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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 04/17/2026 has been entered.
Election/Restrictions
Amendment entered on 04/17/2026 is acknowledged. Claims 1, 3-4, 6-15 and 17-18 are pending and claims 17 and 18 are added by applicant as new claims. Since new claims 17 and 18 does not add further search burden the claims are examined in this office action.
Therefore, claims 1, 3-4, 6-15 and 17-18 along with the elected species of SEQ ID NO:90 as the spacer sequence from claim 12 and SEQ ID NO:83 as the region of amino acid sequence from claim 14 are examined in this office action.
Following 35 USC § 112 -Written Description Requirement rejection has been modified in light of applicant’s amendment of claims 1 and 14 to recite target is in 3’ region of the a sequence having at least 90% identity to SEQ ID NOs:75 and 76.
Claim Rejections - 35 USC § 112 -Written Description Requirement
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, 3-4, 6-15 and 17-18 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.
Analysis of Breadth of Claims
Claims 1 recite large variants of SGR1 gene comprising any of the nucleotide’s sequence having at least 90% sequence identity to SEQ ID NOs: 75 and 77 and SGR2 gene comprising any of the nucleotide’s sequence having at least 90% sequence identity to SEQ ID NOs: 76 and 78.
Claim 12 recites large variants of spacer sequence having at least 90% sequence identity to SEQ ID NO: 90.
Claim 14 recites large variants of nucleotide sequence having at least 90% sequence identity to SEQ ID NOs: 81-82.
Claim 14 recites large variants of amino acid sequence having at least 90% sequence identity to SEQ ID NO: 83.
Claim 1 recite any mutation (i.e. any substitution, an insertion and/or a deletion) generated in the 3’ end of a promoter region of endogenous SGR1 gene and the SGR2 gene with target site of any structures.
Any of the target site mutation would require increase in resistance to soybean rust, delayed senescence and/or chlorophyll retention as compared to a control soybean plant (claim 1).
Guide nucleic acid of SEQ ID NO:90 would bind to various regions of Glycine max plant genome.
What is Described in the Specification
Applicant describes:
SGR1 and SGR2 which are endogenous soybean genes (page 39, lines 22-23).
Specification describes SEQ ID NOs: 115-144 are example of edited endogenous SGR nucleic acid sequences as shown in Table 1 (page 7, lines 19-20) (see table below). Table 1 shows the deletion of different sizes for the wild type SGR1 identified as Locus 28 and SGR2 identified as locus 27.
Applicant describes promoter editing to generate knockdown of SGR1 and SGR2 (Page 73, lines 24-25).
To generate range of promoter alleles with multiple spacer sequences were designed across the promoters of SGR1 and SGR2 and placed into one construct (page 73, lines 28-30).
Vectors encoding the spacer as well as a CRISPR-Cas effector were introduced into soy using Agrobacterium (Table 2) (page 73, lines 32-33).
Molecular screening was employed to identify E0 plants carrying deletions in the promoters of both SGR1 and SGR2 (page 74, lines 1-4)
A dark induced leaf senescence assay was used to identify E1 plants that showed chlorophyll leaf senescence assay was used to identify E1 plants that showed chlorophyll expression (Fig. 4, Table 3) (page 74, lines 5-9).
Expression level of SGR 1/2 was assayed in a subset of lines that showed a range of phenotypes and edit types (Fig. 5) (page 74, lines 9-10).
Lines with SGR1/2 knockdown and minimal senescence delays and chlorophyll retention were selfed to get E2 seeds. Homozygous E2 lines were then subjected to greenhouse screening with a panel of isolates as well as field screening.
Two E1 lines that were homozygous for large deletions in SGR2 but heterozygous for large deletions in SGR1 were grown another generation to isolate homozygous edited lines (Table 3) that contain large deletions in both promoters (page 75, lines 9-12).
Difference Between What was described and What is Claimed
Applicant has not described variants of SGR1 gene comprising any of the nucleotide sequences having at least 90% sequence identity to SEQ ID NOs: 75 and 77 and SGR2 gene comprising any of the nucleotide sequences having at least 90% sequence identity to SEQ ID NOs: 76 and 78 (claim 1).
Applicant has not described variants of spacer sequence having at least 90% sequence identity to SEQ ID NO: 90 (claim 12).
Applicant has not described variants of nucleotide sequences having at least 90% sequence identity to SEQ ID NOs: 79-82 (claims 14).
Applicant has not described variants of amino acid sequence having at least 90% sequence identity to SEQ ID NO: 83 (claim 14).
Applicant has not described any mutation (i.e. any substitution, an insertion and/or a deletion) generated in the 3’ end of a promoter region of endogenous SGR1 gene and the SGR2 gene with target site of any structures would be useful.
Applicant has not described the target site in the 3’ end of the promoter region which would have any structure (claim 1).
Applicant has not described any target site mutation in the 3’ end would cause increase in resistance to soybean rust, delayed senescence and/or chlorophyll retention as compared to a control soybean plant (claim 1).
Applicant has not described guide nucleic acid of SEQ ID NO:90 would bind to a specific endogenous STAYGREEN (SGR) gene (claim 15).
Analysis
The purpose of the written description is to ensure that the inventor had possession at the time the invention was made, of the specific subject claimed. For a broad generic claim, the specification must provide adequate written description to identify the genus of the claim.
Applicant has not described large variants of SGR1 gene comprising any of the nucleotide sequences having at least 90% sequence identity to SEQ ID NOs: 75 and 77 and SGR2 gene comprising any of the nucleotide sequences having at least 90% sequence identity to SEQ ID NOs: 76 and 78. For example SEQ ID NO: 75 is 2298 nucleotide long. A variant of SEQ ID NO: 75 having at least 90% identity would have ~230 nucleic acid (NA) changes (i.e., substitutions, deletions, insertions, or additions) relative to SEQ ID NO: 75, and this encompasses a genus of nucleic acid sequence that includes at least ~4230 different molecules. For this reason, the genus of nucleic acid molecules having at least 90% identity to SEQ ID NO: 75 is an very large genus of molecules. Furthermore, applicant has not described large variants of nucleotides sequence having at least 90% sequence identity to SEQ ID NOs: 75-78. Similarly, Applicant has not described variants of coding sequences (cDNAs) as nucleotide sequence having at least 90% sequence identity to SEQ ID NOs: 81-82 of SGR1 and SGR2 (claim 14). Furthermore, Applicant has not described variants of nucleotide sequence having at least 90% sequence identity to SEQ ID NOs: 79-80 as mRNA sequences of SGR1 and SGR2 respectively as described in Spec, page 7, lines 11-12 (as claimed in claim 14).
It is known in the art that the SGR protein require specific motifs to be functional for specific traits. Jiao et al. (Published: 2020, Journal: Botanical studies, 61(1), 1-9); teaches stay green gene (SGR) homologs are widespread in the plant kingdom and the SGR proteins has specific conserved motifs (page 2, left last paragraph, Page 3, Figure 1). Jiao et al. teaches SGR homologs function in Chl degradation in leaf, seeds (page 6, right paragraph 2), alters expression of ethylene induced genes, ethylene receptor genes, ethylene signaling, carotenoid accumulation (page 6, right paragraph 3), nodule development (page 6, right paragraph 4). Jiao et al. teaches their phylogenetic analysis revealed that specific conserved SGR domain, chloroplast transit peptide, and variable C-terminal region are present in different SGR genes (page2, left last paragraph, see Figures 1-2). Therefore, applicant has not described at least 90% sequence identity to SEQ ID NOs: 75, 76 and 81-82 that would retain such conserved domains or motifs to have uses as a SGR gene to be modified for use as exhibit increase in resistance to soybean rust, delayed senescence and/or chlorophyll retention as compared to a control soybean plant as described in claim 12 and showed in specification.
Claim 12 recites variants of spacer sequence having at least 90% sequence identity to SEQ ID NO: 90. The SEQ ID NO: 90 is 23 nucleotide long. A variant of SEQ ID NO: 90 having at least 90% identity would have ~2 nucleic acid (NA) changes (i.e., substitutions, deletions, insertions, or additions) relative to SEQ ID NO: 90 anywhere in the sequence in any combination. Applicant has not described any spacer sequence having at least 90% sequence identity to SEQ ID NO: 90 would have been found useful for creating mutant SGR1 and SGR2 genes that would exhibit increase in resistance to soybean rust, delayed senescence and/or chlorophyll retention as compared to a control soybean plant other than SEQ ID NO:90 itself.
Applicant has not described variants of amino acid sequence having at least 90% sequence identity to SEQ ID NO: 83 (claim 14). SEQ ID NO: 83 is 141 amino acid long. A variant of SEQ ID NO: 83 having at least 90% identity would have ~14 amino acids (AA) changes (i.e., substitutions, deletions, insertions, or additions) relative to SEQ ID NO: 83, and this encompasses a genus of nucleic acid sequence that includes at least ~2014 different molecules. Applicant has not described SGR gene encoding protein having a region comprising amino acid sequence having at least 90% sequence identity to SEQ ID NO: 83.
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Furthermore, the state of the art at the time of the instant invention was that although the skilled artisan would appreciate the nucleic acid comprising SEQ ID NO: 75-82, and 90 and amino acid sequence of SEQ ID NOs: 83 one would not be able to readily predict function of nucleic acid which has at least 90% identity to SEQ ID NOs: 75-82, and 90 and amino acid sequence having at least 90% sequence identity to SEQ ID NO: 83 and it is impossible to predict such a broad sequence variation will have any required function. For example, Guo et al. (Published Year: 2004, Journal: Proceedings of the National Academy of Sciences, Vol. 101(25), pages: 9205-9210) teaches that while proteins are fairly tolerant to mutations resulting in single amino acid changes, increasing the number of substitutions additively increases the probability that the protein will be inactivated (page 9209, right. col., paragraph 2).
Furthermore, it is known in the art that a variation in promoter region would affect the relative expression of endogenous genes. Rodríguez-Leal et al. (Published:2017, Journal: Cell 171:470–480) teaches deletion of different sizes of conserved region (i.e. CR1, CR2 etc.) in SlCLV3 promoter in different species of tomato (page 475, Figure 4E) provides continuum of locule number variation where relative expression of SICLV3 is reduced in for example in m6 and m3 where there is no changes in m12 and m8 (page 475, Figure 4F). Therefore, targeting different regions of the nucleotides would lead to various results in the expression of the genes leading to variation in the phenotypes.
Applicant has not described wherein any deletion in the promoter of SGR1 or SGR2 significantly increases disease resistance for example Figure 7 left pane shows CE35915 is not different than non-transformed control, furthermore CE35914, CE35906, CE40104, CE39930 also are no different than the non-transformed control (as shown with same mean comparison letters A and B). Thus if the percent severity is similar in these edit call for each edited plant as compared to non-transformed control is observed in other mutation it is not predictable for example for CE35915 has edits on SGR1 and SGR2 sequences ranging from 5D to 70D and 6 insertions (I) (page 77, Table 3) which has mean disease rating not different from the untransformed control, in fact the rating is higher than the control (Figure 7) showing more disease even with the homozygous deletion lines having mutation in promoter regions of SGR1 and SGR2 sequences (Table 3, Figure 7).
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Same lower SPAD chlorophyll reading also seen in CE35915 as compared to control in all the DPI (9, 11, and 14) (see Figure 6), and chlorophyll retention as shown by proportion of green remaining pixels shown in Figure 4 also show CE35915, and CE5842 has pixel proportion same as control (Figure 4).
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Thus, it is not clear whether any mutation in promoter region of the SGR1 and SGR2 would have exhibited increase in resistance to soybean rust, delayed senescence and chlorophyll retention as compared to a control plant or part thereof not comprising the mutation.
Applicant does not describe the function of mutation to any portion of promoter region of STAYGREEN (SGR) genes SGR1 and SGR2 is predictable to have use for example edit type CE35915 for SEQ ID NOs: 130-133 (Spec, page 76, Table 1) shows no significant effect to any traits in soybean compared to non-transformed control (Drawing, Figure 7 and its mean comparison, Figure 4 and Figure 6). Therefore there is dearth of description of such large variants of SGR1 and SGR2 when muted in its 3’ promoter region would have for example increase in ASR resistance delayed senescence and/or chlorophyll retention.
Applicant has amended to recite the target site is in the 3’ region of the for example SEQ ID NO:75 that is 2298 nucleotides long and Applicant has not described which of these sequences are considered 3’ (since a sequence would have 5’ to 3’ orientation). Therefore applicant has not described target sites of 3’ region of a sequence having 90% identity to SEQ ID NOs:75 and 76 that would lead to specific function to exhibit an increase in resistance to soybean rust, delayed senescence and/or chlorophyll retention as compared to a control plant or part thereof not comprising the mutation.
Applicant has not described guide nucleic acid of SEQ ID NO:90 would bind to a specific endogenous STAYGREEN (SGR) gene (claim 15). For example alignment of SEQ ID NO: 90 has 100% identity to various regions for example chromosome 1, 11 and 13 of a Glycine max and Glycine Soja plants (see alignment summary below). If GmSGR1 and GmSGR2 located on chromosome 11 and 1, there is dearth of description of alignment at chromosome 13. Therefore there is dearth of description of guide nucleic acid of SEQ ID NO:90 would bind to a specific endogenous STAYGREEN (SGR) gene leading to have specific changes in function of the gene.
Alignment of SEQ ID NO:90 to GenEmble database summary.
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Given the virtually large structural variable associated with these embodiments of any mutation in the promoter region of large variants of SGR gene in 3’ region and sequences with at least 90% identity, the claims read on an extremely broad and highly diverse structures that needs to have specific function to exhibit an increase in resistance to soybean rust, delayed senescence and/or chlorophyll retention as compared to a control plant or part thereof not comprising the mutation. However, applicant has not reduced to practice any embodiments where a mutation in any portion of promoter region of the variants of the SGR1 and SGR2 can be effective to increase in resistance to soybean rust, delayed senescence and chlorophyll retention as compared to a control plant or part thereof without targeting specific regions of SGR1 and SGR2 genes. Thus, there is dearth of description any mutation in promoter regions of SGR1 and SGR2 that would have any uses or would be effective to increase resistance to soybean rust, causes delayed senescence and/or chlorophyll retention.
Given the large structural diversity associated with the claimed genus, Applicant’s disclosure is not representative of the claimed genus as a whole. This point is particularly relevant because, as discussed above, the prior art speaks to the disconnection between the structure of the broadly claimed variants of SGR1 and SGR2 genes in Soybean plants with no known function or with function of increased resistance to soybean rust, and that they cause delayed senescence and/or chlorophyll retention.
"The test for sufficiency is whether the disclosure of the application relied upon reasonably conveys to one skilled in the art that the inventor had possession of the claimed subject matter as of the filing date." Ariad Pharm, Inc, v EH Lilly & Co., 598 F.3d 1336, 1351 (Fed. Cir. 2010). To satisfy the written description requirement, a patent specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention. Lockwood v. Amer. Airlines, ina, 107 F.3d 1565, 1572, 41 USPQ2d 1961, 1966 (Fed. Cir. 1997). "An applicant shows possession of the claimed invention by describing the claimed invention with all of its limitations. Lockwood, 107 F.3d at 1572, 41 USPG2d at 1966". While the written description requirement does not demand either examples or an actual reduction, actual "possession" or reduction to practice outside of the specification is not enough. Ariad Pharm, Inc. v. Eli Lilly & Co., 598 F,3d 1336,1352 (Fed. Cir. 2010). Rather, it is the specification itself that must demonstrate possession. Id.
The Federal Circuit has clarified the application of the written description requirement to inventions in the field of biotechnology. The court stated that, “A description of a genus of cDNAs may be achieved by means of a recitation of a representative number of cDNAs, defined by nucleotide sequence, falling within the scope of the genus or of a recitation of structural features common to members of the genus, which features constitute a substantial portion of the genus.” See University of California v. Eli Lilly and Co., 119 F. 3d 1559; 43 USPQ2d 1398, 1406 (Fed. Cir. 1997).
Thus, based on the analysis above, Applicant has not met either of the two elements of the written description requirement as set forth in the court's decision in Eli Lilly. As a result, it is not clear that Applicant was in possession of the claimed genus at the time this application was filed.
Response to Argument
Applicants’ arguments filed 04/17/2026 have been fully considered but they are not persuasive.
Applicant argues claim 1 recites structural components including that the mutations are generated in the promoter regions as being in the 3' region of a sequence having at least 90% identity to SEQ ID NOs:75 and 76 and also recite the functional element of increase in resistance to soybean rust, delayed senescence and/or chlorophyll retention (Response to rejection, page 8, paragraph 2). Applicant has repeated argument as provided before that 90% sequence identity to the SGRI and SGR2 genes as including, e.g., homologs can be identified by methods such as computer-based alignments and hybridization (Response to rejection, page 8, paragraph 3)
Applicant argues claim 14 recites that the guide nucleic acid binds to a target site in promoter region which is 3’ region of a sequence having at least 90% identity to SEQ ID NO:75 and/or SEO ID NO:76 (Response to rejection, page 9, paragraph 1).
Regarding argument about teaching of homologs, although one skilled in art would find homologs of recited sequences and Jiao et al. teaches such homologs and conserved motifs the argument were not found persuasive since there is no predictability that mutating any of those homologs would lead to changes in any useful functions or function of increase in resistance to soybean rust, delayed senescence and/or chlorophyll retention as compared to a control soybean plant. Furthermore, applicant has not described targeting which of the motifs or conserved region would lead to the change in which function of the protein. Therefore, there is dearth of description of method of mutating any nucleotide sequences having large genus of 90% sequence identity to the SGR1 and SGR2 genes would have change in any useful function other than the method mutating recited genes of SEQ ID NOs: 75-78 leading to function of increase in resistance to soybean rust, delayed senescence and/or chlorophyll retention. Furthermore, regarding argument on skilled in the art would recognize the members of genus of sequences claimed, the argument was not found persuasive since applicant has not described that the members of genus of sequences that when mutated would have useful function or would increase resistance to soybean rust, delayed senescence and/or chlorophyll retention. Applicant is claiming mutating large number of sequences in any of the 3’ region in promoters Instead applicant’s example only showed mutating at specific region of the promoter would cause increase in resistance to soybean rust, delayed senescence and/or chlorophyll retention by modifying genes of SEQ ID NOs: 75-78.
For example, figure 7 left pane shows CE35915 is not different than non-transformed control, furthermore CE35914, CE35906, CE40104, CE39930 also are no different than the non-transformed control (as shown with same mean comparison letters A and B). Thus if the percent severity similar in these edit call for each edited plant as compared to non-transformed control is observed in other mutation it is not predictable for example for CE35915 has edits on SGR1 and SGR2 sequences ranging from 5D to 70D and 6 insertions (I) (page 77, Table 3) which has mean disease rating not different from the untransformed control, in fact the rating is higher than the control (Figure 7) showing more disease even with the homozygous deletion lines having mutation in promoter regions of SGR1 and SGR2 sequences (Table 3, Figure 7).
Regarding argument on spacers having one, two, three, four, or five mismatches, since applicant does not have examples it is not clear whether such spacers with mismatches would work for SGR1 and SGR2 genes, since some function changes would have been associated with the specific motif changes for example Jiao et al. page 7, left last paragraph teaches RY motif with “CATGCA” in promoters of SGR1 and SGR1 and ABRE motifs which would have been required to be changed in specific positions to have functional changes in the coding region, therefore applicant has not described that any of the changes in the 2 nucleotides (i.e. 90% identity) of the recited spacer sequence as SEQ ID NO:90 would have described functional changes showed by applicant as increase in resistance to soybean rust, delayed senescence and/or chlorophyll retention.
Furthermore, Rodriguez-Leal et al. is clear to show that for the increase or decrease in expression of an associated gene specific mutational changes in specific promoter region of the gene are needed and some mutation causes increase, other causes decrease and other do not have effect to the expression of the gene at all. Furthermore, applicant’s example also showed various changes in expression when SGR1 and SGR2 genes were targeted in different regions of promoter. Therefore, a skilled in art would not be able to readily predict function of nucleic acid which has at least 90% identity to SEQ ID NOs: 75-82, and 90 and amino acid sequence having at least 90% sequence identity to SEQ ID NO: 83 and it is impossible to predict such a broad sequence variation will have any required function. Furthermore, applicant has not shown that the Applicant has not described any mutation in the promoter of SGR1 or SGR2 would significantly increase disease resistance also showed by Applicant’s Figures 4, 6 and 7.
Regarding argument on claim 14 recites that the guide nucleic acid binds to a target site, applicant has not described genus of guide nucleic acid that binds to the large genus of sequence having 90% sequence identity to any one of the nucleotide sequences of 75 and 76 in the 3’ region.
Following 35 USC § 103 obviousness rejection has been modified in light of applicant’s amendment of claims 1 and 14 to recite target is in 3’ region of the a sequence having at least 90% identity to SEQ ID NOs:75 and 76. Furthermore, previous 35 USC § 102 rejection over claim 14 has been reanalyzed as 103 rejection.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
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.
Obvious over Chang et al. and further in view of Gao et al., Shi et al. and Lippman et al.
Claims 1, 3-4, 6-11, 13 and 17 are rejected under 35 U.S.C. 103 as being unpatentable over Chang et al. (Published: 2019, Journal: Plant Physiology, 180: 711–717), and further in view of Gao et al. (Published: 2016, Journal: Molecular Plant 9: 1272–1285) and further in view of Lippman et al. (WIPO International Pub. No.: WO 2018/213547 A1, publication date: 22 November 2018), and further in view of Shi et al. (Published: 2016, Journal: Acta Physiol. Plant 38:97 DOI 10.1007/s11738-016-2105-y).
Claims are drawn to a method of producing a soybean plant comprising mutated endogenous genes SGR1 and SGR2 with a gene editing system wherein the mutation is generated in the promoter region of the genes.
Regarding claims 1, 4, and 10-11, Chang et al. teaches the SDS foliar chlorosis was lowest in the mutant genotype wherein expression level of the GmSGR1 and GmSGR2 increases post inoculation (page 714, Figure 2), the increase was higher for LD01-5907 (wild type) compared to the mutant soybean variety Soybean variety GD2422 (d1d1d2d2 genotype) the mutant Soybean variety GD2422 (d1d1d2d2 genotype) maintained its relative chlorophyll content (see Figure 3 below). Therefore, lower expression of the GmSGR1 and GmSGR2 would have been associated with stay green trait in the mutants.
Chang et al. teaches GmSGR1 (as D3) (Glyma.11G027400) and GmSGR2 (Glyma.01G214600) (as D1) are the STAY-GREEN gene, were found in SoyBase used in their study (page 713, right first paragraph, Supplemental Figure S3) which is the same gene as applicant described as their wildtype genes see Spec, page 76, Table 1 as locus 37 and locus 38 as SGR2 and SGR1 genes as SEQ ID NOs: 76 and 75. Furthermore, NCBI BLAST sequence alignment of the SGR protein elected as SEQ ID NO: 73 and 74 shows 100% sequence identity t0 a D2 and D1 (senescence-inducible chloroplast stay-green protein) of soybean (Glycine max) (see enclosed PDF) which are applicant described SGR1 and SGR2 genes.
Chang et al. teaches the SDS foliar chlorosis was lowest in the mutant genotype wherein expression level of the GmSGR1 and GmSGR2 increases post inoculation (page 714, Figure 2), the increase was higher for LD01-5907 (wild type) compared to the mutant soybean variety Soybean variety GD2422 (d1d1d2d2 genotype) the mutant Soybean variety GD2422 (d1d1d2d2 genotype) maintained its relative chlorophyll content (see Figure 3 below). Therefore, the mutant had delayed senescence or had longer chlorophyll retention.
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Gao et al. teaches NYE1/SGR1 is key regulator of chlorophyll catabolism in diverse plant species (page 1272, Abstract) wherein the mutation in the ABRE motifs of the truncated promoter sequence in Arabidopsis NYE1/SGR1 genes showed significant difference in the ABA responsiveness for the expression of the NYE1/SGR1 genes (see Figure 1 below, page 1275, left first paragraph). Gao et al. teaches ABRE consensus motif A1 core G-box motif (CACGTG) such motif are present in both Applicant’s SEQ ID NOs: 75 and 76 (see sequence listings).
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Furthermore, Shi et al. teaches transcripts of GmSGR1 which has 100% identity to applicant’s SEQ ID NO: 3 (see Shie et al. supplementary Figure 1) were gradually increased in leaves along with the leaf senescent progression which was showed by expression of GUS driven by GmSGR1 promoter showing GmSGR1is involved in senescence signaling and chlorophyl degradation (page 96, Abstract). Shi et al. teaches GmSGR1 promoter harbors a set of critical cis-acting regulatory elements that function in regulating gene transcription, including one of TATA box (motif sequence of TTATTT, locating at-182) that involves the binding with RNA polymerases and five of CAAT boxes (motif sequence of CAAT, locating at-259, -298,-410,-533, and-798, respectively) etc. that control the frequency of gene transcription (see Fig. 2 below). Sinc the motifs are mostly located at the 3’ end of the promote of GmSGR1 gene, someone skilled in the art would make mutation in this 3’ region.
Shi et al. teaches the promoter elements are also involved in ethylene and jasmonic acid response (page 97, right paragraph 2). Therefore someone skilled in the art would target the promoter region of SGR1 and its ortholog SGR2 leading to change in the chlorophyll degradation. Furthermore, Shi et al. teaches over expression of GmSGR1 increase chlorophyl degradation (page 96, right second to last paragraph), therefore someone skilled in the art would obviously try to decrease the expression for delayed chlorophyl degradation.
Shi et al. teaches negatively regulating GmSGR1 via genetic manipulation can be used as a possible strategy for generation of soybean germplasm with improved productivity through modulation of Chl degradation and PSII efficiency at the late growth stages (page97, right first paragraph). Shi et al. teaches presence of D1 and D2 system in soybean involved in Chl degradation metabolism (page 97, left paragraph 2).
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Chang et al., Gao et al. and Shi et al. do not teach the mutation is created by contacting a target site in the promoter region of an endogenous SGR1 and SGR2 genes with a gene editing system comprising a nuclease comprising a cleavage domain and a nucleic acid binding domain binding to the SGR1 and SGR2 genes.
Lippman et al. teaches cis - regulatory elements in gene promoters present an exciting target for creating new, weak alleles with the ultimate goal of modulating crop yield traits (page 66, lines 11-12). Lipman et al. teaches gene editing using CRISPR/Cas9 technology to produce genetic variation that changes the expression -hence the activity of a single gene in a controlled and directed manner wherein the technology can be used to target specific sets of genes with major effects, taking advantage of genomic information regarding the developmental patterns and genes controlling yield traits, pathogen resistance etc. (page 87, lines 1-12). Lipman teaches the method would be used in soybean (page 34, lines 14-18). Lipman et al. teaches an example of a method of generating mutated promoters using CRISPR/Cas9 (i.e. a gene editing system comprising a nuclease comprising a cleavage domain) and eight sgRNAs (i.e. a gene editing system comprising a nucleic acid binding domain) in tomato plant targeting upstream of the transcriptional start site in SlCLV3 gen, wherein the DNA sequencing revealed modification in the transcriptional expression of the gene and range of new phenotypes (page84, lines 23-26, page 85, lines 1-7, See figure 3below). Thus, such method would produce in a soybean plant a mutation in cis-regulatory elements in gene promoters that would change expression of the gene leading to phenotype changes.
Furthermore, Lippman et al. teaches targeting 3’ region of the WUS regulatory element by Cas9 (page 67, lines 21-30). Lippman et al. teaches the regulatory region (i.e. promoter) is located downstream of the 3’ end of coding region (page 9, lines 10-19).
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Thus it would have obvious to someone skill in art from teaching, suggestion and motivation from Chang et al. for a method of altering the expression of the SGR1 and SGR2 for modulation of disease resistance and stay green trait in soybean, furthermore teaching from Gao et al. that the targeting of the promoter region of SGR gene would alter the expression of the SGR gene leading to the change in stay green traits, and further suggestion from Shi et al. that the manipulation of specific region of the promoter of SGR1 gene by decreasing its expression would delay chlorophyl degradation and someone skilled in the art would have carried out by the method described by Lippman et al. to target specific region of the promoters and screen for mutation leading to the change in stay green traits in the soybean plant. Someone skilled in the art would combine arts to develop a recited method for producing a soybean plant comprising a mutated endogenous SGR1 and SGR2 gene and since the domains affecting expression of the genes are located in 3’end of the promoter someone skilled in the art would target the 3’ region of the promoter as taught by Shi et al.
Regarding claim 3, CRISPR Cas9 cleaves the endogenous genes. Furthermore, Chang et al. teaches endogenous SGR1 and SGR2 genes in soybean.
Regarding claims 6-9, Lipman et al. teaches CRISPR Cas9 induced mutation are deletion, insertion etc. (page15, paragraph 0077) or combination of the mutation. A combination of deletion and insertion would equivalent to at least a substitution and that would be for a, G, C or T.
Regarding claim 13, the plant would have been the obvious product of the obvious method of claim 1.
Regarding claim 17, Shi et al. teaches presence of motif such as “AACGTG”, “TCTGGTG” effecting expression of gene which are located in the 1000-2298 region of promoter of SEQ ID NO:75. Furthermore, someone skilled in the art would also target the 100-2260 region of SEQ ID NO:76 as GmSGR2 gene.
Response to Argument
Applicant's arguments filed 04/17/2026 have been fully considered but they are not persuasive.
Applicant argues claim 1 is amended to further define the target site in the promoter region of the endogenous SGRI gene as being in the 3' region of a sequence having at least 90% identity to SEQ ID NO:75, and the target site in the promoter region of the endogenous SGR2 gene as being in the 3' region of a sequence having at least 90% identity to SEQ ID NO:76 that would lead to increase in resistance to soybean rust, delayed senescence and/or chlorophyll retention (Response to Rejection, page 11, paragraph 2).
Applicant assert Gao et al. is directed to regulators of chlorophyll degradation and leaf senescence in Arabidopsis and describes truncations in NYEI promoter fragments to test abscisic acid (ABA) responsiveness, but Gao et al. does not teach or suggest a mutation in the promoter region of an endogenous gene or a phenotype associated with the same (Response to Rejection, page 11, paragraph 3). Applicant argues Gao et al. teach about SDS foliar chlorosis by a mutation in coding sequence of SGR1 and SGR2, and Lippman'547 et al. does not teach SGR1 and SGR2 and the recited traits (Response to Rejection, pages 11-12, last and first paragraphs respectively).
Regarding argument on Chang et al. does not teach gene editing system to produce mutation in promoter region the argument was not found persuasive since the art teaches the SEQ ID NOs: 75 and 76 in soybean and it clearly teaches that the lower expression of the GmSGR1 and GmSGR2 is associated with stay green trait in the mutants. Furthermore, Gao et al. showed the predictable science that the specific deletion in promoter region leads to altered expression of the stay green genes.
Regarding argument on Lippman’547 is random not directed to a particular target site, the argument was not found persuasive since applicant’s claim does not recite targeting specific region of promoter and Lippman’547 does teach targeting 3’ promoter region. Furthermore, Lippman et al. teaches their sgRNA are target putative regulatory region of a gene within 5 kb upstream or downstream of the coding sequence (page8, paragraph 0057), the sgRNA are complementary to a target sequence within a target region (page 10, paragraph 0062), Furthermore, Lippman et al. teaches in FIGs. 2C-2F is a diagram showing that CRISPR-Cas9 targeting the region downstream of WUS containing the le motif in S.pim and S.lyc disrupted a putative AGAMOUS binding site (CArG) using specific sgRNA leading to increase in locule numbers in fruits (page7, paragraph 0043).
Therefore Change et al. and further in view of Gao et al. and Lippman et al. showed the targeting promoter region would alter expression of a stay green genes SGR1 and SGR2 leading to predictable change in function for example alteration of senescence process. Furthermore, applicant does not recite any function changes except claim 2 and recite targeting any site of a promoter region leading to mutated SGR1 and SGR2 gene with any genomic structure. Furthermore, the newly added art of Shi et al. showed since the many of the motif or domains effecting expression of the genes are located at 3’ region of the promoter, a skilled in the art would target 3’ region of the promoter for mutation.
Furthermore, where a rejection of a claim is based on two or more references, a reply that is limited to what a subset of the applied references teaches or fails to teach, or that fails to address the combined teaching of the applied references may be considered to be an argument that attacks the reference(s) individually. Where an applicant’s reply establishes that each of the applied references fails to teach a limitation and addresses the combined teachings and/or suggestions of the applied prior art, the reply as a whole does not attack the references individually as the phrase is used in Keller and reliance on Keller would not be appropriate. This is because the test for obviousness is what the combined teachings of the references would have suggested to a person having ordinary skill in the art (PHOSITA).”
Obvious over Chang et al. and further in view of Lipman et al. and Nowak et al.
Claims 14 and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Chang et al., and further in view of Lipman et al. and Nowak et al. (Published: 2016, Journal: Nucleic Acids Research, 44 (20): 9555–9564 doi: 10.1093/nar/gkw908).
Claim recites a guide nucleic acid that binds to sequence having at least 90% sequence identity to SEQ ID NOs: 81 or 82 and any sequence with 90% identity to any one of SEQ ID NOs: 75-80.
Regarding claim 14, Shi et al. teaches transcripts of GmSGR1 which has 100% identity to applicant’s SEQ ID NO: 3 (see Shie et al. supplementary Figure 1) were gradually increased in leaves along with the leaf senescent progression which was showed by expression of GUS driven by GmSGR1 promoter showing GmSGR1is involved in senescence signaling and chlorophyl degradation (page 96, Abstract). Shi et al. teaches GmSGR1 promoter harbors a set of critical cis-acting regulatory elements that function in regulating gene transcription, including one of TATA box (motif sequence of TTATTT, locating at-182) that involves the binding with RNA polymerases and five of CAAT boxes (motif sequence of CAAT, locating at-259, -298,-410,-533, and-798, respectively) etc. that control the frequency of gene transcription (see Fig. 2 below). Sinc the motifs are mostly located at the 3’ end of the promote of GmSGR1 gene, someone skilled in the art would make mutation in this region.
Shi et al. teaches the promoter elements are also involved in ethylene and jasmonic acid response (page 97, right paragraph 2). Therefore someone skilled in the art would target the promoter region of SGR1 and its ortholog SGR2 leading to change in the chlorophyll degradation. Furthermore, Shi et al. teaches over expression of GmSGR1 increase chlorophyl degradation (page 96, right second to last paragraph), therefore someone skilled in the art would obviously try to decrease the expression for delayed chlorophyl degradation.
Shi et al. teaches negatively regulating GmSGR1 via genetic manipulation can be used a possible strategy for generation of soybean germplasm with improved productivity through modulation of Chl degradation and PSII efficiency at the late growth stages (page97, right first paragraph). Shi et al teaches presence of D1 and D2 system in soybean involved in Chl degradation metabolism (page 97, left paragraph 2).
Lipman teaches the method would be used in soybean (page 34, lines 14-18). Lipman et al. teaches an example of a method of generating mutated promoters using CRISPR/Cas9 (i.e. a gene editing system comprising a nuclease comprising a cleavage domain) and eight sgRNAs (i.e. a gene editing system comprising a nucleic acid binding domain) in tomato plant targeting upstream of the transcriptional start site in SlCLV3 gen, wherein the DNA sequencing revealed modification in the transcriptional expression of the gene and range of new phenotypes (page84, lines 23-26, page 85, lines 1-7, See figure 3below).
Chang et al. teaches primer sequences (i.e. nucleic acid sequence) in Supplemental Table S4 with range of nucleic acid fragment from GmSGR1 and GmSGR2 genes, for example “GGAGCCACCATTACAAGCG” has 100% sequence identity to SEQ ID NO:75 and 81 and it is located on 3’ end of the SEQ ID NO:75. Thus the sequence would bind to the SGR1 gene. Furthermore, the sequence is a guide nucleic acid since the first two nucleotides “GG” would have been used as Protospacer Adjacent Motif (PAM) sequence for Cas9. See for example Lippman et al. teaches that “NGG” where N can be any other nucleotide is a PAM sequence for Cas9 nuclease in a guide RNA (page 25, lines 17-29).
Alignment of Chang et al.’s primer “GGAGCCACCATTACAAGCG” to the SEQ ID NO: 75:
Query: None Query ID: lcl|Query_8062113 Length: 19
>
Sequence ID: Query_8062115 Length: 2298
Range 1: 1946 to 1964
Score:35.6 bits(38), Expect:5e-07,
Identities:19/19(100%), Gaps:0/19(0%), Strand: Plus/Plus
Query 1 GGAGCCACCATTACAAGCG 19
|||||||||||||||||||
Sbjct 1946 GGAGCCACCATTACAAGCG 1964
Furthermore, SEQ ID NO:75 is a UTR region as defined as “untranslated regions of SGR genomic sequences as described herein (e.g., SEQ ID N0:75 or SEQ ID N0:76) (pages 18 and 19, last and first paragraph). There the region is a portion of the promoter region and is located on 3’ end.
Chang et al. does not teach designing gRNA comprising a spacer of a gene.
Nowak et al. teaches combining spacer sequence and repeat sequence to develop guide RNA (page 9556, Figure 1).
Therefore someone skilled in the art before effective date of filling of the invention from teaching suggestions and motivation from Shi et al. would try to mutate the 3’ region of promoter of SEQ ID NO:75 and would select the fragment taught by Chang et al. as target. Furthermore, develop a gRNA as taught by Lippman et al. and Nowak et al. Leading to the invention of the guide RNA that would bind to any of sequence with 90% identity to SEQ ID NOs: 75 or 76. The target site would have been at 3’ end of the SGR1 sequence as taught by Shi et al. that would comprise domains to decrease expression of the gene leading to delayed chlorophyl degradation.
Regarding claim 18, Shi et al. teaches presence of motif such as “AACGTG”, “TCTGGTG” effecting expression of gene which are located in the 1000-2298 region of promoter of SEQ ID NO:75. Furthermore, someone skilled in the art would also target the 100-2260 region of SEQ ID NO:76 as GmSGR2 gene located on 3’ end.
Response to Argument
Applicant's arguments filed 04/17/2026 have been fully considered but they are not persuasive.
Applicant argues ordinarily skilled artisan would understand that the repeat sequence of a guide nucleic acid could not be any sequence or fragment, as it must be a sequence that will function in the CRISPR-Cas system being used. See, e.g., specification at page 66, lines 18 to 24. Applicant argues Chang’s sequence comprising "GG," alleged to be evidenced by Lippman as a PAM sequence for Cas9, is not the same as a repeat sequence, both as described in the specification and in view of the knowledge of the ordinarily skilled artisan (Response to rejection, page 10, paragraph 2).
Applicant argues the cited primer sequence of Chang was used in a PCR reaction to amplify genomic DNA encoding GmSGR1. See, Chang, Supplemental Methods, lines 51-58. Applicant submits that a primer is not, nor would it be construed by one of ordinary skill in the art to be, a guide nucleic acid. Applicant argues the specification (and general knowledge in the art) specifies that a guide nucleic acid has at least two components, namely at least one spacer sequence that is complementary to (and hybridizes to) a target DNA and at least one repeat sequence (e.g., of a CRISPR-Cas system). See, e.g., the paragraph spanning pages 65 and 66 of the specification. Applicant argues there is no additional sequence associated with the primer of Chang that would constitute a repeat sequence. Applicant argues therefore, Chang fails to teach a guide nucleic acid as claimed.
Applicant's arguments filed have been fully considered but they are not persuasive since applicant defines “A "guide nucleic acid," "guide RNA," "gRNA," "CRISPR RNA/DNA" "crRNA" or "crDNA" as used herein means a nucleic acid that comprises at least one spacer sequence, which is complementary to (and hybridizes to) a target DNA (e.g., protospacer), and at least one repeat sequence (e.g., a repeat of a Type V Cas12a CRISPR-Cas system, or a fragment or portion thereof; a repeat of a Type II Cas9 CRISPR-Cas system, or fragment thereof; a repeat of a Type V C2c 1 CRISPR Cas system, or a fragment thereof; a repeat of a CRISPR-Cas system of, for example, C2c3, Cas12a (also referred to as Cpfl), Casl2b, Cas12c, Casl2d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Casl, CaslB, Cas2, Cas3, Cas3', Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csx12), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, C1m·3, Crm4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG), and/or Csf5, or a fragment thereof), wherein the repeat sequence may be linked to the 5' end and/or the 3' end of the spacer sequence” (Spec, page 64 and 66). Therefore the main component is a spacer sequence that is complementary to SGR1 or SGR2 and repeat sequence would comprise a fragments of repeat of for example of Cas9 wherein since fragment of the repeat sequence could be any sequence. For example applicant defines “As used herein with respect to nucleic acids, the term "fragment" or "portion" refers to a nucleic acid that is reduced in length relative (e.g., reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 340, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, or 900 or more nucleotides or any range or value therein) to a reference nucleic acid and that comprises, consists essentially of and/or consists of a nucleotide sequence of contiguous nucleotides identical or almost identical (e.g., 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to a corresponding portion of the reference nucleic acid (page 16, lines 4-13). Therefore it is not clear what does it mean to be “consists essentially of” or “almost identical” in the fragments of repeat sequence of any of a CRISPR-cas effector protein therefore the repeat sequence would have been any sequence few to many sequences in the disclosed sequence of Chang et al. for example if the sequence was required by any of the larger numbers of 340 -900 or more or any range or value therein. This would lead to some fragments which would be as small as single nucleotides. This makes the main components of guide RNA is spacer sequence and PAM sequences that is discloses by Chang et al. wherein the 90% identity would allow small fragment to be associated with the guide RNA.
Furthermore, the new art of Nowak et al. has been analyzed that teaches designing a guide RNA for a specific spacer sequence for a gene, see analysis above.
Conclusion
No claim is allowed.
Claims 12 and 15 are free of prior art. closest prior art is Gao et al. which does not expressly teach spacer sequence SEQ ID NO:90 binding to the SGR1 and SGR2 leads to mutation of the SGR1 and SGR2 by a nuclease. Furthermore, Gao et al. does not teach guide nucleic acid which would also comprise repeat sequence (see definition above) and comprise SEQ ID NO:90. There is no specific teachings to choose sequence of SEQ ID NO:90 as spacer sequence in the method of mutating a soybean plant and making it a guide nucleic acid.
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/SANTOSH SHARMA/Examiner, Art Unit 1663
/Amjad Abraham/SPE, Art Unit 1663
