012) BMC Plant Biology. 12: 196. (published 11/1/2012, hereafter Sharma) taken with the evidence of NCBI Reference Sequence NP_001141292.2 (available 9/3/2021, after the priority filing date of the instant application).
Claims 1, 3-4 & 27 are drawn to a method comprising modifying the expression of a CYP75B3 gene at least 95% identical to SEQ ID NO: 31.
Sharma teaches that the maize red aleurone1 (pr1) gene encodes a CYP450-dependent flavonoid 3’-hydroxylase (ZmF3’H1) required for biosynthesis of anthocyanin pigments (abstract). Pr1 is also likely involved in the biosynthetic pathway for apigeninidin and luteolinidin (figure 1). Sharma teaches pr1 mutant maize lines (table 1). Sharma also teaches that Zmf3’h1 is regulated by P1 (page 5, right column, paragraph 2) and teaches mutants of P1 that have reduced luteolinidin glycosides and anthocyanidin levels (table 1, figure 6B).
Sharma teaches a method of crossing pr1-MGS14273 plants with P1-ww, P1-rr-4B2-P1-ww and p-del2 lines and selfing the progeny to recover populations segregating for purple and red aleurones; homozygous stocks were developed by six subsequent cycles of self-pollination and selection (page 10, left column, paragraph 1).
Sharma teaches that the pr1 locus is used as a phenotypic marker in maize genetics because of the role in determining kernel aleurone color (page 7, right column, paragraph 2). Sharma teaches that a functional Zmf3’h1 participates in the accumulation of 3’-hydroxylated C-glycosyl flavones (page 7, left column, paragraph 2 & right column, paragraph 1; figure 7).
NCBI Reference Sequence NP_001141292.2 provides evidence that red aleurone1 is a CYP75B3 gene with a sequence that is identical to SEQ ID NO: 31. See alignment below.
Flavonoid 3'-monooxygenase CYP75B3 [Zea mays]
Sequence ID: NP_001141292.2Length: 515Number of Matches: 2
Alignment statistics for match #1
Score
Expect
Method
Identities
Positives
Gaps
1035 bits(2677)
0.0
Compositional matrix adjust.
515/515(100%)
515/515(100%)
0/515(0%)
Query 1 MDVPLPLLLGSVAVSLVVWCLLLRRGGAGKGKRPLPPGPRGWPVLGNLPQVGAKPHHTMC 60
MDVPLPLLLGSVAVSLVVWCLLLRRGGAGKGKRPLPPGPRGWPVLGNLPQVGAKPHHTMC
Sbjct 1 MDVPLPLLLGSVAVSLVVWCLLLRRGGAGKGKRPLPPGPRGWPVLGNLPQVGAKPHHTMC 60
Query 61 AMAREYGPLFRLRFGSAEVVVAASARVAAQFLRAHDANFSNRPPNSGAEHVAYNYQDLVF 120
AMAREYGPLFRLRFGSAEVVVAASARVAAQFLRAHDANFSNRPPNSGAEHVAYNYQDLVF
Sbjct 61 AMAREYGPLFRLRFGSAEVVVAASARVAAQFLRAHDANFSNRPPNSGAEHVAYNYQDLVF 120
Query 121 APYGSRWRALRKLCALHLFSAKALDDLRGVREGEVALMVRELARQGERGRAAVALGQVAN 180
APYGSRWRALRKLCALHLFSAKALDDLRGVREGEVALMVRELARQGERGRAAVALGQVAN
Sbjct 121 APYGSRWRALRKLCALHLFSAKALDDLRGVREGEVALMVRELARQGERGRAAVALGQVAN 180
Query 181 VCATNTLARATVGRRVFAVDGGEGAREFKEMVVELMQLAGVFNVGDFVPALAWLDPQGVV 240
VCATNTLARATVGRRVFAVDGGEGAREFKEMVVELMQLAGVFNVGDFVPALAWLDPQGVV
Sbjct 181 VCATNTLARATVGRRVFAVDGGEGAREFKEMVVELMQLAGVFNVGDFVPALAWLDPQGVV 240
Query 241 GRMKRLHRRYDDMMNGIIRERKAAEEGKDLLSVLLARMREQQPLAEGDDTRFNETDIKAL 300
GRMKRLHRRYDDMMNGIIRERKAAEEGKDLLSVLLARMREQQPLAEGDDTRFNETDIKAL
Sbjct 241 GRMKRLHRRYDDMMNGIIRERKAAEEGKDLLSVLLARMREQQPLAEGDDTRFNETDIKAL 300
Query 301 LLNLFTAGTDTTSSTVEWALAELIRHPDVLRKAQQELDAVVGRDRLVSESDLPRLTYLTA 360
LLNLFTAGTDTTSSTVEWALAELIRHPDVLRKAQQELDAVVGRDRLVSESDLPRLTYLTA
Sbjct 301 LLNLFTAGTDTTSSTVEWALAELIRHPDVLRKAQQELDAVVGRDRLVSESDLPRLTYLTA 360
Query 361 VIKETFRLHPSTPLSLPRVAAEECEVDGFRIPAGTTLLVNVWAIA RDPEAWPEPLEFRPA 420
VIKETFRLHPSTPLSLPRVAAEECEVDGFRIPAGTTLLVNVWAIA RDPEAWPEPLEFRPA
Sbjct 361 VIKETFRLHPSTPLSLPRVAAEECEVDGFRIPAGTTLLVNVWAIA RDPEAWPEPLEFRPA 420
Query 421 RFLPGGSHAGVDVKGSDFELIPFGAGRRICAGLSWGLRMVTLMTATLVHALDWDLADGMT 480
RFLPGGSHAGVDVKGSDFELIPFGAGRRICAGLSWGLRMVTLMTATLVHALDWDLADGMT
Sbjct 421 RFLPGGSHAGVDVKGSDFELIPFGAGRRICAGLSWGLRMVTLMTATLVHALDWDLADGMT 480
Query 481 ADKLDMEEAYGLTLQRAVPLMVRPAPRLLPSAYAE 515
ADKLDMEEAYGLTLQRAVPLMVRPAPRLLPSAYAE
Sbjct 481 ADKLDMEEAYGLTLQRAVPLMVRPAPRLLPSAYAE 515
Before the filing of the instant application, it would have been obvious to one of skill in the art to generate pr1 null mutants in various maize backgrounds, including maize comprising a red aleurone1 gene encoding a protein identical to SEQ ID NO: 31. One of ordinary skill in the art would have been motivated to generate a pr1 null mutant to be used as a phenotypic marker. One of ordinary skill in the art would have had reasonable expectation of success, because the pr1 gene was known as well as null mutants, and some maize cultivars would have comprised a gene encoding a sequence of SEQ ID NO: 31.
Instant claim 1 is a method of increasing the ability of a crop plant to assimilate atmospheric nitrogen comprising modifying the expression of a CYP75B3 gene to reduce or eliminate its expression. Crossbreeding to recover populations with a pr1 null mutations reads on eliminating expression of this gene, which has a sequence of >95% identity to instant SEQ ID NO: 31 in at least some maize lines. Red aleurone1 is involved in flavone biosynthesis or degradation in cells of the plant such that the plant produces an increased amount of one or more flavones. Although Sharma is silent regarding exudation of the flavones and biofilm formation in N2-fixing bacteria present in the soil in proximity to the plant’s roots, these are intended results which would be inherent to the only listed active step of the method, which is to reduce or eliminate expression of the CYP75B3 gene. Similarly, the biofilm formation leading to an increase in the ability of the bacteria to fix atmospheric nitrogen and the nitrogen assimilated by the plant (instant claim 3) is a result that would follow from the positive active steps of Sharma. At least one of the flavones is glycosylated (claim 4). The plant is corn (instant claim 27).
Thus, the method of producing pr1 null mutant lines of maize reads on a method of instant claims 1, 3-4 & 27, and it would have been obvious to perform this method in other common maize lines such as those comprising the gene encoding SEQ ID NO: 31.
Claim(s) 1, 3-6, 9, 13 & 27-30 are rejected under 35 U.S.C. 103 as being unpatentable over Lam et al (2019). New Phytologist. 223: 204–219 (published 3/18/2019, hereafter Lam 2019) in view of Casas et al (2014) Frontiers in Plant Science. 5. 440. (Published 9/8/2014, hereafter Casas) and Spini et al (2016) Plant Soil. 399: 159–178 (published 9/30/2015, hereafter Spini), taken with the evidence of NCBI Reference Sequence NP_001141292.2 (available 9/3/2021, after the priority filing date of the instant application) and NCBI reference Gene ID 100273383 (accessed 7/6/2026).
This is a new rejection, necessitated by Applicant’s amendments. Applicant’s remarks filed 3/24/2026 have been considered below as they pertain to the instant rejection, but they are not persuasive.
Claims 1, 3-6, 9, 13 & 27-30 are drawn to a method of increasing the ability of a crop to assimilate atmospheric nitrogen comprising modifying the expression of a CYP75B3 gene and the plant made from this method.
Lam 2019 teaches a transfer DNA insertion mutant in rice for the cyp75b4 gene, as well as a cyp75b3 mutant and a cyp75b3 cyp75b4 double mutant generated by introducing a binary vector comprising single-guide RNAs targeting the genes and a Cas9 gene into rice calli (page 206, left column, paragraph 2). Lam 2019 teaches that these genes are involved in flavone biosynthesis (figure 1) and that mutant cyp75b3 plants produce an increased amount of apigenin and apigenin C-hexosides and apigenin C-hexosides C pentosides, which are flavone C-glycosides, while cyp75b4 mutant plants accumulate luteolin compared to wild-type plants (page 208, right column, paragraph 2-page 209, left column, paragraph 2; figure 3 & figure S5). Double mutants had significantly more extractable apigenin (figure S8). Lam 2019 discloses that the rice CYP75B3 gene groups phylogenetically with a maize gene GRMZM2G025832 (figure S18). Lam teaches that CYP75B3-like proteins are highly conserved in grasses and teaches that a maize Zmf3’h1 mutant was depleted in 3’-hydroxylated flavone C-glycosides and may function similar to the role of CYP75B3 in rice (page 213, right column, paragraph 3-page 214, left column, paragraph 1).
Lam 2019 teaches a single guide RNA targeting 5’-GTACACAAGGTACCACACGG-3’ for the CRISPR/Cas9 mutation of cyp75b3 (page 206, left column, paragraph 2). The sequence of this target is found on the reverse of instant SEQ ID NO: 9, which reads on the guide RNA comprising a target sequence that is at least 95% identical to a sequence within SEQ ID NO: 9 (claim 15). See alignment below.
Score
Expect
Identities
Gaps
Strand
40.1 bits(20)
1e-07
20/20(100%)
0/20(0%)
Plus/Minus
Query 100 CCGTGTGGTACCTTGTGTAC 119
||||||||||||||||||||
Sbjct 20 CCGTGTGGTACCTTGTGTAC 1
Lam 2019 does not teach the method in maize, or a crop plant comprising a CYP75B3 gene encoding a protein of SEQ ID NO: 31, or a guide RNA with a target sequence of instant SEQ ID NO: 13. Lam 2019 does not explicitly teach that exuded flavones lead to biofilm formation in nitrogen fixing bacteria in proximity to the plant’s roots.
Casas teaches a motivation to increase flavone production in maize, because flavones are associated with the beneficial effects of the Mediterranean diet. Apigenin especially exhibits potent anti-angiogenic, anti-inflammatory and anti-carcinogenic activities (page 2, left column, paragraph 1). Casas teaches that maize is one of the most important cereal crops worldwide, but breeding has lost many of the phytochemicals important for plant protection, human, and livestock nutrition (page 1, right column, paragraph 2). Casas teaches biosynthetic pathways of flavone and flavonoids in maize, including apigenin, apigenin-7-O-glucoside, and luteolin (figure 1).
Spini teaches that Ensifer meliloti is a nitrogen-fixing bacterium found in temperate soils worldwide (page160, right column, paragraph 3). Plant roots release a cocktail of nodulation-inducing molecules at the start of symbiosis, and luteolin is an active inducer of E. meliloti (page 160, right column, paragraph 3-page 161, left column, paragraph 1). Spini teaches that E. meliloti 3001 has a significant increase in biofilm formation in the presence of luteolin on LB medium (page 169, right column, paragraph 2; figure 6a). Spini teaches that the biofilm formation can increase survival under unfavorable conditions and plays a role in colonization and host invasion (page 169, right column, paragraph 2). Spini also teaches that luteolin induces expression of iron metabolism genes and stimulates siderophore production, which is important to nitrogen fixation in E. meliloti because enzymes related to nitrogen fixation contain iron cofactors (page 173, right column, paragraph 3-page 174, left column, paragraph 1 & page 170, left column, paragraph 2). Spini mentions that Ensifer meliloti was formerly known as Sinorhizobium meliloti (page 160, right column, paragraph 2).
NCBI Gene ID 100273383 provides evidence that the locus known as GRMZM2G025832 encodes a protein with a reference sequence of NP_001141292.2.
NCBI Reference Sequence NP_001141292.2 provides evidence that the protein encoded by GRMZM2G025832 comprises a CYP75B3 gene with a sequence that is identical to SEQ ID NO: 31. See alignment above.
Before the filing of the instant application, it would have been obvious to one of skill in the art to modify the method of Lam 2019 to knockout a CYP75B3 and CYP75B4 gene in maize. One of ordinary skill would have been motivated to knockout a maize CYP75B3 and CYP75B4 gene in order to increase flavonoids like apigenin, which has health benefits. One of ordinary skill would have had reasonable expectation of success, because Lam 2019 taught a homologous maize gene and that CYP75B3 proteins are highly conserved in grasses.
Use of a maize plant of one genetic background over another would have been obvious, and so a maize CYP75B3 knockout having a CYP75B3 gene encoding a sequence at least 95% identical to instant SEQ ID NO: 31 or even 100% identical would have been obvious.
Before the time of filing of the instant application, it would have been obvious to one of ordinary skill in the art to grow a cyp75b4/cyp75b3 mutant plant in soil, where Ensifer meliloti is found naturally. One of ordinary skill in the art would have been motivated to do so, because growing in soil would be a simple substitution of one known element for another to obtain predictable results. One of ordinary skill would have had reasonable expectation of success because maize is grown in soil in the field routinely. Because the cyp75b4 mutant produces increased luteolin, it would have been obvious to one of ordinary skill in the art that the flavone luteolin produced by the plant would induce biofilm formation in N2-fixing bacteria present in the soil.
Furthermore, before the time of filing of the instant application, it would have been obvious to one of ordinary skill in the art that the biofilm formation would increase the ability of the bacteria to fix atmospheric nitrogen if the bacteria and plant were present in stressful conditions, because biofilm formation was known to increase survival under stress. Living bacteria has a higher ability to fix atmospheric nitrogen than dead bacteria. It would likewise have been obvious that the fixed atmospheric nitrogen would be assimilated by the plant.
While Lam 2019 does not measure root exudate directly, an increase in apigenin C-hexosides, apigenin C-hexosides C-pentosides, or apigenin O-conjugates in mutant plants with reduced expression of cyp75b3 and cyp75b4 would inherently lead to exudation from the plant roots. No additional step in the biosynthetic pathway is required for excess apigenin to be exuded from roots. The instant specification demonstrates that excess apigenin and apigenin-7-glucoside in roots from a knockout of the cyp75b3 and cyp75b4 genes leads to increased apigenin in the root exudates (page 34, paragraph [0104]). Thus, a method of creating mutant cyp75b3 maize plants reads on the method of claims 1, 3, 4, 5, & 27 and the genetically modified crop plant of claims 28-30.
The method of creating the CRISPR/Cas9 mutant plants of Lam 2019 also reads on the method of claim 6, wherein expression is modified by introducing into the plant a guide RNA and an RNA-guided nuclease, and claims 9 & 13.
Thus, claims 1, 3-6, 9, 13 & 27-30 are obvious in view of Lam 2019, Casas, and Spini.
Applicant urges that the present claims recite the element of a polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 31 which is not disclosed nor suggested by Lam, Spini, or Zhang (Remarks, page 8, paragraphs 1-4).
This argument is unpersuasive, because Casas teaches a motivation to apply the method of Lam to maize to increase apigenin. Although Lam, Spini, and Casas do not teach the sequence of SEQ ID NO: 31, they do teach a maize gene that is a homolog of the CYP75B3 rice gene, and this gene encodes a polypeptide of SEQ ID NO: 31 in at least some cultivars of maize. Because the choice of maize cultivar is a design choice, the instant application would have been obvious prior to the filing of the instant application. In order to show that the instant application would not have been obvious in view of the prior art, Applicant would need to provide evidence that methods and maize plants comprising a CYP75B3 gene encoding a sequence 95% identical to SEQ ID NO: 31 provides unexpected results in comparison to methods and maize plants comprising a CYP75B3 gene that is not 95% identical to SEQ ID NO: 31.
Claims 15 is rejected under 35 U.S.C. 103 as being unpatentable over Lam 2019, Casas, and Spini, as applied to claims 1, 3-6, 9, 13 & 27-30 above, and further in view of NCBI reference sequence NM_001147820.1 (available 4/23/2017).
Claim 15 is drawn to the method wherein the guide RNA comprises a target sequence that is at least 95% identical to a 20-nucleotide sequence within SEQ ID NO: 9.
The teachings of Lam 2019, Casas, and Spini are presented above. They do not teach a guide RNA sequence comprising a target sequence that is at least 95% identical to a 20-nucleotide sequence within SEQ ID NO: 9 used in a method of modifying a maize endogenous CYP75B3 gene.
NCBI Reference Sequence NM_001147820.1 teaches a sequence of the GRMZM2G025832 locus that comprises sequences longer than 20 nucleotides with 100% identity to sequences in instant SEQ ID NO: 9. See examples in bold in the alignment below.
Sequence ID: Query_3932175Length: 1572Number of Matches: 577
Range 1: 27 to 693GraphicsNext MatchPrevious Match
Alignment statistics for match #1
Score
Expect
Identities
Gaps
Strand
599 bits(302)
1e-174
576/667(86%)
9/667(1%)
Plus/Plus
Query 189 TGGCCCGTGCTGGGCAACCTGCCGCAGCTCGGCGACAAGCCGCACCACACCATGTGCGCC 248
||||| ||||||||||||||||||||| |||||| ||||||||||||||||||||| ||
Sbjct 27 TGGCCGGTGCTGGGCAACCTGCCGCAGGTCGGCGCCAAGCCGCACCACACCATGTGTGCT 86
Query 249 CTGGCGCGGCAGTACGGCCCGCTGTTCCGGCTCCGGTTCGGCTGCGCCGAGGTGGTGGTG 308
|||||||| |||||||||||||||||||||||||||||||| |||||||||||||||||
Sbjct 87 ATGGCGCGGGAGTACGGCCCGCTGTTCCGGCTCCGGTTCGGCAGCGCCGAGGTGGTGGTG 146
Query 309 GCCGCGTCGGCGCCCGTGGCTGCGCAGTTCCTGCGCGGGCACGATGCCAACTTCAGCAAC 368
||||||||||||| ||||| || ||||||||||||| ||||| |||||||||||||||
Sbjct 147 GCCGCGTCGGCGCGGGTGGCGGCCCAGTTCCTGCGCGCCCACGACGCCAACTTCAGCAAC 206
Query 369 CGCCCGCCCAACTCGGGCGCCGAGCACGTCGCGTACAACTACCAGGACCTCGTCTTCGCG 428
|||||||||||||| || ||||||||||| |||||||||||||||||||| || |||||
Sbjct 207 CGCCCGCCCAACTCCGGGGCCGAGCACGTGGCGTACAACTACCAGGACCTGGTGTTCGCC 266
Query 429 CCCTACGGTGCTCGCTGGCGCGCCCTGCGGAAGCTGTGCGCGCTCCACCTCTTCTCGGCC 488
|| ||||| | || |||||||| || ||||||||||||||||||||||||||||| |||
Sbjct 267 CCGTACGGCTCCCGGTGGCGCGCGCTACGGAAGCTGTGCGCGCTCCACCTCTTCTCCGCC 326
Query 489 AAGGCGCTCGACGACCTCCGAGCAGTCCGGGAGGGCGAGGTCGCGCTCATGGTGAGGAAC 548
||||| || |||||||| || | ||| | ||||||||||| ||||||||||||||| |
Sbjct 327 AAGGCCCTGGACGACCTGCGCGGCGTCAGAGAGGGCGAGGTAGCGCTCATGGTGAGGGAG 386
Query 549 CTCGCTCGGCAG-------CAGG--CGGCGTCAGTGGCGCTGGGGCAGGAAGCGAACGTC 599
||||| |||||| ||| ||| | ||||||||||| |||| || ||||||
Sbjct 387 CTCGCCCGGCAGGGAGAGCGAGGACGGGCCGCCGTGGCGCTGGGCCAGGTGGCCAACGTC 446
Query 600 TGCGCCACGAACACGCTGGCCCGCGCCACCATCGGTCACCGGGTGTTCGCCGTCGACGGC 659
||||| || |||||||||||||| || ||| | || | || |||||||||||||||||
Sbjct 447 TGCGCGACCAACACGCTGGCCCGGGCGACCGTGGGCCGGCGCGTGTTCGCCGTCGACGGA 506
Query 660 GGGGAAGGCGCAAGGGAGTTCAAGGAGATGGTTGTGGAGCTGATGCAGCTCGCCGGCGTT 719
||||| ||||| |||||||||||||||||||| ||||||||||||||||||||||| ||
Sbjct 507 GGGGAGGGCGCCAGGGAGTTCAAGGAGATGGTGGTGGAGCTGATGCAGCTCGCCGGGGTC 566
Query 720 TTCAACGTCGGGGACTTCGTGCCGGCGCTCCGGTGGCTCGACCCGCAGGGCGTCGTGGCA 779
|||||||||||||||||||||||||||||| ||||||||||||||||||||| || |
Sbjct 567 TTCAACGTCGGGGACTTCGTGCCGGCGCTCGCGTGGCTCGACCCGCAGGGCGTGGTCGGC 626
Query 780 AAGATGAAGAGGCTGCACCGTCGGTACGACAACATGATGAACGGATTCATCAACGAAAGG 839
| ||||||| |||||||||| |||||||| ||||||||||||| |||||| || |||
Sbjct 627 AGGATGAAGCGGCTGCACCGCAGGTACGACGACATGATGAACGGGATCATCAGGGAGAGG 686
Query 840 AAGGCCG 846
|||||||
Sbjct 687 AAGGCCG 693
Before the filing of the instant application, it would have been obvious to one of ordinary skill in the art to modify the method of Lam 2019 to use a guide RNA comprising a target sequence at least 95% identical to a 20-nucleotide sequence within SEQ ID NO: 9. One of ordinary skill in the art would have been motivated to adjust the sequence of the guide RNA in order to target a maize CYP75B3 gene and through routine optimization could have arrived at a target sequence such as one of the sequences found in the sequence of the maize gene in bold above. One of ordinary skill in the art would have had reasonable expectation of success, because design of guide RNA target sequences was routine in the art prior to the instant filing and the maize homologous gene was known. Instant claim 15 would have been obvious.
Claims 31 & 35-36 are rejected under 35 U.S.C. 103 as being unpatentable over Lam 2019 Casas and Spini, as applied to claims 1, 3-6, 9, 13 & 27-30 above, and further in view of Zhang et al (2017) Plant Physiology and Biochemistry. 111: 30-38 (available online 11/18/2016, hereafter Zhang).
This is a new rejection in light of Applicant' s amendment of the claims. Applicant' s arguments filed 3/24/2026 in response to the Office action mailed 11/25/2025 have been fully considered as they apply to the new rejection, but they are not persuasive.
Claims 31 & 35-36 are drawn to a method of increasing assimilation of atmospheric nitrogen in a grain crop plant grown under reduced inorganic nitrogen conditions
The teachings of Lam 2019, Casas, and Spini are presented above. These references do not teach growing a crop plant in soil with a lower than recommended amount of inorganic nitrogen.
Zhang teaches that nitrogen limitation is a type of abiotic stress that plants respond to by synthesizing flavonoids (page 30, right column, paragraph 2-page 31, left column, paragraph 1). Zhang teaches that 6-C-glycosides of luteolin and apigenin are common in wheat, maize, barley, and other crops (page 36, right column, paragraph 4). Zhang teaches a motivation for plants to express flavonoids, in that the flavonoid pathway is related to abiotic stress, including in rice, and may play a role in defense (page 36, right column, paragraph 2). Flavonoids increase resistance to or reduce damage caused by abiotic stress and their derivatives play a role in resistance to abiotic stresses (page 36, right column, paragraph 1).
Zhang teaches a method of growing wheat under a low-nitrogen treatment (fertilized with 120kg/hm2) and under a normal-nitrogen treatment (225 kg/hm2) in soil with 56.0mg/kg available nitrogen, 0.83g/kg total nitrogen (page 31, left column, paragraph 6-right column, paragraph 1). Zhang teaches that low nitrogen conditions led to higher relative contents of apigenin 6-C glycoside, luteolin-6-C glucoside, Apigenin-6-C-arabinoside 8-C-hexoside, and other metabolites (page 36, left column, paragraph 4; table 2 & figure 3).
Before the effective filing date of the instant application, it would have been obvious to one of ordinary skill in the art to modify the method of Lam 2019 growing crop plant with a mutation in a cyp75b3 gene to grow in the field under low nitrogen conditions, as taught by Zhang. One of ordinary skill would have been motivated to grow the crop plant in the field under low nitrogen conditions, because both low nitrogen conditions and mutations in cyp75b3 lead to increased production of flavone C-glycosides. The rationale to make this modification would be combining prior art elements according to known methods to yield predictable results. One of ordinary skill would have been motivated to increase flavonoids in the plant because Zhang teaches that flavonoids provide protection against abiotic and biotic stresses. One of ordinary skill would have had reasonable expectation of success because 6-C-glycosides of luteolin and apigenin are common in cereal crops.
Thus, the method of providing a genetically modified rice crop plant with a mutation in cyp75b3 and growing the plant in soil comprising inorganic nitrogen lower than the recommended amount for the crop plant to produce an increased amount of one or more flavones (claims 31, 35 & 36) is obvious in view of Lam 2019, Casas, Spini, and Zhang. Claims 1, 3-6, 9, 13 & 27-30 are also obvious as presented above.
Applicant urges that Lam, Spini, and Zhang do not teach a gene encoding a polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 31, and so the references fail to teach all claim elements and the claims are not obvious (Remarks, page 8, paragraph 1-paragraph 4).
This argument is unpersuasive, because Casas teaches a motivation to increase flavonoid and apigenin in maize. Lam 2019 teaches the homologous maize gene to the rice CYP75B3 gene and teaches that the gene is conserved in grasses. Thus, the knockout of the maize gene would have been obvious prior to the instant filing date. Although the sequence of SEQ ID NO: 31 was not taught, this sequence is the sequence of the polypeptide encoded by the maize gene taught by Lam 2019 in at least some cultivars of maize. Because the choice of maize cultivar is a design choice, the instant application would have been obvious prior to the filing of the instant application.
In order to show that the instant application would not have been obvious in view of the prior art, Applicant would need to provide evidence that methods and maize plants comprising a CYP75B3 gene encoding a sequence 95% identical to SEQ ID NO: 31 provides unexpected results in comparison to methods and maize plants comprising a CYP75B3 gene that is not 95% identical to SEQ ID NO: 31.
Claim 32 is rejected under 35 U.S.C. 103 as being unpatentable over Lam 2019, Casas, Spini, and Zhang as applied to claims 1, 3-6, 9, 13, 27-31 & 35-36 above, and further in view of Morris et al (2018) Agronomy Journal. 110:1-37 (published 12/14/2017, hereafter Morris).
Claim 32 is drawn to the method wherein the amount of inorganic nitrogen is less than 50% of the standard or recommended amount for the crop plant.
The teachings of Lam 2019, Casas, Spini, and Zhang are presented above. They do not teach the amount of inorganic nitrogen is less than 50% of the recommended amount for a maize plant.
Morris teaches that most of the N fertilizer used in the United States is applied to corn, but too much N fertilizer pollutes air and water through volatilization, denitrification, leaching, and runoff (abstract). Morris teaches a motivation to reduce nitrogen applications, depending on prices of corn or nitrogen, because higher N application lowers profitability (page 13, left column, paragraph 1). The economic optimum nitrogen rate can differ greatly across sites (figure 10).
Before the filing of the instant application, it would have been obvious to grow a maize plant with reduced CYP75B3 in soil with less than 50% of the recommended inorganic nitrogen. One of ordinary skill in the art would have been motivated to grow the plant in soil comprising this much nitrogen in order to maintain profitability in response to high N prices or low maize prices. One of ordinary skill in the art would have had reasonable expectation of success, because maize is grown over a range of soil nitrogen.
Claims 1, 3-6, 9, 13, 27-32 & 35-36 are obvious over Lam 2019, Spini, Casas, Zhang, and Morris.
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Lam 2019, Casas, and Spini with the as applied to claims 1, 3-6, 9, 13 & 27-30 above, and further in view of Li et al (2016) Nature Plants. 2(10): 1-6 (published 9/12/2016, hereafter Li).
This is a new rejection in light of Applicant' s amendment of the claims. Applicant' s arguments filed 3/24/2026 in response to the Office action mailed 11/25/2025 have been fully considered as they apply to the new rejection, but they are not persuasive.
Claim 8 is drawn to a method wherein a donor template comprising sequences homologous to the genomic region surrounding the target site is introduced.
The teachings of Lam 2019, Casas, and Spini are presented above. They do not teach a donor template and the DNA repaired using said donor template.
Li teaches a method of genetically modifying rice plants by creating Cas9 and single guide RNA expression vectors, a donor plasmid containing a DNA fragment with point mutations, and introducing the targeting and donor vectors into rice embryogenic calli with biolistic bombardment (page 5, left column, paragraph 2-right column, paragraph 1). Li teaches that this method successfully led to insertion of donor sequence between target cut sites, but no off-target mutations were detected (page 3, left column, paragraph 2).
Li teaches a motivation to use homologous recombination for CRISPR/Cas9 methods rather than non-homologous end joining, because non-homologous end joining is error prone while homologous recombination is high-fidelity (page 1, left column, paragraph 1).
Before the time of filing of the instant application, it would have been obvious to one of ordinary skill in the art to modify the CRISPR/Cas9 method of Lam 2019 to incorporate a donor template as taught by Li. One of ordinary skill would have been motivated to add a donor template because Li teaches that the method of homologous recombination to induce gene mutations is high fidelity compared to the error-prone method used by Lam 2019. One of ordinary skill would have had reasonable expectation of success, because CRISPR/Cas9 genome modification methods were routine prior to the filing of the instant application.
Applicant urges that Lam, Spini, Zhang, and Li do not teach a gene encoding a polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 31, and so the references fail to teach all claim elements and the claims are not obvious (Remarks, page 8, paragraph 4-page 9, paragraph 1).
This argument is unpersuasive, because Casas teaches a motivation to increase flavonoid and apigenin in maize. Lam 2019 teaches the homologous maize gene to the rice CYP75B3 gene and teaches that the gene is conserved in grasses. Thus, the knockout of the maize gene would have been obvious prior to the instant filing date. Although the sequence of SEQ ID NO: 31 was not taught, this sequence is the sequence of the polypeptide encoded by the maize gene taught by Lam 2019 in at least some cultivars of maize. Because the choice of maize cultivar is a design choice, the instant application would have been obvious prior to the filing of the instant application.
In order to show that the instant application would not have been obvious in view of the prior art, Applicant would need to provide evidence that methods and maize plants comprising a CYP75B3 gene encoding a sequence 95% identical to SEQ ID NO: 31 provides unexpected results in comparison to methods and maize plants comprising a CYP75B3 gene that is not 95% identical to SEQ ID NO: 31.
Conclusion
No claims are allowed.
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/VICTORIA L DELEO/Examiner, Art Unit 1662
/Anne Kubelik/Primary Examiner, Art Unit 1663
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