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 Claims
Claims 1,3-6,8-9,13-15,27-33 and 35-36 are under examination on the merits.
The objections to the drawings are withdrawn in light of the substitute drawings filed 9/19/2025.
The objections to the specification are withdrawn in light of Applicant’s amendments.
The objections to claims 13, 14 & 35 are withdrawn in light of Applicant’s amendments.
The rejections of claims 3, 8 & 13-15 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph are withdrawn in light of Applicant’s amendments.
The rejection of claim 36 under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, is withdrawn in light of Applicant’s amendments.
The rejection of claims 1, 5, & 27-30 under 35 U.S.C. 102(a)(1) as being anticipated by Lam et al (2015). Plant Physiology. 168: 1527–1536 is withdrawn in light of Applicant’s amendments.
The rejection of claims 1, 4-6, 9, 13, 15 & 27-30 under 35 U.S.C. 102(a)(1) as being anticipated by Lam et al (2019). New Phytologist. 223: 204–219, taken with the evidence of NCBI GenBank accession AK070442 and NCBI GenBank accession AK064736 is withdrawn in light of Applicant’s amendments.
The rejection of claims 1, 4-6, 9, 13-15 & 27-30 under 35 U.S.C. 103 as being unpatentable over Lam et al (2019). New Phytologist. 223: 204–219, taken with the evidence of NCBI GenBank accession AK070442 and NCBI GenBank accession AK064736, is withdrawn in light of Applicant’s amendments.
The rejection of claims 2-3 under 35 U.S.C. 103 as being unpatentable over Lam 2019 as applied to claims 1, 4-6, 9, 13 & 27-30, and further in view of Spini et al (2016) Plant Soil. 399: 159–178, is withdrawn in light of Applicant’s amendments.
The rejection of claims 31 & 35-36 and 1, 4-6, 9, 13 & 27-30 under 35 U.S.C. 103 as being unpatentable over Lam 2019 further in view of Zhang et al (2017) Plant Physiology and Biochemistry. 111: 30-38 is withdrawn in light of Applicant’s amendments.
The rejection of claims 32 & 33 under 35 U.S.C. 103 as being unpatentable over Lam 2019 and Zhang as applied to claims 1, 4-6, 9, 13, 27-31 & 35-36, 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 PLoS ONE. 15(7): e0235975 is withdrawn in light of Applicant’s amendments.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 claim 8 is under 35 U.S.C. 103 as being unpatentable over Lam 2019 as applied to claims 1, 4-6, 9, 13 & 27-30, and further in view of Li et al (2016) Nature Plants. 2(10): 1-6 is withdrawn in light of Applicant’s amendments.
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.
Claims 1,3-6,8-9,13-15,27-33 and 35-36 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 set forth in the Office action mailed 4/22/2025, as applied to claims 1-6, 8-9, 13-15, 27-33 & 35-36. Applicant' s arguments filed 9/19/2025 have been fully considered but they are not persuasive.
Claims 1,3-6,8-9,13-15,27-33 and 35-36 require a CYP75B3 gene or homolog or ortholog thereof, wherein the gene encodes a polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 31. Claim 13 further requires the gene to encode SEQ ID NO: 31. Claim 14 requires a target sequence at least 95% identical to SEQ ID NO: 13. Claim 15 requires a target sequence at least 95% identical to a sequence within SEQ ID NO: 9.
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. However, the instant specification does not define any lower limit to the amino acid identity of homologs or orthologs of such a gene, so the claims broadly encompass any gene, of any sequence identity, that is a homolog or ortholog to a gene encoding a polypeptide with 95% identity to SEQ ID NO: 31.
Genes with a target sequence at least 95% identical to the 20 bp-long SEQ ID NO: 13 encompass genes with 19 conserved consecutive nucleotides relative to the 1,545 nucleotides required to encode the 515 amino acid-long SEQ ID NO: 31. Because the length of the target sequence of claim 15 is undefined by the specification, genes with a target sequence at least 95% identical to a sequence within SEQ ID NO: 9 encompass genes with as little as 2 consecutive nucleotides also found within SEQ ID NO: 9, encompassing effectively any CYP75B3 gene or homolog or ortholog.
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. SEQ ID NO: 40 has only 23% coverage of SEQ ID NO: 31, yet the amino acid sequence still has 77% local identity to SEQ ID NO: 31. See alignment below. Thus, the specification does not describe species over the full scope of the proteins and does not describe the full scope of the claimed homologs and orthologs of the gene.
Score
Expect
Method
Identities
Positives
Gaps
190 bits(483)
6e-64
Compositional matrix adjust.
91/117(78%)
99/117(84%)
1/117(0%)
Query 399 VNVWAIA RDPEAW-PEPLEFRPARFLPGGSHAGVDVKGSDFELIPFGAGRRICAGLSWGL 457
+NVWAIA RDP +W P+PLEFRP RFLPGG H DVKG D+ELIPFGAGRRICAGL WGL
Sbjct 1 MNVWAIA RDPASWGPDPLEFRPVRFLPGGLHESADVKGGDYELIPFGAGRRICAGLGWGL 60
Query 458 RMVTLMTATLVHALDWDLADGMTADKLDMEEAYGLTLQRAVPLMVRPAPRLLPSAYA 514
RMVTLMTATLVHA DW L DG T +KL+MEEAYG TLQRAVPL+V+P PRLL SAY
Sbjct 61 RMVTLMTATLVHAFDWSLVDGTTPEKLNMEEAYGQTLQRAVPLVVQPVPRLLSSAYT 117
The instant specification teaches that CYP75B3 is a hydroxylase involved in the conversion of apigenin to luteolin (paragraph [0063]).
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).
CYP75B genes in Asteraceae include flavonoid 3’,5’-hydroxylases (F3’5’H), although F3’5’H genes are typically CYP75A genes (Xiao page 2, paragraphs 3-4). The rice CYP75B4 gene also has F3’5’H function (Xiao page 2, paragraph 4). F3’5’H activity and flavonoid 3’-hydroxylase activity can be exchanged through amino acid substitutions (Xiao page 2, paragraph 4). CYP genes with identity greater than 55% sequence identity are divided into subfamilies, and CYP75B is one such subfamily (page 2, paragraph 2-3). Thus, the art discloses that CYP75B genes typically have greater than 55% sequence identity. This encompasses a great deal of variation for homologs and orthologs.
The structural features that distinguish homologs or orthologs to CYP75B3 genes from CYP75B genes that are not homologs or orthologs to CYP75B3 differ across species and are not described in the specification. The structural features that distinguish homologs and orthologs of CYP75B3 specifically are not described in the 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 homologs and orthologs of 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 with regard to the structural and functional characteristics of the compositions used in the claimed methods, Applicant does not appear to have been in possession of the claimed genus at the time this application was filed.
Applicant urges that amended claims require a gene that encodes SEQ ID NO: 31 or an amino acid at least 95% identical in sequence, and a target sequence at least 95% identical to SEQ ID NO: 13 and a target sequence at least 95% identical to a sequence within SEQ ID NO: 9, and that structure responsible for the function of CYP75B3 has been provided by incorporation of references of journal articles in paragraph [0062] of the specification (Remarks, page 10, paragraph 1-page 11, paragraph 1).
This argument is unpersuasive, because the amended claims still encompass homologs or orthologs of CYP75B3 that do not themselves necessarily have 95% identity to the amino acid sequence of SEQ ID NO: 31 or the structural features of CYP75B3, even if the structural features of CYP75B3 could be considered sufficiently described by the incorporation by reference of scientific articles. The specification does not define a homolog or ortholog of a CYP75B3 gene to require any specific amino acid identity or structural feature. Thus, the examples provided by the instant specification would not demonstrate to one of skill in the art that Applicant was in possession of homologs and orthologs of a gene encoding an amino acid at least 95% identical to SEQ ID NO: 31 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.
Claims 1, 3-6, 9, 13-15 & 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 Spini et al (2016) Plant Soil. 399: 159–178 (published 9/30/2015, hereafter Spini) taken with the evidence of NCBI GenBank accession AK064736 (available 12/4/2008).
This is a new rejection in light of Applicant' s amendment of the claims. Applicant' s arguments filed 9/19/2025 in response to the Office action mailed 4/22/2025 have been fully considered as they apply to the new rejection, but they are not persuasive.
Claims 1, 3-6, 9, 13-15 & 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 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
In addition, the sequence of the cyp75b3 gene targeted by Lam 2019 is available from NCBI GenBank accession AK064736 (table S1).
Lam 2019 does not teach 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.
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).
GenBank accession AK064736 provides evidence of an expressed mRNA encoding a protein similar to a flavonoid 3'-monooxygenase Cytochrome P450 75B2 (CDS lines 2-4) and comprising a sequence that is 100% identical to instant SEQ ID NO: 13. See alignment below.
AK064736.1
Sequence ID: Query_1048870Length: 1863Number of Matches: 9
Range 1: 732 to 751GraphicsNext MatchPrevious Match
Alignment statistics for match #1
Score
Expect
Identities
Gaps
Strand
40.1 bits(20)
4e-08
20/20(100%)
0/20(0%)
Plus/Plus
Query 1 ACTTCGTGCCGGCGCTCCGG 20
||||||||||||||||||||
Sbjct 732 ACTTCGTGCCGGCGCTCCGG 751
Before the time of filing of the instant application, it would have been obvious to one of ordinary skill in the art to grow the cyp75b4/cyp75b3 rice mutant plant taught by Lam 2019 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 rice 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.
Before the time of filing of the instant application, it would also have been obvious to one of ordinary skill in the art to substitute the guide RNA taught by Lam 2019 for one of instant SEQ ID NO: 13. One of ordinary skill would have been motivated to do so because the target sequence of instant SEQ ID NO: 13 was present in the cyp75b3 gene targeted by Lam 2019. One of ordinary skill in the art would have had reasonable expectation of success because CRISPR/Cas9 editing and guide RNA design was routine at the time of filing of the instant application.
Before the time of filing of the instant application, it would also have been obvious to one of ordinary skill in the art to substitute the guide RNA target taught by Lam 2019 to target cyp75b3 for its reverse complement, which is identical to a sequence within instant SEQ ID NO: 9 (see alignment above). One of ordinary skill would have been motivated to make the substitution based on simple substitution, because the sequence of the gene was known and the target site had been demonstrated in Lam 2019’s CRISPR/Cas9 method. One of ordinary skill in the art would have had reasonable expectation of success because CRISPR/Cas9 editing and guide RNA design was routine at the time of filing of the instant application.
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, the method of creating the double mutant cyp75b3 cyp75b4 rice plants taught by Lam 2019 reads on the method of claims 1, 4, 5, 13, & 27 and the genetically modified crop plant of claims 28-30.
The method of creating the CRISPR/Cas9 double 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, 13-15 & 27-30 are obvious in view of Lam 2019 and Spini and taken with the evidence of GenBank accession AK064736.
Applicant urges that the prior art references do not disclose or suggest all of the claim limitations because Lam 2019 does not disclose mutant rice plants with a CYP75B3 gene at least 95% identical to instant SEQ ID NO: 31 or that flavones induce biofilm formation in N2-fixing bacteria in proximity to the plant’s roots, that the gene encoding the polypeptide comprising the amino acid sequence is at least 95% identical to SEQ ID NO: 31, and a guide RNA with a target sequence of SEQ ID NO: 13 or a target sequence within SEQ ID NO: 9, and the secondary references fail to cure these deficiencies. Applicant urges one of skill in the art would not have been motivated to arrive at the present invention (Remarks, page 11, paragraphs 2-3 & page 12, paragraph 3 & page 12, paragraph 4-page 13, paragraph 1).
This argument is unpersuasive, because Lam 2019 discloses that cyp75b3 is phylogenetically similar to, which reads on a homolog to, a maize gene GRMZM2G025832. GenBank accession AQK72244.1 (available 2/6/2017) provides evidence that the maize protein from the locus synonymous with GRMZM2G025832 that is homologous to rice CYP75B3 has an amino acid sequence with 100% identity to instant SEQ ID NO: 31. See alignment below. Thus, the CYP75B3 rice gene of Lam 2019 still reads on a homolog or ortholog of a gene encoding an amino acid with a sequence at least 95% or 100% identical to SEQ ID NO: 31.
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
These rice genes have sequences that include sequences within or at least 95% identical to instant SEQ ID NOs: 9 & 13 or their reverse. Because design of guide RNA was routine prior to the time of filing of the instant application, design of a guide RNA with the sequence of SEQ ID NO: 13 or a sequence of SEQ ID NO: 9 would have been obvious to one of skill in the art even if the exact sequences were not taught as the guide RNA used by Lam 2019.
Applicant urges Spini does not cure the deficiencies of Lam 2019 because luteolin, taught by Spini, is not the only flavone described in the present application and Spini does not suggest that other flavones would behave in the same way as luteolin. Applicant urges that not all nitrogen-fixing bacteria would behave in the same manner and have the same niches and habitats as Ensifer meliloti, the nitrogen-fixing bacteria taught by Spini. Applicant points out that Spini does not teach modification of the CYP75B3 gene (Remarks, page 13, paragraph 2).
This argument is unpersuasive, because the claims require one or more flavone, and luteolin is one flavone. The behavior of other flavones is irrelevant to the rejection. Likewise, Ensifer meliloti is a nitrogen fixing bacteria that forms biofilm; knowledge of the behavior, niches, and habitats of all other nitrogen fixing bacteria does not change the facts taught by Spini that render claim 3 obvious. Any embodiment of a claim limitation that is known in the art is sufficient for the claim to be obvious.
Lam 2019 teaches modification of a CYP75B3 gene homolog, so the fact that this limitation is not taught by Spini does not make the combination non-obvious. Individual references do not need to teach all claim limitations, if all limitations are taught by the references in combination.
Additionally, flavone exudation by the plant’s roots is an inherent result of the step of modifying the expression of a CYP75B3 gene and does not require a separate step practiced by one of skill in the art, so one of skill in the art would have required no motivation to observe the recited result from the method taught by Lam 2019. Likewise, the induction of biofilm formation in N2-fixing bacteria present in soil in proximity to the plant’s roots by the increased flavones reads on an intended result of the method rather than an active step. Baldani et al (2014) Plant Soil. 384: 413-431 (published 7/25/2014, hereafter Baldani) provides evidence that rhizosphere soil (which reads on soil in proximity to the plant’s roots) supports an enormous bacterial population from which N2-fixing bacteria may be isolated (page 418, left column, paragraph 1). So, the rice plants of Lam 2019 would be expected to have N2-fixing bacteria in proximity to the roots.
A “wherein” clause may limit a process claim when the clause gives "meaning and purpose to the manipulative steps" (Griffin v. Bertina, 285 F.3d 1029, 1034, 62 USPQ2d 1431 (Fed. Cir. 2002)). The courts have held that a "whereby clause in a method claim is not given weight when it simply expresses the intended result of a process step positively recited." (Hoffer v. Microsoft Corp., 405 F.3d 1326, 1329, 74 USPQ2d 1481, 1483 (Fed. Cir. 2005), quoting Minton v. Nat’l Ass’n of Securities Dealers, Inc., 336 F.3d 1373, 1381, 67 USPQ2d 1614, 1620 (Fed. Cir. 2003)). Since the “wherein” clause of claim 1 relating to biofilm formation describes expected results, and the anticipated results of the claimed processes are inherent to the positively recited steps, they are not given additional weight as a limitation.
Claims 31 & 35-36 are rejected under 35 U.S.C. 103 as being unpatentable over Lam 2019 and Spini, taken with the evidence of GenBank accession AK064736, as applied to claims 1, 3-6, 9, 13-15 & 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 9/19/2025 in response to the Office action mailed 4/22/2025 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 and Spini are presented above. These references do not teach growing a grain 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 rice with a mutation in a cyp75b3 and/or cyp75b4 gene (which are homologs of a cyp75b3 gene encoding an amino acid sequence of SEQ ID NO: 31) to grow in the field under low nitrogen conditions, as taught by Zhang. One of ordinary skill would have been motivated to grow the rice in the field under low nitrogen conditions, because both low nitrogen conditions and mutations in cyp75b3 or cyp75b4 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, such as wheat and rice.
Thus, the method of providing a genetically modified rice crop plant with a mutation in cyp75b3 or cyp75b4 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, Spini, and Zhang and taken with the evidence of GenBank accession AK064736. Claims 1, 3-6, 9, 13-15 & 27-30 are also obvious as presented above.
Applicant urges that the art references do not teach each and every element of the present application, because Zhang does not mention modification of the CYP75B3 gene or root exudates or suggest a connection between flavones, biofilm production, or nitrogen-fixing bacteria, so one skilled in the art would not be motivated to arrive at the present invention (Remarks, page 13, paragraph 3).
This argument is unpersuasive, because amended claims require only a homolog of a gene encoding an amino acid sequence 95% identical to instant SEQ ID NO: 31, and Lam 2019 does teach that the mutated rice CYP genes are phylogenetically related, which reads on a homolog or ortholog, to a maize gene that encodes instant SEQ ID NO: 31. Additionally flavone exudation by the plant’s roots is an inherent result of the step of modifying the expression of a CYP75B3 gene and does not require a separate step practiced by one of skill in the art, so one of skill in the art would have required no motivation to observe the recited result from the method taught by Lam 2019. As presented above, Spini teaches biofilm formation in N2-fixing bacteria present in soil and nitrogen fixation in response to a flavone, which reads on the limitations of the amended claims.
Claims 32 & 33 are rejected under 35 U.S.C. 103 as being unpatentable over Lam 2019, Spini, and Zhang taken with the evidence of GenBank accession AK064736 as applied to claims 1, 3-6, 9, 13-15, 27-31 & 35-36 above, and further in view of Shao et al (2020). PLoS ONE. 15(7): e0235975 (published 7/10/2020, hereafter Shao).
This is a new rejection in light of Applicant' s amendment of the claims. Applicant' s arguments filed 9/19/2025 in response to the Office action mailed 4/22/2025 have been fully considered as they apply to the new rejection, but they are not persuasive.
Claims 32-33 are drawn to a method of increasing the assimilation of atmospheric nitrogen in a grain crop, wherein the amount of inorganic nitrogen is less than 50% of the standard amount or 50ppm for rice.
The teachings of Lam 2019, Spini, and Zhang are presented above. They do not teach growing rice in soil with less than 50 ppm inorganic nitrogen or less than 50% of the recommended amount of nitrogen.
Shao teaches a method of cultivating rice under control solution (10 mM NO3- and 0.065 µM NH4) and under nitrate deficient solution (0 mM NO3- and 0.065 µM NH4) (page 3, paragraph 3). Thus, Shao teaches a nitrogen-deficient nutrient solution for cultivating rice with less than half the nitrogen contained in the full-strength solution and approximately (0.065 µM NH4 * 6 * 18.04g/mol * 1000) 0.0070 ppm inorganic nitrogen. Shao teaches planting rice seeds on paddy soil before transplanting to the nutrient solution (page 3, paragraph 3). Shao teaches that rice cultivated in the nitrogen deficient solution had increased expression of genes involved in the flavonoid biosynthesis process, including chalcone synthase (page 13, paragraph 4; figure 5).
Shao also teaches a motivation for growing rice under lower inorganic nitrogen regimes, because nitrogen-use efficiency is low and nitrogen lost from rice fields can severely pollute the environment and water system (page 2, paragraph 2).
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 method of Lam 2019 and Zhang to grow the rice with a mutation in a cyp75b3 and/or cyp75b4 gene under conditions of nitrate starvation as taught by Shao in order to increase flavonoid and flavone production. One of ordinary skill in the art would have been motivated to do so to increase flavone production in the rice, and the rationale would have been 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 the rice in Shao’s method grew for 4 weeks under nitrogen deficiency. Therefore, a method of increasing the assimilation of nitrogen in a grain crop wherein the plant is grown in soil comprising less than 50% of the standard amount or less than 50ppm inorganic nitrogen would have been obvious in view of Lam 2019, Spini, Zhang, and Shao and with the evidence of GenBank accession AK064736. Claims 1, 3-6, 9, 13-15, 27-33 & 35-36 are obvious in view of Lam 2019, Spini, Zhang, and Shao.
Applicant urges that Lam 2019 and Zhang 2017 do not teach every element of the present application and Shao does not cure these deficiencies (Remarks, page 14, paragraph 1).
This argument is unpersuasive, for the reasons presented above. In short, Spini teaches nitrogen fixation in a bacterium and the formation of a biofilm in response to a flavone. Therefore, these claims limitations would be met by the mutant plants of Lam 2019 when grown in soil where nitrogen fixing bacteria are present. In addition, the connection between flavones, biofilm production and nitrogen-fixing bacteria is an intended and inherent result of the methods that are taught by Lam 2019. Lam 2019 does in fact teach that the double mutants had significantly more extractable flavone. Because the connection between flavone production, flavone exudation, and biofilm production in the nitrogen-fixing bacteria is inherent and requires no additional active step other than the creation of the plant with the modified CYP75B3/CYP75B4 genes as taught by Lam 2019, one of ordinary skill in the art would not have required any additional motivation to arrive at the present invention beyond the motivation presented above to combine Shao with Lam 2019, Spini, and Zhang to increase flavone production.
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Lam 2019 and Spini with the evidence of GenBank accession AK064736 as applied to claims 1, 3-6, 9, 13-15 & 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 9/19/2025 in response to the Office action mailed 4/22/2025 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 are presented above. Lam 2019 does 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 both Li and Lam 2019 performed the methods in rice.
Applicant urges that one of skill in the art would not be motivated to arrive at the present invention because, Applicant urges, Lam 2019 does not teach each and every element of the present application and Li does not mention modification of the CYP75B3 gene or root exudates or a connection between flavones, biofilm production, and nitrogen-fixing bacteria (Remarks, page 14, paragraph 2).
This argument is unpersuasive, for the reasons presented above. In short, Spini teaches a connection between a flavone and nitrogen-fixing bacteria and biofilm production. Additionally, the connection between flavones, biofilm production and nitrogen-fixing bacteria is an intended and inherent result of the methods that are taught by Lam 2019 and Spini. Lam 2019 does teach that the double mutants had significantly more extractable flavone and that the mutated genes are homologs of a maize CYP75B3 gene. Because the connection between flavone production, flavone exudation, and biofilm production in the nitrogen-fixing bacteria is inherent and requires no additional active step other than the creation of the plant with the modified CYP75B3/CYP75B4 genes as taught by Lam 2019 and Spini, it is not necessary that Li teach these limitations. Instead, the motivation to combine Li with Lam 2019 and Spini would be to produce CRISPR/Cas9 mutations in the CYP75B3/CYP75B4 genes that are less error prone than the method taught by Lam 2019.
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.
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/VICTORIA L DELEO/Examiner, Art Unit 1662
/Anne Kubelik/Primary Examiner, Art Unit 1662
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