DETAILED ACTION
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 08/05/2026 has been entered.
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 .
Claim Status
Claims 1, 4, 11, 12, 16, 18, 31, 52, 57, 59, 60, 64, 66, 68, 79-81, 83, 89 and 98-99 are pending.
Claims 1, 16, 31, 52, 60, 66, 83, and 98 are currently amended.
Claim 99 is newly added.
Claims 1, 4, 11, 12, 16, 18, 31, 52, 57, 59, 60, 64, 66, 68, 79-81, 83, 89 and 98-99 are examined on the merits.
Claim Rejections - 35 USC § 112
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.
Written Descriptions
Claim 1, 4, 11, 12, 16, 18, 31, 52, 57, 59, 60, 64, 66, 68, and 98 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.
The Federal Circuit has clarified the application of the written description requirement. The court stated that a written description of an invention "requires a precise definition, such as by structure, formula, [or] chemical name, of the claimed subject matter sufficient to distinguish it from other materials". University of California v. Eli Lilly and Co., 119 F.3d 1559, 1568; 43 USPQ2d 1398, 1406 (Fed. Cir. 1997). The court also concluded that "naming a type of material generally known to exist, in the absence of knowledge as to what that material consists of, is not description of that material". Id. Further, the court held that to adequately describe a claimed genus, Patent Owner must describe a representative number of the species of the claimed genus, and that one of skill in the art should be able to "visualize or recognize the identity of the members of the genus". Id.
The claims are broad in the following aspects:
Claim 1 and its dependent claims recite a soybean plant comprising a base deletion in the region of an endogenous BZR1 gene encoding the PEST domain, wherein the deletion disrupts phosphorylation sites in the PEST domain, and the plant exhibits an improved yield trait relative to a soybean plant lacking the mutation.
Claim 1 does not limit the deletion of a particular position within the PEST-domain-encoding region, a particular deletion size, a particular number or identity of phosphorylation sites, or a particular resulting change in the BZR1 polypeptide. Accordingly, claim 1 encompasses numerous structurally different deletion throughout the PEST-domain-encoding region, provided that the deletion produces the two recited functional results: disruption of phosphorylation sites and improvement of a yield-associated trait.
BZR1 is a transcription factor whose activity is controlled by phosphorylation and dephosphorylation within regulatory regions that include the PEST domain. Active, dephosphorylated BZR1 accumulates in nucleus and regulates downstream brassinosteroid-responsive genes involved in plant growth and reproductive development (p739) (Baoqiang Wang, Genome-wide identification, structural analysis, and expression profiles of the BZR gene family in tomato, Journal of Plant Biochemistry and Biotechnology, Volume 31, pages 739–750, 2022). Therefore, whether a PEST-region deletion produces increased BZR1 activity and improved yield depends on the particular position and size of the deletions, the number and identity of the phosphorylation-associated residues affected, and the resulting effect on BZR1 phosphorylation, localization, and transcriptional activity.
The specification describes an editing strategy directed to the PEST-domain regions of four soybean BZR1 genes: Glyma·17g248900 (SEQ ID NO:69), Glyma·14G076900 (SEQ ID NO:72), Glyma·06G034000 (SEQ ID NO:75), and Glyma·04G033800 (SEQ ID NO:78). Example 2 and Table 1 provide specific edited alleles (alleles A-J), each defined by particular small deletions (e. g., 7-49 bp) round the PEST region in those same four BZR1 genes, with the resulting amino acid changes set forth in SEQ ID Nos: 117, 119, 121, 123, 125, 127, 129, 131, 133, and 135 (Example 2 and Table 1).
However, these examples do not establish a common relationship between deletion position or size, the particular phosphorylation sites disrupted, and the resulting yield phenotype. The disclosed deletions differ in their locations and lengths and therefore remove or alter different numbers and combinations of amino-acid residues within the PEST regulatory region. The specification does not identify which individual phosphorylation sites must be disrupted, how many sites must be disrupted, or which deleting boundaries consistently shift BZR1 toward its active, dephosphorylated states.
The phenotypic results in Tables 2-6 further demonstrate that the effect is dependent on the particular deletion. Although some disclosed PEST-region deletions are associated with increased pod or seed number, other closely related deletions produce little improvement or an apparent reduction in one or more yield-associated traits (Tables 2-6). Thus, merely locating a deletion within the PEST-domain-encoding regions does not predict whether that deletion will disrupt the relevant phosphorylation regulation in a manner that increases BZR1 activity and produces improved yield.
The state of the art likewise establishes that the PEST region is functionally sensitive to the particular mutation introduced. Song teaches that phosphorylation-associated residues within the PEST region regulate the activity and localization of soybean BZR1/BZL proteins (Li Song et. al., BMC Plant Biology (2019) 19:86, pp1-15). Tang demonstrates that deletion of amino acids 232-251 from the BZR1 PEST region disrupts its phosphorylation-regulatory interaction (Wenqiang Tang et. al., Natural Cell Biology (2011)Volume 13, Number 2, pp124-131). Zhang demonstrate that mutation of a particular residue, P216 of soybean GmBZL2, increases the ration of active, dephosphorylated protein and produces gain-of-function effect (Yu Zhang et. al., Scientific Reports (2016) volume 6, Article number: 31134, pp1-14). Collectively, these references show that the functional results depend on which residue or residues are altered and on how the particular alteration affects phosphorylation regulation; they do not establish that deletions of different sizes or at different positions through the PEST region would predictably produce the same gain-of-function and improved-yield phenotype.
Accordingly, the specification describes only a limited set of particular PEST-region deletions and does not disclose either representative species across the claimed genus of deletion positions, deletion sizes, and affected phosphorylation sites or common structural features by which a POSITA could identify which PEST-region deletions will produce both required functional results. The specification therefore does not reasonable convey that Applicant possessed the full genus of PEST-region base deletion that disrupts phosphorylation sites and produces an improved-yield phenotype at the time of filing.
Scope of Enablement
Claim 1, 3, 4, 8, 11, 12, 16, 18, 31, 52, 57, 59, 60, 64, 66, 68, and 98 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specifications, while being enabling a soybean plant (or part) comprising one of the specifically described edited alleles A-J (SEQ ID NO: 116-135) does not reasonably provide enablement for the full scope of the claimed inventions. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims.
An “analysis of whether a particular claim is supported by the disclosure in an application requires a determination of whether that disclosure, when filed, contained sufficient information regarding the subject matter of the claims as to enable one skilled in the pertinent art to make and use the claimed invention.” MPEP 2164.01. “A conclusion of lack of enablement means that. . . the specification, at the time the application was filed, would not have taught one skilled in the art how to make and/or use the full scope of the claimed invention [i.e. commensurate scope] without undue experimentation.” In re Wright, 999 F.2d 1557,1562, 27 USPQ2d 1510, 1513 (Fed. Cir. 1993); MPEP 2164.01.
In In re Wands, 858 F.2d 731,8 USPQ2d 1400 (Fed. Cir. 1988), several factors implicated in determination of whether a disclosure satisfies the enablement requirement and whether any necessary experimentation is “undue” are identified. These factors include, but are not limited to:
(A) The breadth of the claims;
(B) The nature of the invention;
(C) The state of the prior art;
(D) The level of one of ordinary skill;
(E) The level of predictability in the art;
(F) The amount of direction provided by the inventor;
(G) The existence of working examples; and
(H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure. In re Wands, 858 F.2d 731,737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988). No single factor is independently determinative of enablement; rather “[i]t is improper to conclude that a disclosure is not enabling based on an analysis of only one of the above factors while ignoring one or more of the others.” MPEP 2164.01. Likewise, all factors may not be relevant to the enablement analysis of any individual claim.
Claims 1 and the dependent claims recite soybean plant comprising a base deletion in the region of an endogenous BZR1 gene encoding the PEST domain, wherein the deletion disrupts phosphorylation sites in the PEST domain, and the plant exhibits an improved yield trait relative to a soybean plant lacking the mutation.
The specification provides working examples (examples 1-3 paragraph 0333-0337) in soybean (Glycine max), describes an editing strategy focused on the PEST domain of four endogenous soybean BZR1 genes. Examples 1-3 and Table 1 disclose alleles A-J containing particular deletions of approximately 7-49 base pairs within or around those PEST regions, together with resulting amino-acid changes and phenotype measurements (pa0333-0337; Tables 1-6).
However, claim 1 is not limited to the particular deletions exemplified in the specification. The claim encompasses base deletions of different sites and at different nucleotide positions throughout the PEST-domain-encoding region. It also does not identify which phosphorylation sites must be disrupted, how many phosphorylation sites must be disrupted, or which deletion boundaries will produce the required improved-yield phenotype.
BZR1 is a transcription factor whose activity is controlled by phosphorylation and dephosphorylation. Active, dephosphorylated BZR1 accumulated in the nucleus and regulated downstream brassinosteroid-responsive genes involved in plant growth and reproduction development (Wang, pages 739–750). Accordingly, whether a PEST-region deletion produces the claimed result depends on the precise position and size of the deletion, the number and identity of the phosphorylation-associated residues affected, and the resulting effect on BZR1 phosphorylation, localization, stability, and transcriptional activity.
The specification does not provide sufficient guidance for predicting these results from the location of deletion within the PEST region alone. It does not identify a particular phosphorylation site or combination of sites whose disruption consistently increased active, dephosphorylated BZR1, or does it define a deletion size or positional range that constantly produces improved yield.
The state of the art confirms this sensitivity to the particular mutation. Song teaches that soybean BmBZL3 (SEQ ID NO:75 in instant claims) contains multiple BIN2/GSK3 phosphorylation sites, and a C-terminal PEST domain that contains a highly conserved proline residue. Song further demonstrates that a single P219L substitution withing the PEST region converts GmBAL3 into a dominant gain-of-function alleles, whereas increased expression of wild-type GmBZL3 does not produce the same functional result (p4-5). Thus, song demonstrates that BZR1 activity depends on alteration of a particular residue within PEST region, rather than merely the presence of an unspecified mutation somewhere in that region.
Tang identifies amino acids 215-251 as a regulatory region containing the BAR1 PEST domain and demonstrates that deletion of amino acids 232-251 abolishes PP2A interaction and substantially alters BZR1 phosphorylation regulation (p128, Fig. 4a-c). Zhang similarly demonstrates that a particular P216L mutation in the conserved PEST mutation in the conserved PEST region of soybean GmBZL2 increases the dephosphorylated-to phosphorylated ration and produces increased BmBZL2 activity (Fig. 2b-c; p5, pa3). Theres references confirm that the biochemical effect depends on the particular residue or residues altered and the resulting change in phosphorylation regulation. They do not provide a general rule by which a POSITA could predict the effect of deletions having different sizes and positions throughout the PEST region.
Practicing claim 1 throughout its full scope would therefore require a POSITA to design and introduce numerous deletions at different positions and of different sizes within the PEST-domain-encoding region. For each deletion, the POSITA would then need to determine which phosphorylation sites were affected, measure the resulting phosphorylation state and activity of BZR1, regenerated soybean plants, and evaluated the plants for the required improved-yield phenotype.
Indeed, the specification itself describes generating BZR1 variation, regenerating plants, assaying progeny for one or more improved-yield traits, selecting progeny exhibiting the desired phenotype, and repeating the process as necessary. This constitutes a generate-and-screen approach for discovering successful members of the claimed functional genus, rather than predictive guidance identifying which PEST-region deletions will satisfy both the phosphorylation-disruption and improved-yield limitations.
The experimental results further demonstrate the need for such screening. Alleles A-J contain different deletions within the PEST-region-encoding sequence, but the phenotype data in Tables 2-6 show heterogeneous results, including increases in certain yield-associated trats for some alleles and little change or reductions for other alleles. Thus, even among the expressly tested PEST-region deletions, the desired improved-yield phenotype cannot be reliably predicted from the presence of a deletion withing the PEST region. Of experimentation required to identify which deletion positions, sizes, and affected phosphorylation sites produce both claimed functional results would be substantial. The specification provides only limited working examples, the relationship between deletion structure and yield phenotype is unpredictable, and each candidate deletion requires empirical biochemical and plant-level testing. Considering the breadth of the claimed deletion genus, the limited predictive guidance, the heterogenous experimental results, and the amount of require screening, practicing the full scope of claim 1 would require undue experimentation.
Accordingly, the specification does not enable the full scope of claim 1 and its dependent claims. Therefore, the claims are rejected under 35 U.S.C. 112(a) for lacking enablement.
Response to Applicant’s Remarks:
Written Description:
Applicant argues that the amended claims are sufficiently supported because the specification discloses SEQ ID BI: 71, 74, 77, and 80, the corresponding PEST-domain sequences, and working examples showing PEST-domain sequences, and working examples showing PEST-region deletions that disrupt phosphorylation sites and produce improved yield traits.
The argument has been considered but is not persuasive. Although the amendments narrow the claims and the specification provides support for the specifically exemplified BZR1 sequences and edited alleles, the claims remain broader than those examples and encompass numerous PEST-region deletions across BZR1 polypeptides withing the recited sequence-identity scope. As discussed above, the specification does not provide representative species or common structural features sufficient to demonstrate possession of the full genus of deletions that will both disrupt phosphorylation sites and produce the recited improved-yield phenotype. Accordingly, the written description rejection is maintained.
Enablement:
Applicant argues that the specification enables the claims because a person of ordinary skill can identify PEST-region deletions by sequence analysis and screen resulting mutants for improved yield traits. The argument has been considered but is not persuasive. The issue is not whether the specifically exemplified edits can be generated or whether mutants can ultimately be identified by screening, but whether the full scope of the claimed genus can be practiced without undue experimentation. The specification does not provide a predictive relationship between deletion position or size within the PEST region and the resulting phosphorylation and yield phenotype. Rather, the specification’s’ own examples show heterogeneous phenotypic outcomes among different PEST-region deletions and instruct the skilled artisan to generate mutations, regenerate plans, assay progeny, and select those having the desired phenotype. Thus, practicing the full scope of the scope would require mutation-by-mutation generation and empirical screening rather than routine confirmation of profitable embodiments.
Accordingly, the enablement region is maintained.
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.
Claims 1, 4, 11, 12, 16, 18, and 31 are rejected under 35 U.S.C. §103 as being unpatentable over Song (2019) in view of Tang (2011), Lu (Qing Shi Mimmie Lu et. al., BMC Biotechnology (2022) 22:7) and Zhang (2016).
Claim 1 recites a soybean plant or part thereof comprising at least one mutation in an endogenous BRASSINAZOLE-RESISTANT 1 (BZRI) gene encoding a BZR1 polypeptide, wherein the endogenous BZR1 gene: encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 71, 74, 77, or 80, wherein the at least one mutation comprises a base deletion in the region of the endogenous BZR1 gene that encodes the PEST domain of the BZR1 polypeptide, and wherein the at least one mutation disrupts phosphorylation sites in the PEST domain, and wherein the soybean plant or part thereof comprising the at least one mutation exhibits a phenotype of improved yield traits as compared to a soybean plant that is devoid of the at least one mutation.
Under the broadest reasonable interpretation consistent with the specification, the limitation “a base deletion in the region of the endogenous BZR1 gene that encodes the PEST domain” encompasses deletion of one or more nucleotide bases located within the portion of the endogenous BZR1 gene encoding the PEST domain. The specification defines mutations to include base deletions and explains that deletions may involve one or more nucleotides and may be in-frame or frameshifting. The specification further describes base deletions ranging from a single nucleotide to multiple consecutive nucleotides within and endogenous BZR1 gene. Accordingly, the claim does not require deletion of the entire PEST domain, a particular deletion size, or an in-frame deletion. However, claim 1 separately requires that the mutation “disrupts phosphorylation sites in the PEST domain”, and therefore the claimed deletion must also affect one or more phosphorylation-associated sites within PEST domain.
The limitation “exhibits improved yield traits” is likewise interpreted broadly consistent with the specification. The specification defines “improved yield traits” to include measurable improvements in traits associated with plant productivity or growth, including, for example, increased seed number, seed weight, biomass, branching, pod production, and related yield components. The specification further explains that an improved yield trait is determined relative to a control plant and my result from differences in organ number or size, plant architecture, flowering, nutrient uptake, or other characteristics contributing to yield (pa 0098). Thus, claim 1 does not require an increase in total commercial soybean yield, but encompasses an improvement in a yield-associated component, such as increased seed number, relative to a soybean plant lacking the mutation.
Song teaches GmBZL3 (sequence shows in fig 1) and GmBZL3 is SEQ ID NO:77 in instant claims, Song further teaches GmBZL1, 2, 4 are also shown to be SEQ ID 71, 74, 80 respectively (see alignment below). Song further teaches that soybean BZR1-line/BZL proteins contain a PEST domain with multiple phosphorylation-related residues, and that the phosphorylation state of this regulatory region controls BZR/BZL activity and BR-related phenotypes (page 3, right column). Song also teaches naturally occurring soybean allelic variation, including a nucleic acid deletion in soybean lines PI594599 and PI603154 causing a frameshift and premature stop codon (page 9, left column), and further teaches a PEST-region mutation, P129L, associated with altered localization, phosphorylation-related behavior, and bzr1-1D-like phenotypes (page 4, paragraph 2). Thus, song teaches the claimed soybean BZR1/BZL proteins, identifies the PEST region as a phosphorylation-regulatory region, and demonstrates that mutations of this region alter BZR1/BZL activity.
Song does not explicitly teach a soybean plant having an endogenous BZR1 base deletion specifically in the region encoding the PEST domain that disrupts phosphorylation sites and exhibits the claimed improved-yield phenotype.
Tang teaches that the BZR1 PEST region is a functionally significant regulatory region and identifies amino acids 215-251 as a PP2A-binding region containing the PEST domain. Tang further expressly generates a ΔPEST mutant by deleting amino acids 232-251 from full-length BZR1 and shows that deletion of this region abolishes PP2A interaction and substantially alters BZR1 phosphorylation regulation (p128, Fig. 4a-c). Tang therefore demonstrates that deletion within the PEST region disrupts the regulatory interaction controlling the phosphorylation state of BZR1.
Lu teaches a CRISPR/Cas genome-editing system for inducing heritable mutations in soybean via stable transformation and states that the system can be employed to edit other genes for soybean trait improvement (Abstract). Lu further teaches guide-RNA-directed targeting of selected soybean genomic loci and production of deletions and other indels in endogenous soybean genes (p2, left column; p3, right column; p10-11, Fig. 7). Lu therefore provides an established method for introducing the claimed based deletion into the PEST-domain-encoding region of an endogenous soybean BZR1/BZL gene.
Zhang teaches that GmBZL2 is a soybean BZR1 orthologue containing a conserved PEST domain and identifies P216 within the PEST domain as corresponding to P234 of Arabidopsis BZR1 (Fig. 2b-c; p5, pa3). Zhang further teaches that the P216 mutation increases the dephosphorylated/phosphorylated ratio of GmBZL2 and enhances GmBZL2 activity (Fig. 3d-e). Thus, Zhang directly demonstrates that mutation of the conserved PEST region increases the active, dephosphorylated form of the soybean BZR1 orthologue and thereby produces a gain-of-function effect.
Zhang further teaches that GmBZL2 is a transcription factor. Increased active, dephosphorylated GmBZL2 accumulates in the nucleus and regulates downstream brassinosteroid-responsive genes involved in plant growth and reproductive development.
Zhang demonstrates that expression of mutated GmBZL2P216L enhances BR signaling and affects seed number per silique, and express suggests that increased GmZRL2 activity could increase seed number per pod and thereby increase soybean-seed yield (Figs. 4 and 6; p11, pa2-3).
It would have been obvious to a person of ordinary skill in the art at the time of the invention to introduce a base deletion into the PEST-domain-encoding region of the endogenous soybean BZR1 gene in view of the combined teachings of Song, Tang, and Lu. Song teaches the functional importance of the soybean BZR1 PEST region and that alteration of this region affects BZR1 activity; Tang expressly demonstrates that deletion within the corresponding BZR1 PEST region alters BZR1 regulation; and Lu provides an established soybean genome-editing method for introducing targeted deletions into endogenous genes.
A POSITA would have been motivated to introduce the base deletion to disrupt phosphorylation-associated site within the PEST region and thereby alter the phosphorylation state and activity of the BZR1/BZL protein. Zhang provides a reasonable expectation that mutation of the corresponding soybean PEST region would increase the active, dephosphorylated form of GmBZL2 and produce a gain-of-function protein. Because GmBZL2 is a transcription factor, the increased active protein would regulate downstream brassinosteroid-responsive genes controlling plant growth and reproductive development. Zhang further provides a reasonable expectation that this enhanced BZR1/BZL activity would increase seed number or another yield-associated trait and thereby improve soybean yield.
Accordingly, claim 1 is obvious over Song in view of Tang, Lu and Zhang.
Claim 4 recites the soybean plant or part thereof of claim 1, wherein at least one mutation results in a mutated BZR1 polypeptide having increased activity or constitutive activity.
For the same reason set forth with respect to claim 1, Song teaches that a PEST-region mutation such as GmBZL3P219L can produce bzr1-1D-like phenotypes indicative of enhanced or constitutive BZR1-related activity (page 4, paragraph 2). Tang further teaches that the BZR1 PEST region is a functionally significant regulatory region (Tang, p128, Fig. 4). Zhang further teaches that mutation of the conserved P216 residue within the GmBZL2 PEST domain increases GmBZL2 activity and increases the dephosphorylated/phosphorylated ration of the protein (Fig. 3d-e).
Claim 4 is obvious over Song in view of Tang, Lu and Zhang.
Claim 11 recites the soybean plant or part thereof of claim 1, wherein the base deletion is an in-frame deletion. Claim 12 recites the soybean plant or part thereof of claim 1, wherein the base deletion is an out-of-frame deletion.
For the same reason set forth with respect to claim 1, Tang further expressly teaches deletion of amino acids 232-251 within the BZR1 PEST region while retaining an expressed BZR1 ΔPEST protein (p128, Fig. 4a-c). Lu teaches CRISPR/Cas editing in soybean producing coding-region deletions, including deletions resulting in frameshift mutations (p10-11, Gig. 7).
Therefore, claims 11 and 12 are obvious over Song in view of Tang, Lu and Zhang.
Claim 16 recites the soybean plant or part thereof of claim 1, wherein PEST domain is a region of the BZR1 polypeptide having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 97, 98, 101, 102, 105, 106, or 109.
Song teaches the soybean BZR1-like protein GmBZL3 and its conserved PEST-domain region. Sequence alignment shows that the GmBZL3 region spanning amino acids 225-260 is 100% identical to SEQ ID NO: 105 of the instant application (see below). Thus, Song teaches a BZR1 polypeptide comprising a region meeting the recited requirement of at least 80% sequence identity to one of SEQ ID NO: 97, 98, 101, 102, 105, 106, or 109.
For the same reason set forth with respect to claim 1, claim 16 is obvious over Song, Tang, Lu and Zhang.
Claim 18 recites the soybean plant or part thereof of claim 1, wherein the at least one mutation results in a mutated BZR1 gene having at least 90% sequence identity to a nucleotide sequence of SEQ ID NO:116, 118, 120, 122, 124, 126, 128, 130, 132, or 134 and/or a mutated BZR polypeptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:117, 119, 121, 123, 125, 127, 129, 131, 133, or 135.
For the same reason set forth with respect to claim 1, Song teaches natural soybean alleles exist with small deletions in the BZR1-faimily gene with a 1-bp deletion causing a frameshift and premature stop (page 9, paragraph 1). Tang further teaches a deletion within the BZR1 PEST region (p128, Fig. 4a-c).
Song does not teach the specific SEQ ID NOs:116, 118, 120, 122, 124, 126, 128, 130, 132, 134 and SEQ ID NOs:117, 119, 121, 123, 125, 127, 129, 131, 133, 135, nor the requirement that the mutated gene/polypeptides have “≥90% identity” to those particular sequences.
In view of Song’s teaching that small deletions/frameshifts in a soybean BZR1 family gene exist and alter the protein, Tang’s teaching of deletion within he BZR1 PEST region, and Lu’s teaching of targeted deletions in endogenous soybean genes, a POSITA would have been motivated to generate soybean plants having a mutated BZR1 gene/polypeptide with at least 90% sequence identity to the listed SEQ ID NO. In soybean, naturally occurring allelic variation among cultivars and closely related BZR1 sequences is expected, therefore implementing Song’s taught mutation in BZR1 coding sequences that remain highly homologous (≥90% identity) would have been a predictable, routine approach to obtain the same activity-shifted BZR1 while preserving core BZR1 structure and function. Accordingly, a POSITA would reasonably expect that BZR1 variants retaining ≥90% identity to the disclosed sequences, still carrying the taught mutation, would function as mutated BZR1 genes/polypeptides as recited in claim 18.
For the improved-yield limitation inherited from claim 1, Zhang further provides the teachings discussed above concerning activation of the soybean BZR1 orthologue GmBZL2 and the expected effect on seed number and soybean seed yield.
Accordingly, claim 18 is obvious over Song, Tang, Lu and Zhang.
Claim 31 recites a soybean plant regenerated from the plant part of claim 1 and comprising the mutation in the endogenous BZR1 gene, wherein the plant that is regenerated exhibits a phenotype of improved yield traits, compared to a plant that is devoid of the at least one mutation.
For the same reason set forth with respect to claim 1 and 16, Zhang teaches the relationship between increased activity of the soybean BZR1 orthologue GmBZL2 and increased seed production and expressly suggests application of this relationship to increasing soybean seed yield, and Lu teaches regeneration of genome-edited soybean plants. Accordingly, claim 31 is obvious over Song in view of Tang and Lu.
Accordingly, claim 31 is obvious over Song, Tang, Lu and Zhang.
Claims 52, 57, 59, 60, 68, 79-81, 83, 89 and 98-99 are rejected under 35 U.S.C. §103 as being unpatentable over Song (2019), in view of Tang (2011) and Lu (2022).
Claim 52 recites method for producing a soybean plant/part thereof having a mutation in an endogenous BZR1 by contacting a target site in the BZR1 gene with a nuclease (cleavage + nucleic acid binding domain) that binds to a target site in BZR1; BZR1 defined by identity to the SEQID NOs; wherein the target site comprises a nucleotide sequence encoding an amino acid sequence having at least 80% sequence identity to SEQ ID NOs: 97, 98, 101, 102, 105, 106, or 109.
Song teaches that BZR1 activity is regulated by phosphorylation, and specifically describes that BIN2 phosphorylates BZR1 and that BZR1 contains a phosphorylated PEST domain that includes conserved phosphorylation sites (page 3, right column). Song teaches that alterations in /around PEST region are functionally meaningful, describing a P219L mutation and stating that this mutation affects nuclear localization, protein stability, and phosphorylation / dephosphorylation (page 4, left and right column). Song teaches yield-relevant phenotypes tied to BZR1 variants, reporting that BmBZL2 (P216L)/GmBZL3(P219L) behave like bzr1-1D, and that overexpression of these variants increased seed number (and plant height) in the reported system (page 2, left column, paragraph 3).
Tang further teaches that the BZR1 PEST region is a functionally significant regulatory region and specifically deletes amino acids 232-251 within the PEST region, thereby altering BZR1 regulation (p128, Fig. 4a-c).
Song and Tang do not teach a soybean genome editing method that “contacts a target site” with a nuclease system to create mutations in an endogenous soybean BZR1 gene.
Lu teaches a CRISPR/Cas genome editing system for inducing heritable mutations in soybean via stable transformation, and explicitly states the system “can be employed to edit other genes for soybean trait improvement”(page1, abstract). Lu teaches that in engineered CRISPR/Cas9, Cas9 is guided to a specific genomic locus by a single guide RNA (sgRNA) containing a 20-nt target sequence adjacent to a PAM (page 2, left column). Lu teaches design/selection of guide sequences and routine specificity checking using CRISPR design tools and BLAST against soybean genomic resources (page 3, right column).
Given Song’s teaching that the soybean BZR1 PEST region is functionally important, Tang’s teaching that deletion within the BZR1 PEST region alters BZR1 regulation, and Lu’s established soybean CRISPR/Cas system, one of ordinary skill would have been motivated to target the PEST-domain-encoding region of endogenous soybean BZR1 using Lu’s genome-editing system, with a reasonable expectation of successfully generating the mutation.
Accordingly, claim 52 would have been obvious over Song, Tang and Lu.
Claim 57 recites the method of claim 52, wherein the mutation is a base substitution. Claim 59 recites the method of claim 52, wherein the mutation is a base deletion or a base insertion of one or more base pairs. Claim 60 recites the method of claim 59, wherein the mutation is an in-frame deletion or an out-of-frame deletion.
Lu teaches that CRISPR/Cas editing in soybean yields mutations including deletions/insertions, and further reports that substitutions can also occur among observed outcomes (page 5, left column, paragraph 2; page 7, left column, paragraph 1).
Lu teaches that soybean edits are short deletions (one to several nucleotides), longer deletions (~60 nucleotides), insertions (commonly 1 nt insertion), which in target coding regions, resulting frameshifting or amino acid altering (in-frame) (page 10, left and right column; page 11, fig 7).
For the same reasons set forth with respect to claim 52, Tang further teaches deletion within the BZR1 PEST region (p128, Fig. 4a-c).
Thus claim 57, 59 and 60 is obvious over Song, Tang and Lu.
Claim 68 recites the method of claim 52, wherein the mutation results in a mutated BZR1 gene having at least 90% sequence identity to a nucleotide sequence of SEQ ID NO:116, 118, 120, 122, 124, 126, 128, 130, 132, or 134 and/or a mutated BZR polypeptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:117, 119, 121, 123, 125, 127, 129, 131, 133, or 135.
Song teaches the functional importance of specific PEST/phosphorylation related alterations in soybean BZR1-type proteins. Tang further teaches deletion within the BZR1 PEST region.
Song does not teach producing soybean plants having a mutation that results in a mutated BZR1 gene or BZR1 polypeptides with ≥90% sequence identity to the specific edited sequence recited in claim 68, nor does Song disclose generating those specific edited alleles by genome editing.
Lu teaches that CRISPR/Cas9 in soybean produces a range of allele sequences, including deletion s extending to dozens of nucleotides, and that such edits can be inherited.
Accordingly, the recited “mutated BZR1 gene/polypeptide≥90% identity” to a set of edited sequences would have been obvious as it encompasses the expected spectrum of closely related edited alleles produced when applying Lu’s soybean CRISPR mutagenesis to the Song and Tong motivated BZR1 target region. Accordingly, claim 68 is obvious over Song, Tang and Lu.
Claim 81 recites a modified BRASSINAZOLE-RESISTANT 1 (BZR1) polypeptide comprising a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:117, 119, 121, 123, 125, 127, 129, 131, 133, or 135.
Song teaches soybean BZR1 family protein GmBZL3 (SEQ ID NO:77 in instant case) containing a PEST domain, identifying a conserved proline in the PEST region that affects protein localization and phosphorylation status, and describing a P219L PEST region mutation that produces bzr1-1D-like BR-response phenotypes when expressed in Arabidopsis (fig 1, figs2-3; page 4, right column and left column). Tang further teaches a modified BZR1 polypeptide containing a deletion within the PEST region, BZR1 ΔPEST, in which amino acids 232-251 are deleted.
Song and Tang do not teach using soybean CRISPR methods to generate endogenous edited alleles, nor does it explicitly disclose the applicants’ SEQ ID NOs as written.
Lu teaches a stable, heritable CRISPR-Cas9 workflow in soybean (Agrobacterium transformation using “half-seed” explants), producing indels with high efficiencies and transmission to progeny (abstract/results, construct description and mutation outcomes)
In view of Song’s identification of the PEST region and conserved residue affecting phosphorylation/localization and BR signaling, a POSITA would have been motivated to use Lu’s established soybean CRISPR system to generate high-identity BZR1 variants(e. g., sing-amino acid substitutions or small in-frame indels) in the endogenous soybean BRZR homologs with reasonable expectation of success, thereby meeting the broad ≥90%identity scope of claim 81. Accordingly claim 81 is obvious in view of Song, Tang and Lu.
Claim 79 recites a nucleic acid encoding the BRASSINAZOLE- RESISTANT 1 (BZR1) polypeptide of claim 81, wherein the nucleic acid comprises a mutation, further wherein the mutation is an in-frame base deletion or out-of-frame base deletion.
Song teaches BZR1-family proteins with a PEST domain and putative phosphorylation sites by GSK-3 kinase (sequence/feature mapping in fig.1) and that mutation in the PEST motif (P219L) alters BR response phenotypes (abstract). Tang further teaches deletion within he BZR1 PEST region.
Lu teaches CRISPR -Cas9 editing in soybean routinely yields insertions/deletions at targeted loci, including coding-region indels that can be in-frame depending on size(e.g., multiples of 3), and such edits can be inherited (Fig 7).
Accordingly, it would have been obvious to generate a soybean BZR1 nucleic acid comprising an in-frame or out-of-frame base deletion in the BZR1 target region.
Accordingly claim 79 is obvious in view of Song, Tang and Lu.
Claim 80 recites a nucleic acid of claim 79, wherein the nucleic acid comprises a mutated BZR1 gene having at least 90% sequence identity to a nucleotide sequence of SEQ ID NO:116, 118, 120, 122, 124, 126, 128, 130, 132, or 134.
For the same reasons set forth with respect to claim 79, Song teaches soybean BZR1-family genes and functional alteration of the conserved PEST region, Tang further teaches deletion within the BZR1 PEST region, and Lu teaches targeted soybean CRISPR/Cas editing producing localized deletions in endogenous gene.
Accordingly, the resulting mutated BZR1 gene would reasonably be expected to retain at least 90% sequence identity to the corresponding BZR1 sequence because the edit is localized, and the remainder of the gene sequence is retained.
Accordingly claim 80 is obvious in view of Song, Tang and Lu.
Claim 83 recites a soybean plant or part thereof comprising the nucleic acid of claim 79.
For the same reasons set forth with respect to claim 79, and because Lu teaches production of genome-edited soybean plants comprising heritable mutations, claim 83 is obvious in view of Song, Tang and Lu.
Claim 89 recites a plant genome or plant genomic DNA comprising a nucleotide sequence encoding the modified BRASSINAZOLE-RESISTANT 1 (BZR1) polypeptide of claim 81, wherein the nucleotide sequence is NO:116, 118, 120, 122, 124, 126, 128, 130, 132, or 134.
For the same reasons set forth with respect to claim 81, and because Lu teaches targeted editing of endogenous soybean genomic DBA, claim 89 is obvious in view of Song, Tang and Lu.
Claim 98 recites a guide nucleic acid that binds to a target site within an endogenous soybean gene encoding a BZR1 polypeptide, wherein the endogenous gene and encoded polypeptide satisfy the recited ≥80% sequence identity limitations, and wherein the target site comprises a nucleotide sequence encoding an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 97, 98, 101, 102, 105, 106, or 109.
Song teaches identification of soybean BZR1 family proteins and specific functional regions/feature (PEST region, phosphorylation-related sites) suitable for targeted modification.
Tang further teaches that the BZR1 PEST region is a functionally significant regulatory region and expressly demonstrates deletion within that region.
Lu teaches construction and use of sgRNA/guide RNAs to target specific soybean genomic loci for CRISPR editing using stable transformation and regeneration, including the standard guide/PAM targeting framework and successful recovery of edited plants.
Given Song’s and Tang’s teachings regarding the functional significance of the BZR1 PEST region, a POSITA would have been motivated to select that region as the target and use Lu’s routine guide-design methodology to design a guide nucleic acid direct to the corresponding endogenous soybean BZR1 PEST-domain-encoding region.
Accordingly, claim 98 is obvious in view of Song, Tang and Lu.
Claim 99 recites a guide nucleic acid of claim 98, wherein the guide nucleic acid comprises a spacer sequence having the nucleotide sequence of SEQ ID NO:112, 113, 114, or 115.
For the same reasons set forth with respect to claim 98, Lu teaches that CRISPR guide sequence are selected based on complementarity to a selected genomic target sequence and the presence of an appropriate PAM (p2, left column, pa1). SEQ ID NO: 112-115 corresponds to target sequences within the Song-disclosed soybean BZR1 gene region. Therefore, once the BZR1 PEST-domain region was selected as the target, selection of the recited spacer sequences would have been a routine matter of guide design.
Accordingly, claim 99 is obvious in view of Song, Tang and Lu.
Claims 64 and 66 are rejected under 35 U.S.C. §103 as being unpatentable over Song (2019), in view of Tang (2011), Lu (2022) as apply to claim 52, and Zhang (2016)
Claim 52 as the teachings of Song, Tang and Lu are discussed above.
Claims 64 and 66 are interpreted as dependent of claim 52.
Claim 64 recites the method of claim 52, wherein the mutation in the endogenous BZR1 gene produces a BZR1 polypeptide having reduced phosphorylation.
Song teaches that BZR1 is phosphorylated by BIN2, and that BZR1 contains a phosphorylated PEST domain with conserved phosphorylation sites(page 2, left column, paragraph 1; and page 3, left and right column). Song further teaches that changes in this region (e. g., P219L) are associated with altered phosphorylation /dephosphorylation behavior (page 10, left and right column).
Song does not teach how to generate endogenous BZR1 gene produces a BZR1 polypeptide having reduced phosphorylation.
Tang teaches that alteration of the BZR1 PEST region affects phosphorylation regulation and that the bzr1-1D mutation within the PEST region increases PP2A binding and increases the unphosphorylated form of BZR1 (p128, Fig 4d-e).
Lu teaches routine, targeted creation of indels/substitutions in soybean coding regions via CRISPR/Cas9.
Zhang teaches that the soybean BZR1 orthologue GmBZL2 containing a conserved PEST domain and that mutation of the conserved P216 residue to leucine results in a higher dephosphorylated/phosphorylated ratio than wild-type BmBZL2, demonstrating reduced phosphorylation and increased GmBZL2 activity (Fig. 3d-e).
Thus, for the same reasons set forth with respect to claim 52, a POSITA is motivated to modifying the BZR1 region implicated by Song and Tang using Lu’s method to generate endogenous BZR1 gene produces a BZR1 polypeptide. Zhang further provides a reasonable expectation that alteration of the conserved PEST regulatory region of a soybean BZR1 orthologue can reduce phosphorylation and increase BZR1 activity.
Accordingly, Claim 64 is obvious over Song, Tang, Lu and Zhang.
Claim 66 recites the method of claim 52, wherein the soybean plant that is produced exhibits an improved yield trait as compared to a soybean plant that is devoid of the mutation.
For the same reasons set forth with respect to claim 52, Song teaches that BZR1-type variants linked to increased reproductive output, such as increased seed number in the reported system (abstract). Tang further teaches that the BZR1 PEST region is a functionally significant regulatory region. Lu teaches targeted genome editing in soybean and recovery of heritable edited soybean plants.
Zhang further teaches that mutation of the soybean BZR1 orthologue GmZRL2 at the conserved PEST-domain residue P216 enhances GmBRL2 activity and BR signaling, and that expression of GmBAL2P216L results in increased seed number per silique. Zhang further states that these findings suggest potential application of GmBZL2 to increasing soybean seed yield.
Combining Song and Tang with Lu’s soybean editing method, a POSITA would be motivated to make BZR1 mutants in soybean expecting improvements in yield components (more seeds, pods, biomass, etc.). Accordingly, claim 66 is obvious over Song, Tang and Lu.
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Response to Applicant’s Remarks:
Applicant argues that Song does not teach a base deletion in the region of an endogenous soybean BZR1 gene encoding the PEST domain that disrupts phosphorylation sites, and that Song’s P219L gene encoding the PEST domain that disrupts phosphorylation sites, and that Song’s P219L PEST-region mutation is a substitution rather than a deletion. Applicant further argues that Song does not provide a reasonable expectation that such a mutation would result in the claimed improved yield traits.
Applicants’ arguments have been considered but are not persuasive in view of the region as presently formulated. The rejection does not rely on Song alone for the claimed PEST-domain deletion. Tang is additionally relied upon for teaching the functional significance of the BZR1 PEST region and for expressly demonstrating deletion within the PEST region, including deletion of amino acid 232-251, which alters BZR1 regulation (p128, Fig 4a-c). Song is relied upon for the soybean BZR1/BZL context, the functional significance of alteration of the PEST region, and BZR1-related growth and reproductive phenotypes, while Lu teaches targeted CRISRP/Cas genome editing in soybean. Zhang further teaches that mutation of the PEST region of the soybean BZR1 orthologues GmBZL2 alters its phosphorylation/activity and that enhanced GmBAL2 activity is associated with increased seed number and is suggested for improving soybean seed yield. Thus, the cited reference collectively provides both a reason to modify the soybean BZR1 PEST region and a reasonable expectation that an appropriately selected modification affecting BZR1 regulation could produce an improved yield-associated phenotype.
Applicant also argues that Lu merely demonstrates editing of PDS and does not teach editing BZR1 or provide a reasonable expectation of successfully producing the claimed BZR1mutaton.
This argument is not persuasive because Lu is relied upon for its established soybean genome-editing methodology, not for teaching BZR1 itself. In view of Song and Tang’s teachings identifying the BZR1 PEST region as a functionally significant target, a person of ordinary skill would have had reason to apply Lu’s soybean genome-editing method to that selected BZR1 region for the reasons discussed in the rejection above. Zhang further supports the expectation that altering regulation of a soybean BZR1 orthologue can affect seed-related traits.
Accordingly, Applicant’s arguments do not overcome the rejection under 35 U.S.C. §103.
Conclusion
No claims are allowed.
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/YANXIN SHEN/ Examiner, Art Unit 1663
/WEIHUA FAN/ Primary Examiner, Art Unit 1663
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