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 .
Election/Restrictions
Applicant’s election of Group I in the reply filed on 7/30/2026 is acknowledged.
The Examiner withdraws the species election and rejoins all the species.
The Applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Claim Status
Claims 1-2, 7-10, 13-15, 18-19, 21, 23-25, 44-45, 47-53 are pending.
Claims 23-25 and 47-48 are withdrawn from examination as being part of non-elected inventions.
Claims 49-53 are newly added by the Applicant.
Claims 1-2, 7, 9-10, 13-15, 18-19, 21, 44-45 and 49-53 are being examined.
Examiner’s Note
In the claim listing, the status of every claim must be indicated after the claim number by using one of the following identifiers in a parenthetical expression: (Original), (Currently amended), (Canceled), (Withdrawn), (Previously presented), (New), and (Not entered). See 37 C.F.R. 1.121.
Claims 23-25 and 47-48 are withdrawn from examination as being part of non-elected inventions. However, the claims do not indicate “withdrawn”. The Applicant needs to clearly mention the status of claims 23-25 and 47-48.
Claim Objections
Claims 44-45 are objected to because of the following informalities: Claims 44-45 depend from withdrawn claim 23.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1, 7, 9-10, 13-15, 18-19, 21 and 44 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 recites, “…WRKY transcription factor, or an ortholog thereof…” (line 3-4).
The Applicant describes, “The term "ortholog" refers to genes in different species that evolved from a common ancestral gene by speciation. Normally, orthologs retain the same function in the course of evolution. Identification of orthologs is critical for reliable prediction of gene function in newly sequenced genomes”.
WRKY transcription factors are not described or defined as per its functions, per se., but as presence of a 60 amino acid WRKY domain that is defined by a highly conserved WRKYGQK heptapeptide at the N-terminus and a zinc-finger-like motif at the C-terminus (Tang et al., Characterization and co-expression analysis of WRKY orthologs involved in responses to multiple abiotic stresses in Pak-choi (Brassica campestris ssp. chinensis), 2013, BMC Plant Biology, 13:188; p.1, bridging paragraph between left and right columns). WRKY TFs are known to be involved in various functions including various abiotic stresses, including salinity, drought, cold, heat, and abscisic acid (ABA) signaling (Tang et al.; p.1, right column, para 2, line 3-5).
It is unclear to the Examiner which genes or proteins would qualify as the orthologs of a WRKY family of transcription factors. It is not clear if other transcription factors would qualify as orthologs of WRKY family of transcription factors.
Claims 7, 9-10, 13-15, 18-19, 21 and 44 inherit the indefiniteness of claim 1.
Claim Rejections - 35 USC § 112(a)
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.
Scope of Enablement
Claims 1-2, 7, 9-10, 13-15, 18-19, 21, 44-45 and 49-53 are 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 Arabidopsis and canola WRKY47 transcription factors (i.e., SEQ ID NO: 19-20), does not reasonably provide enablement for any other WRKY transcription factors or its orthologs. 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 use the invention commensurate in scope with these claims.
The Applicant describes WRKY47 genes in Arabidopsis (spec, para 00286-00287; Example 1), canola (spec, para 00291; Example 2) and pennycress (spec, Example 4) while knocking-out the WRKY47 transcription factors in canola (BnaCnng01360D) (spec, Example 3) and pennycress (spec, Example 4).
Currently, there are 72 known members of the WRKY family in Arabidopsis and more than 100 members in rice, soybean or poplar; 68 in Sorghum; 38 in Physcomitrella patens; 35 in Sellaginella moellendorffii; 80 in pinus and about 45 in barley (Bakshi et al.; p.1, left column, para 1, line 7-11). Two of the members of the WRKY family, WRKY9 (Krishnamurthy et al., WRKY9 transcription factor regulates cytochrome P450 genes CYP94B3 and CYP86B1, leading to increased root suberin and salt tolerance in Arabidopsis, 2021, Physiologia Plantarum, 172:1673–1687; abstract, line 21-22; p. 1674, right column, para 2, line 16-18) and WRKY33 (Krishnamurthy et al., p.1674, left column, last para, line 47-49) are known to be involved in suberin production via AtCYP94B3. However, unlike WRK47, WRKY9 and WRKY33 need to be upregulated to increase suberin production, as discussed below.
The Applicant does not provide examples of the WRKY family of transcription factors comprising many species other than WRKY47.
The Applicant describes only downregulation of WRKY47 protein by knocking out two separate WRKY in canola (spec, p.70-71, example 3) and Pennycress (spec, p. 71-72), example 4) plants to increase suberin in a plant.
The Applicant also does not provide any guidance for a skilled artisan to alter (in a specific way) the expression of a specific member of the WRKY family other than WRKY47, with suberin production.
The Applicant describes, “The term "ortholog" refers to genes in different species that evolved from a common ancestral gene by speciation. Normally, orthologs retain the same function in the course of evolution. Identification of orthologs is critical for reliable prediction of gene function in newly sequenced genomes” (spec, p.22, para 0095). However, the Applicant does not describe which genes or proteins would qualify as “orthologue” and how a skilled artisan would identify/define an orthologue of a WRKY gene/protein including the one comprising instant SEQ ID NOs: 19-20, as recited in claim 2. The Applicant does not describe or define if any protein having at least 95% sequence identity (as recited in claim 2) to any one of the instant sequences comprising the recited SEQ ID NOs: 19-20 would be considered as an ortholog of a WRKY protein. Moreover, the phylogenetic tree in Fig. 7A-B describing “orthologs” of canola and Arabidopsis WRKY47 protein, respectively, are having less than 95% sequence identity (79.27% and 76.09%, respectively).
Current status of the art describes that WRKY proteins play central roles in abiotic stress, biotic stress, growth and development, and leaf senescence in multiple plants (Cui et al.; p.1, right column, para 2, line 4-7).
Current status of the art, and the instant specification, do not teach which WRKY protein(s), other than WRKY47, WRKY9, and WRKY33, is/are involved in suberin production. Current status of the art also does not teach which gene(s)/protein(s) would be qualified as “orthologs” of WRKY47. Current status of the art does not describe what type of “altering expression” of the WRKY transcription factor would increase suberin production (other than the three discussed above), which would depend on the specific WRKY protein.
In the absence of further guidance, undue trial and error experimentations would be needed to identify specific WRKY gene(s)/protein(s) that are orthologs of Arabidopsis WRKY47 or canola WRKY47 which are involved in suberin production and the type of “altering” (other than downregulation) of gene expression or regulation needed to increase suberin production in a plant.
Based on breadth of the claims, lack of any working example, lack of guidance in the instant description or in prior art, the specification at the time of the application filed would not have taught one skilled in the art how to make and use the full scope of the claimed invention without performing undue experiments.
Written Description
Claims 1-2, 7, 9-10, 13-15, 18-19, 21, 44-45 and 49-53 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 claims 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.
Claim 1 recites, “… altering expression of at least one endogenous WRKY transcription factor, or an ortholog thereof, wherein said altering causes increased production of suberin in the plant, plant part, or plant cell.”
The Applicant describes WRKY47 genes in Arabidopsis (spec, para 00286-00287; Example 1), canola (spec, para 00291; Example 2) and pennycress (spec, Example 4) while knocking-out the WRKY47 transcription factors in canola (BnaCnng01360D) (spec, Example 3) and pennycress (spec, Example 4).
Currently, there are 72 known members of the WRKY family in Arabidopsis and more than 100 members in rice, soybean or poplar; 68 in Sorghum; 38 in Physcomitrella patens; 35 in Sellaginella moellendorffii; 80 in pinus and about 45 in barley (Bakshi et al.; p.1, left column, para 1, line 7-11). Besides WRKY47, two other members of the WRKY family of transcription factors, WRKY9 (Krishnamurthy et al., WRKY9 transcription factor regulates cytochrome P450 genes CYP94B3 and CYP86B1, leading to increased root suberin and salt tolerance in Arabidopsis, 2021, Physiologia Plantarum, 172:1673–1687; abstract, line 21-22; p. 1674, right column, para 2, line 16-18) and WRKY33 (p.1674, left column, last para, line 47-49) are known to be involved in suberin production via AtCYP94B3. However, unlike the instant invention and the recited claims, WRKY9 and WRKY33 need to be upregulated to increase suberin production, as discussed below.
The Applicant does not describe species of the broad genus of the WRKY family of transcription factors other than WRKY47, WRKY9 and WRKY33, that are involved in suberin production.
The Applicant also does not describe the structure function relationship between the members of the WRKY family of transcription factors, other than WRKY47, with suberin production.
The Applicant describes only downregulation of WRKY47 protein by knocking out two separate WRKY in canola (spec, p.70-71, example 3) and Pennycress (spec, p. 71-72), example 4) plants to increase suberin in a plant while claiming the broad genus comprising all types of altering of gene expression in claim 1.
The Applicant describes, “The term "ortholog" refers to genes in different species that evolved from a common ancestral gene by speciation. Normally, orthologs retain the same function in the course of evolution. Identification of orthologs is critical for reliable prediction of gene function in newly sequenced genomes” (spec, p.22, para 0095). However, the Applicant does not describe which genes or proteins would qualify as “orthologue” and how a skilled artisan would identify/define an orthologue of a WRKY gene/protein including the one comprising instant SEQ ID NOs: 19-20, as recited in claim 2. The Applicant does not describe or define if any protein having at least 95% sequence identity (as recited in claim 2) to any one of the instant sequences comprising the recited SEQ ID NOs: 19-20 would be considered as an ortholog of a WRKY protein. Moreover, the phylogenetic tree in Fig. 7A-B describing “orthologs” of canola and Arabidopsis WRKY47 protein, respectively, are having less than 95% sequence identity (79.27% and 76.09%, respectively).
Current status of the art describes that WRKY proteins play central roles in abiotic stress, biotic stress, growth and development, and leaf senescence in multiple plants (Cui et al.; p.1, right column, para 2, line 4-7).
Current status of the art does not teach which other WRKY protein(s) (other than WRKY9, WRKY33 and WRKY47) is/are involved in suberin production and/or the structure function relationship between the WRKY family of transcription factors with suberin production. Current status of the art does not teach which gene(s)/protein(s) would be qualified as “orthologs” of WRKY47. Current status of the art also does not teach what type of “altering expression” of the WRKY transcription factor would increase suberin production, which would depend on the specific WRKY protein, as discussed above. As discussed above, decreasing expression of WRKY47causes increased suberin production, whereas decreasing expression of WRKY9 and WRKY33 reduces suberin production.
Considering the breadth of the claims, lack of representative species of the broad genus claimed, lack of structure function relationship of the broad genus claimed, the Applicant does not appear to have been in possession of the claimed genus at the time this application was filed.
Claim Rejections - 35 USC § 102(a)(1)
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1-2, 7, 9-10, 13-15 and 44 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Feng et al. (Repression of transcription factor AtWRKY47 confers tolerance to boron toxicity in Arabidopsis thaliana, 2021, Ecotoxicology and Environmental Safety 220:112406).
Feng et al. identified the AtWRKY47 gene from the T-DNA insertion line Salk_046806 (p.3, right column, 1, line 3) and describes overexpressing AtWRKY47 using a 35S:WRKY47 cloned in an expression vector (p.4, left column, para 1, line 2-5). The AtWRKY47 protein (Phytozome Gene ID: AT4G01720; GenBank Accession No. NP_192081) is known in the prior art and as implied by Feng et al. (p.4, left column, para 1, line 1-4), comprises 100% sequence identity to instant SEQ ID NO: 20 (as recited in claim 2), as shown below.
Title: US-18-840-877-20
Perfect score: 2585
Sequence: 1 MEEHIQDRREIAFLHSGEFL..........GSSNGDSPQLPQSCTTFSTN 489
Searched: 1 seqs, 489 residues
RESULT 1
AASEQ2_09012026_105509
Best Local Similarity 100.0%; Query Match100.0%; Score 2585; DB 1; Length 489;
Matches 489; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MEEHIQDRREIAFLHSGEFLHGDSDSKDHQPNESPVERHHESSIKEVDFFAAKSQPFDLG 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MEEHIQDRREIAFLHSGEFLHGDSDSKDHQPNESPVERHHESSIKEVDFFAAKSQPFDLG 60
Qy 61 HVRTTTIVGSSGFNDGLGLVNSCHGTSSNDGDDKTKTQISRLKLELERLHEENHKLKHLL 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 HVRTTTIVGSSGFNDGLGLVNSCHGTSSNDGDDKTKTQISRLKLELERLHEENHKLKHLL 120
Qy 121 DEVSESYNDLQRRVLLARQTQVEGLHHKQHEDVPQAGSSQALENRRPKDMNHETPATTLK 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 DEVSESYNDLQRRVLLARQTQVEGLHHKQHEDVPQAGSSQALENRRPKDMNHETPATTLK 180
Qy 181 RRSPDDVDGRDMHRGSPKTPRIDQNKSTNHEEQQNPHDQLPYRKARVSVRARSDATTVND 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 RRSPDDVDGRDMHRGSPKTPRIDQNKSTNHEEQQNPHDQLPYRKARVSVRARSDATTVND 240
Qy 241 GCQWRKYGQKMAKGNPCPRAYYRCTMAVGCPVRKQVQRCAEDTTILTTTYEGNHNHPLPP 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 GCQWRKYGQKMAKGNPCPRAYYRCTMAVGCPVRKQVQRCAEDTTILTTTYEGNHNHPLPP 300
Qy 301 SATAMAATTSAAAAMLLSGSSSSNLHQTLSSPSATSSSSFYHNFPYTSTIATLSASAPFP 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 SATAMAATTSAAAAMLLSGSSSSNLHQTLSSPSATSSSSFYHNFPYTSTIATLSASAPFP 360
Qy 361 TITLDLTNPPRPLQPPPQFLSQYGPAAFLPNANQIRSMNNNNQQLLIPNLFGPQAPPREM 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 TITLDLTNPPRPLQPPPQFLSQYGPAAFLPNANQIRSMNNNNQQLLIPNLFGPQAPPREM 420
Qy 421 VDSVRAAIA MDPNFTAALAAAISNIIGGGNNDNNNNTDINDNKVDAKSGGSSNGDSPQLP 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 VDSVRAAIA MDPNFTAALAAAISNIIGGGNNDNNNNTDINDNKVDAKSGGSSNGDSPQLP 480
Qy 481 QSCTTFSTN 489
|||||||||
Db 481 QSCTTFSTN 489
Feng et al. describes that downregulation (as recited in claim 9) leading to abolishing expression (as recited in claim 13) of the transcription factor AtWRKY47 gene via T-DNA insertion mutation in Arabidopsis thaliana (abstract, line 2) (as recited in claims 14-15) increases tolerance to Boron toxicity (abstract, line 2-4; p.8, left column, last para, line 2-5). Feng et al. also describes that overexpression of AtWRKY47 results in aggravated or lower level of tolerance for Boron toxicity (p.8, left column, last para, line 5-6; Fig. 3). It is an inherent property of the mutant Arabidopsis plant of Feng et al. to have increased production of suberin (as recited in claims 1, 10 and 44) when AtWRKY47 expression is downregulated, as evidenced by the instant specification, which teaches increased suberin production in Arabidopsis plants in which AtWRKY47 expression was downregulated (spec, p.69, para 00289, Fig. 2).
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1, 7, 10, 14-15, 18, 21 and 44 are rejected under 35 U.S.C. 103 as being unpatentable over Krishnamurthy et al. (WRKY9 transcription factor regulates cytochrome P450 genes CYP94B3 and CYP86B1, leading to increased root suberin and salt tolerance in Arabidopsis, 2021, Physiologia Plantarum, 172:1673–1687) in view of Chen et al. (CRISPR/Cas Genome Editing and Precision Plant Breeding in Agriculture, 2019, Annu. Rev. Plant Biol., 70:667-97).
Krishnamurthy et al. teaches increase in suberin production in the roots (as recited in claim 10) of an Arabidopsis thaliana (p.1674, right column, last para, line 5-6) (as recited in claims 14-15) plant having the atcyp94b3 mutation by overexpressing AoCYP94B3 gene from the mangrove tree Avicennia officinalis using 35S CaMV promoter (abstract, line 3-4 and line 14-17; p. 1674, right column, para 2, line 9-13). Krishnamurthy et al. also teaches that the endogenous AtWRKY9 (a WRKY transcription factor) controls (reads on to “altering expression”) suberin deposition (reads on to “production”) (as recited in claim 1) by regulating AtCYP94B3 (abstract, line 21-22; p. 1674, right column, para 2, line 16-18), leading to salt tolerance (abstract; p. 17674, right column, para 2, last 4 lines). AtWRKY9 is an upstream regulator of AtCYP94B3 in Arabidopsis, and atwrky9 mutant with reduced or no expression (i.e., altered expression) of active AtWRKY9 protein suppressed expression of AtCYP94B3 transcripts resulting in reduced suberin in the roots (abstract, line 18-20; p.1685, left column, para 2, line 19-26), as recited in claim 10. Krishnamurthy et al. concluded that WRKY9 has a role in suberin biosynthesis (p.1685, left column, para 2, last 2 lines).
Krishnamurthy et al. also describes that another WRKY protein, WRKY33, controls suberin production by regulating CYP94B1 leading to enhanced salt tolerance of Arabidopsis and rice (p.1674, left column, last para, line 47-49).
Thus, it becomes obvious that overexpressing the WRKY transcription factors WRKY9 and/or WRKY33 would increase CYP94B3 expression which, in turn, would increase suberin production in rice and/or in Arabidopsis plants including in the Arabidopsis atcyp94b3 mutant plants, as recited in claim 1. Increased suberin production by altering expression including over-expression of WRKY33 and/or WRKY9 is a part of the inherent salt tolerance mechanism in crop plants and can be used for generating salt-tolerant crops (title; abstract; p.1685, left column, para 2, last 10 lines).
However, Krishnamurthy et al. does not explicitly describe any targeted gene editing.
Chen et al. teaches precision plant breeding by targeted gene editing (as recited in claim 7) using CRISPR-Cas system (as recited in claims 18-19) (Title and abstract). Chen et al. also teaches different Cas endonucleases including Cas9 (abstract, line 4-6; p.671, para 1, last 4 lines) and Cas12a (p.671, para 2, first 3 lines), as recited in claim 21. It describes that CRISPR-Cas system creates gene knockouts that abolish expression of any specific endogenous gene (as recited in claim 13), chromosomal deletions and translocations, and gene knock-ins (which can replace a gene comprising a stronger promoter resulting in overexpression of the target gene) (p.673, para 2, line 4-5). Chen at al. also describes producing transgene-free plants using the targeted gene editing technique (p.676, para 4, line 7-9; p.676, last para, line 1-3) which significantly reduces the time and labor needed for producing and identifying the target (off target mutation(s) and/or) transgene-free plants.
Before the effective filing date of the invention, it would have been obvious to an ordinarily skilled artisan to alter the expressions of the WRKY9 and/or WRKY33 gene(s) in a plant, as described by Krishnamurthy et al., using the targeted gene editing technique via CRISPR-Cas9, as described by Chen at al.
Before the effective filing date, an ordinarily skilled artisan would have been motivated to overexpress the WRKY9 and/or WRKY33 transcription factor(s) in any commercially important plants using CRISPR-Cas9 based targeted gene editing technique to replace the upstream promoter regions(s) in the respective WRKY9 and/or WRKY33 gene(s) to increase suberin production with a reasonable expectation of success to produce salt-tolerant and commercially useful crop plants. CRISPR-Cas based alteration of gene expression offers an added benefit (compared to traditional methods) to produce transgene-free plants that greatly decrease the time and labor needed for identifying the target (mutated or genome edited) plants, as described by Chen et al.
Regarding claim 44, crossing a plant with a useful trait with itself (selfing) to produce and/or maintain inbred homozygous line or with a different plant of an elite crop variety with a different useful trait followed by selecting a progeny with the desired trait (i.e., increased suberin production resulting in higher salt tolerance, in this case) is a well-known and routine process in the art to introgress different traits from two different parents.
Claims 1-2, 7, 9-10, 13-15, 18-19, 21, 44-45 and 49-53 are rejected under 35 U.S.C. 103 as being unpatentable over Ghanati et al. (Deposition of suberin in roots of soybean induced by excess boron, 2005, Plant Science, 168:397-405) in view of Feng et al. (Repression of transcription factor AtWRKY47 confers tolerance to boron toxicity in Arabidopsis thaliana, 2021, Ecotoxicology and Environmental Safety 220:112406), Chen et al. (CRISPR/Cas Genome Editing and Precision Plant Breeding in Agriculture, 2019, Annu. Rev. Plant Biol., 70:667-97) and Anderson et al. (Systematic analysis of CRISPR–Cas9 mismatch tolerance reveals low levels of off-target activity, 2015, Journal of Biotechnology 211:56–65).
Ghanati et al. describes that treating plants cells, including tobacco and soybean cells,
with excess boron led to increase in production of suberin (as recited in claim 1) in their cell walls (p.397, right column, para 1, line 8-13). Suberized cell walls, especially in the root (as recited in claim 10) tip (abstract, line 3-4), hinder radial movement of water and solutes from root surface toward tissues in central cylinder (i.e., stele, which is responsible for transporting water, minerals and other solutes in the plant) (p.397, right column, para 1, line last 4 lines).
However, Ghanati et al. does not describe any WRKY transcription factor. Ghanati et al. also does not describe any targeted gene editing including CRISPR-Cas technique.
Feng et al. describes that downregulation (as recited in claim 9) leading to abolishing expression (as recited in claim 13) of the transcription factor AtWRKY47 gene via T-DNA insertion mutation in Arabidopsis thaliana (abstract, line 2) (as recited in claims 14-15) increases tolerance to Boron toxicity (abstract, line 2-4; p.8, left column, last para, line 2-5). Feng et al. also describes that overexpression of AtWRKY47 results in aggravated or lower level of tolerance for Boron toxicity (p.8, left column, last para, line 5-6; Fig. 3). AtWRKY47 protein (Phytozome Gene ID: AT4G01720; GenBank Accession No. NP_192081) is known in the prior art and as implied by Feng et al. (p.4, left column, para 1, line 1-4) (discussed above), and comprises 100% sequence identity to instant SEQ ID NO: 20 (as recited in claim 2), as shown above.
Chen et al. teaches precision plant breeding by targeted gene editing (as recited in claim 7) using CRISPR-Cas system (as recited in claims 18-19) (Title and abstract). Chen et al. also teaches using different Cas endonucleases including Cas9 (abstract, line 4-6; p.671, para 1, last 4 lines) and Cas12a (p.671, para 2, first 3 lines), as recited in claim 21. It describes that CRISPR-Cas system can create gene knockouts that abolish expression of specific endogenous gene(s) (as recited in claim 13), chromosomal deletions and translocations, and gene knock-ins (which can replace a gene comprising a stronger promoter resulting in overexpression of the target gene) (p.673, para 2, line 4-5).
Before the effective filing date of the invention, it would have been obvious to an ordinarily skilled artisan to alter, including downregulating and/or knocking-out expression (using CRISPR-Cas system, as described by Chen et al), of WRKY47, as described by Feng et al. Instant SEQ ID NO: 13 (as recited in claim 19) has only one base pair mismatch (data not shown) with the transcript (cDNA) sequence of the AtWRKY47 gene and would work with reasonable expectation of success as a gRNA for the AtWRKY47 gene, as described by Anderson et al.(abstract, line 9-15). However, using any specific gRNA sequence to downregulate/knockout the endogenous WRKY47 gene in Arabidopsis is an experimental design choice of the artisan without affecting the outcome because designing gRNAs is well known and a routine process in the art, as described by Chen et al. (p.686, last para, last 5 lines).
Before the effective filing date, an ordinarily skilled artisan would have been motivated to downregulate including knocking-out expression of AtWRKY47 with a reasonable expectation of success to increase suberin production resulting in increased tolerance to Boron toxicity, as described by Ghanati et al., which is an economically important trait.
Regarding claims 44-45, crossing a plant with a useful trait with itself (selfing) to produce and/or maintain inbred homozygous line or with a different plant of another elite crop variety with a different useful trait followed by selecting a progeny with the desired trait (i.e., increased suberin production resulting in higher salt tolerance, in this case) is a well-known and routine process in the art to introgress different traits from two different patents. The transcript (cDNA) sequence of the AtWRKY47 gene (Gene ID: AT4G01720) comprises 100% sequence identity to the molecular marker comprising SEQ ID NO: 21 (data not shown), as recited in claim 45.
Regarding claims 49-53; Brassica napus WRKY47 gene and the protein encoded of it are known in the prior art comprising more than 95% (97.45%) sequence identity to instant SEQ ID NO: 19 (as recited in claims 49 and 52), as shown below.
RESULT 3
A0A078G9D7_BRANA
ID A0A078G9D7_BRANA Unreviewed; 490 AA.
AC A0A078G9D7;
DT 29-OCT-2014, integrated into UniProtKB/TrEMBL.
DT 29-OCT-2014, sequence version 1.
DT 10-JUN-2026, entry version 42.
DE SubName: Full=BnaA09g00350D protein {ECO:0000313|EMBL:CDY22006.1};
GN Name=BnaA09g00350D {ECO:0000313|EMBL:CDY22006.1};
GN ORFNames=GSBRNA2T00018363001 {ECO:0000313|EMBL:CDY22006.1};
OS Brassica napus (Rape).
OX NCBI_TaxID=3708 {ECO:0000313|EMBL:CDY22006.1, ECO:0000313|Proteomes:UP000028999};
RN [1] {ECO:0000313|EMBL:CDY22006.1, ECO:0000313|Proteomes:UP000028999}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=cv. Darmor-bzh {ECO:0000313|Proteomes:UP000028999};
RX PubMed=25146293; DOI=10.1126/science.1253435;
RA Chalhoub B., Denoeud F., Liu S., Parkin I.A., Tang H., Wang X., Chiquet J.,
RA Belcram H., Tong C., Samans B., Correa M., Da Silva C., Just J.,
RA Falentin C., Koh C.S., Le Clainche I., Bernard M., Bento P., Noel B.,
RA Labadie K., Alberti A., Charles M., Arnaud D., Guo H., Daviaud C.,
RA Alamery S., Jabbari K., Zhao M., Edger P.P., Chelaifa H., Tack D.,
RA Lassalle G., Mestiri I., Schnel N., Le Paslier M.C., Fan G., Renault V.,
RA Bayer P.E., Golicz A.A., Manoli S., Lee T.H., Thi V.H., Chalabi S., Hu Q.,
RA Fan C., Tollenaere R., Lu Y., Battail C., Shen J., Sidebottom C.H.,
RA Wang X., Canaguier A., Chauveau A., Berard A., Deniot G., Guan M., Liu Z.,
RA Sun F., Lim Y.P., Lyons E., Town C.D., Bancroft I., Wang X., Meng J.,
RA Ma J., Pires J.C., King G.J., Brunel D., Delourme R., Renard M., Aury J.M.,
RA Adams K.L., Batley J., Snowdon R.J., Tost J., Edwards D., Zhou Y., Hua W.,
RA Sharpe A.G., Paterson A.H., Guan C., Wincker P.;
RT "Plant genetics. Early allopolyploid evolution in the post-Neolithic
RT Brassica napus oilseed genome.";
RL Science 345:950-953(2014)
DR EMBL; LK032127; CDY22006.1; -; Genomic_DNA.
DR STRING; 3708.A0A078G9D7; -.
DR PaxDb; 3708-A0A078G9D7; -.
DR Gramene; CDY22006; CDY22006; GSBRNA2T00018363001.
DR KEGG; bna:106430080; -.
DR Gene3D; 2.20.25.80; WRKY domain; 1.
DR InterPro; IPR003657; WRKY_dom.
DR InterPro; IPR036576; WRKY_dom_sf.
DR InterPro; IPR044810; WRKY_plant.
DR PANTHER; PTHR31429; WRKY TRANSCRIPTION FACTOR 36-RELATED; 1.
DR PANTHER; PTHR31429:SF59; WRKY TRANSCRIPTION FACTOR 47-RELATED; 1.
DR Pfam; PF03106; WRKY; 1.
DR SMART; SM00774; WRKY; 1.
DR SUPFAM; SSF118290; WRKY DNA-binding domain; 1.
DR PROSITE; PS50811; WRKY; 1.
PE 4: Predicted;
KW Coiled coil {ECO:0000256|SAM:Coils};
KW DNA-binding {ECO:0000256|ARBA:ARBA00023125};
SQ SEQUENCE 490 AA; 53940 MW; AB169CB2244BA85E CRC64;
Best Local Similarity 97.4%; Query Match 95.9%; Score 2507.5; Length 490;
Matches 482; Conservative 2; Mismatches 6; Indels 5; Gaps 3;
Qy 1 MEEHSQDGREIAFLHSGDFLQRDSTSKDHQPNESSVEHHHKQSIKEVDFFAVKSQPYDLG 60
||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||
Db 1 MEEHSQDGREIAFLHSGDFLQRDSTSKDHQPNESSVEHHHKPSIKEVDFFAVKSQPYDLG 60
Qy 61 HMRTTIVGSYGFNDELAPVNSCLRTSSDDDVNDKTKAQINRLRLELERLHEENHKLKHLL 120
||||||||| || |||||||||||||||| ||||||||:||||||||||||||||||||
Db 61 HMRTTIVGSSSFNSELAPVNSCLRTSSDDDGNDKTKAQISRLRLELERLHEENHKLKHLL 120
Qy 121 DEISERYNDLQSRVLLARPTQVEGLQQHEDIPQAVSSQALEDRKPMDMSNDIPATTLKRR 180
||||||||||||||||||||||||||||||||||||||||||||||||:|||||||||||
Db 121 DEISERYNDLQSRVLLARPTQVEGLQQHEDIPQAVSSQALEDRKPMDMNNDIPATTLKRR 180
Qy 181 SPDDVDDRDLHRDSPKAPRLYQNKSANHEEQQNPHDQLPFRKARVSVRARSDATTVNDGC 240
||||||||| ||||||||||||||| ||||||||||||||||||||||||||||||||||
Db 181 SPDDVDDRD-HRDSPKAPRLYQNKSTNHEEQQNPHDQLPFRKARVSVRARSDATTVNDGC 239
Qy 241 QWRKYGQKMAKGNPCPRAYYRCTMAVGCPVRKQVQRCAEDTTILTTTYEGNHNHPLPPSA 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 240 QWRKYGQKMAKGNPCPRAYYRCTMAVGCPVRKQVQRCAEDTTILTTTYEGNHNHPLPPSA 299
Qy 301 TAMAATTSAAAAMLLSGSNTSNFHQTLSTPSAMSSSSSSLYQNFPYTSTIATLSATAPFP 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 300 TAMAATTSAAAAMLLSGSNTSNFHQTLSTPSAMSSSSSSLYQNFPYTSTIATLSATAPFP 359
Qy 361 TITLDLTNPPRPLQPPPPPHFMSQYGPGAYLTNANQTRSINDNYQQLLLPNLSGPQVPPR 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||
Db 360 TITLDLTNPPRPLQPPPPPHFMSQYGPGAYLTNANQTRSINDNYQQLLLPNLSGPQV-PR 418
Qy 421 DMVDTVRAAIA TDPNFTAALAAAISNIIGGGNNNNNANTNDNNNNNNNKVDAKSGGSSNG 480
||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||
Db 419 DMVDTVRAAIA TDPNFTAALAAAISNIIGGGNNNNNANTND---NNNNKVDAKSGGSSNG 475
Qy 481 DSPPLPQSCTTFSTT 495
|||||||||||||||
Db 476 DSPPLPQSCTTFSTT 490
Genomic sequence (gene ID: BnaA09g00350D), cDNA sequence, and the protein sequence encoded by the WRKY47 gene in Brassica napus (as recited in claim 53) are known in the prior art (GenBank accession No. LK032127). The cDNA sequence of the BnWRKY47 gene comprises 100% sequence identity to instant SEQ ID NO: 9 (as recited in claim 51), as shown below.
Title: US-18-840-877-9
Perfect score: 20
Sequence: 1 gaaattgcgttcttacactc 20
Searched: 1 seqs, 3250 residues
Database : NASEQ2_09012026_124249.fasta:*
RESULT 1
NASEQ2_09012026_124249
Best Local Similarity 100.0%; Query Match 100.0%; Score 20; DB 1; Length 3250;
Matches 20; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 GAAATTGCGTTCTTACACTC 20
||||||||||||||||||||
Db 28 GAAATTGCGTTCTTACACTC 47
Before the effective filing date of the invention, it would have been obvious to an ordinarily skilled artisan to introduce genetic modification to downregulating and/or knocking-out expression using CRISPR-Cas system (as described by Chen et al) of the endogenous WRKY47 gene encoding the WRKY47 protein, as described by Feng et al., in the commercially important crop, Brassica napus. Instant SEQ ID NO: 9 (as recited in claim 51) would work with a reasonable expectation of success as a gRNA for the WRKY47 gene in B. napus. However, using any specific gRNA sequence to downregulate/knockout the endogenous BnWRKY47 gene in B. napus is an experimental design choice of the artisan without affecting the outcome, as designing gRNAs is well known and a routine process in the art, as described by Chen et al. (p.686, last para, last 5 lines).
Before the effective filing date, an ordinarily skilled artisan would have been motivated to introduce genetic modification to downregulate including knocking-out the expression of BnWRKY47 in a commercially important B. napus cultivar with a reasonable expectation of success to increase suberin production resulting in increased tolerance to Boron toxicity, as described by Ghanati et al., which is an economically important trait.
Conclusion
No claim is allowed.
Communication
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J.C.
/Jay Chatterjee/Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Units 1661 & 1662
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