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 .
Claim Status
Claims 1-3 and 5-19 are pending.
Claims 1-3 and 5-19 are examined on the merits.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 2-3, 6-9, and 18-19 rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claims 2-3, and 6-8 recite optional limitations using the term “preferably”. For example, claim 2 recites “and preferably, the mutant mirR396 is selected from…”; claim 3 recites “and preferably, an amino acid sequence of the Cas12i…”; claim 6 recites “and preferably, the mutant miR396 is introduced….”; claim 7 reacts “and preferably, an amino acid sequence of the Cas12i….”; and claim 8 recites “wherein preferably, an amino acid sequence of the Cas12i…”. the use of “preferably” renders the scope of the claims unclear because it is uncertain whether the limitations following “preferably” are required limitations of the claimed invention or merely optional embodiments. Therefore, the metes and bounds of the claims cannot be determined with reasonable certainty.
Dependent claims 9, 18, and 19 are included in this rejection because they do not include additional limitations to resolve the ambiguity.
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
Claims 1-3, and 5-19 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 rejected for lacking adequate written description support regarding the broad scope of the following.
Claim 1 is directed to a mutant miR396, wherein the mutant miR396 undergoes a base mutation relatives to parental wild-type soybean miR396, the miR396 is selected from any one or more of miR396a, miR396b, miR396c, miR396d, miR396f, and miR396i, and the mutant miR396 is able to improve seed size, seed weight, number of seeds per plant, or yield and soybean.
The scope of claim 1 is broad because it encompasses any base mutation, including a base deletion, base insertion, or base substitution, in any one or more of six soybean miR396 family members. The specification states that “the base mutation included a base deletion, a base insertion, or a base substitution” (p43, pa2). The specification also identifies soybean miR396a, miR396b, miR396c, miR396d, miR396f, and miR396i as SEQ ID NO: 2-7 (p64, pa1).
However, the working examples are limited to particular deletion events at particular positions in selected edited combinations. For example, the specification describes edited plant acd as having miR396a deletions at bases 229-233 relative to SEQ ID NO: 2, miR396c deletions at bases 229-230 relative to SEQ ID NO: 4, and miR396d deletions at bases 19-29 relative to SEQ ID NO: 5 (p71). The specification describes edited plant acdf as having a miR396a deletion at base 229, miR396b deletions at bases 30-100, miR396c deletion at base 229, miR396d deletions at bases 18-26, and miR396f deletions at bases 264-265 (p72).
The specification also provides additional specific deletion embodiments in the summary, including miR396a deletions at base 229-233, miR396 deletions at bases 228-253 or 229-230, miR369d deletions at bases 18-26 or 19-29, miR396f deletions at base 265 or base 264-265, and miR396i deletions at bases 200-201 (p43-46, Summary).
Thus, the specification provides support for certain specific deletion mutants and selected edited combinations, but claim 1 is not limited to those disclosed deletion positions or edited combinations. Claim 1 instead covers any deletion, insertion, or substitution at any position in any one or more of six miR396 family members, so long as the mutant provides improve seed size, seed weight, number of seeds per plant, or yield. The specification does not describe a representative number of species across that full genus, and does not identify structural features common to mutant miR396s that would distinguish operative mutants from non-operative mutants.
Accordingly, the specification does not reasonably convey to one of ordinary skill in the art that applicant was in possession of the full scope of the claimed mutant miR396 genus.
Claim 3 is deficient for the same reason. Claim 3 recites a gene editing reagent able to cause generation of the mutant miR396 according to claims 1-2 in soybean. The specification describes Cas12i/gRNA reagents and states that the gRNA targets miR396 in soybean and guides Cas12i to genomic locus to mutate miR396 (p47-52). The specification further describes Cas 12i variants having N369R/S433R mutations and S7R/D233R/D267R/N369R/S433R mutations (p47-50, 65-68). The specification also identifies four gRNA guide sequences as SEQ ID NO: 8-11 and the DR sequence as SEQ ID NO: 12 (p51, p64).
However, claim 3 is not limited to the disclosed guide sequences, disclosed target sites, disclosed Cas12i variants, or the specific deletion mutants shown in the examples. Because the mutant miR396 genus of claims 1-2 is not adequately described for its full scope, the broader gene-editing reagent genus for generating that mutant miR396 genus is likewise not adequately described.
Claims 5-19 are deficient for the same reasons because they recite uses, gene-editing reagents, methods of introducing mutant mirR396, methods of gene editing, regeneration, breeding, hybridization, or downstream applications involving the mutant miR396 of claims 1-2 and/or the gene editing reagent of claim 3. Claims 11 and 13 recite additional combinations of mutant miR396 family members undergoing base deletion, but these combinations remain within the same broadly claimed genus discussed above and are not limited to t the particular deletion positions or edited combinations expressly described in the specification. Claims 12 and 18-19 further recite functional performance characteristics or downstream breeding methods that likewise rely upon the same broadly claimed mutant miR396 genus. Because the speciation does not adequately describe the full scope of the claimed mutant miR396 genus or the full scope of reagents capable of producing that genus, the specification also does not reasonably convey possession of the cull scope of the claimed gene-editing reagent, method, and breeding claims.
Scope of Enablement
Claims 1-3, 5-19 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 for making and using the specific soybean miR396 genes explicitly identified as miR396a, miR396b, miR396c, miR396d, miR396f, and miR396i (SEQ ID NO: 2-7), the disclosed Cas12i gene-editing system including the specific Cas12i variants and guide RNAs (SEQ ID NO: 8-12), and the particular edited soybean plants containing the specific deletion mutations expressly described in the specification (including the disclosed acd, acdf, cdf, abdcf, ad, adf, abcd, acfi, and bcdfi mutant combinations); does not reasonably provide enablement for the full scope of the claimed invention, namely any mutant miR396 that undergoes a base mutation relative to parental miR396 and is able to improve seed size, seed weight, number of seeds per plant, or yield in soybean. 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.
Claim 1 broadly encompasses any base mutation in any one or more of soybean miR396a, miR396b, miR396c, miR396d, miR396f, and miR396i, provided that the mutant improves seed size, seed weight, number of seeds per plant, or yield in soybean. The specification states that a base mutation includes “a base deletion, a base insertion, or a base substitution” (p43, pa2), and identifies soybean miR396a, miR396b, miR396c, miR396d, miR396f, and miR396i as SEQ ID NO: 2-7 (p64).
The specification defines the claimed functional results by percentage increases over parental soybean carrying wild-type miR396. For example, improved seed size is defined as at least 3% larger than parental soybean, improved seed weight is defined as at least 5% higher than parental soybean, and improved number of seeds per plant and improved yield are similarly defined by percentage increases over parental soybean (p48-49).
However, the working examples are limited to selected deletion mutants and selected edited combinations. The specification describes edited plants acd, acdf, cdf, abdcf, aci, adf, abcd, acfi, and bcdfi, each having specific deletion events at particular miR396 family members and positions (p71-73). For example, adc contains deletions in miR396a, miR396c, and miR396d, while acdf contains deletions in miR396a, miR396c, miR396d, miR396f, and abcdf contains deletions in miR396a, miR396b, miR396c, miR396d, and miR396f (p71-72).
The specification does not show that the claimed functional results are predictable for any base mutation in any one or more of the six miR396 family members. To the contrary, the specification reports different trait outcomes for different edited combinations. The specification states that edited plants acd and cdf did not increase per-plant yield or number of seeds per plant, although hundred-seed weight was enhanced, while edited plants acdf and abcdf had per-plant yield, number of seeds per plant, and hundred-seed weight significantly improved (p72).
Therefore, a person of ordinary skill in the art would need to generate and screen numerous mutant miR396 alleles and combinations to determine which mutations actually improve seed size, seed weight, number of seeds per plant, or yield. Such experimentation would increase designing or obtaining mutations, regenerating soybean plants, growing the plants, and measuring the claimed agronomic traits. The specification does not provide sufficient guidance to enabled the full scope of substitutions, insertion, deletion, mutation positions, and miR396 member combinations encompassed by claim 1 without undue experimentation.
Claim 3 is also not enabled for its full scope. The specification describes Cas12i/gRNA editing and specific Cas12i mutant proteins, including N369R/S433R and S7R/D233R/D267R/N369R/S433R variants (p47-50, 65-68), and identifies guide sequences SEQ ID NO: 8-11 and DR sequence SEQ ID NO: 12 (p51, p64). However, claim 3 broadly covers a gene editing reagent able to generate the mutant miR396 of claims 1-2. Because claims 1-2 encompass a broad functional genus of mutant miR396s, a skilled artisan would need to design and test additional guide RBAs and editing conditions generate edited soybean plants, and screen the resulting plants for the claimed agronomic improvements.
Claims 5-19 are not enabled for the same reasons because they require using, producing, editing, regenerating, breeding hybridizing, or otherwise employing the broadly claimed mutant miR396 and/or gene-editing reagent. Claims 11 and 13 additionally encompass numerous combinations of deleted soybean miR396 family members, while claims 12 and 18-19 further require achieving the claimed agronomic improvements or producing downstream breeding products using those broadly claimed mutants. A person of ordinal skill would still be required to generate and evaluate numerous additional mutant alleles and combinations to determine which mutants achieve the claimed functional improvements. Accordingly, the specification does not enable the full scope of claims 4-19 without undue experimentation.
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 1-2 are rejected under 35 U.S.C. §103 as being unpatentable over Zhang (Jinshan Zhang et. al., National Science Review (2019) Sep 27;7(1):102–112), in view of Noon (Jason B. Noon et. al., Journal of Experimental Botany (2019) Vol. 70, No. 5 pp. 1653–1668).
Claim 1 recites a mutant microRNA396 (miR396), wherein the mutant miR396 undergoes a base mutation relative to parental miR396; the parental miR396 is wild-type (WT) miR396 derived from soybean; the miR396 is selected from any one or more of miR396a, miR396b, miR396c, miR396d, miR396f, and miR396i; and the mutant miR396 is able to improve a seed size, a seed weight, a number of seeds per plant, or a yield for the soybean.
Zhang teaches that miR396 directly represses growth-regulating factors (GRFs), and that disrupting MIR396 genes can improve yield-related traits (p102 Abstract). Specifically, Zhang states that knockout of MIR396e and MIR396f enhanced grain size and panicle branching, resulting in increased grain yield (Abstract). Zhang further teaches that CRISPR lines with mutations in mature miR396 regions were selected for further study, and that mir396ef mutants showed increase grain length, width, and thickness by 8.4%, 7.95%, and 8.16%, respectively, relative to wild type, and increased 100-grain weight from 25.12 +/- 0.30g in wild type to 31.78 +/- 0.55g in mir396ef plants (p103, right column, pa3-4; Fig 1B-1D). Zhang also teaches that mir396ef plants had longer panicles, more primary branches, higher grain numbers per pinnacle, and about 4% increase in grain yield under conventional nitrogen fertilization (p109, left column, pa3; Fig 2I-2L). Zhang further teaches that mir396ef plants showed about a 15% increase in grain yield under nitrogen-deficient conditions and a 25% increase in above-ground dry biomass (p109, left column, pa3; Fig 3E-3G).
Zhang does not expressly teach soybean miR396 genes.
Noon teaches the soybean miR396-GRF (growth-regulating factors) network (Title and Abstract). Noon further identifies nine canonical soybean pre-miR396 genes (p1656, right column, pa1). Pre-miR396 sequence information are listed in supplemental materials (Table S1 and Table S2; also see below). Deep sequencing efforts have revealed that miRNAs in soybean are differentially expressed during seed development, flowering time, and the shoot apical meristem (p1654, right column, pa4).
Sequence alignment analysis shows that the disclosed pre-miR386a sequence is a 191-nucleotide sequence that exhibits 100% sequence identity to a corresponding fragment of soybean locus LOC100527871 (see below). Further, SEQ ID NO: 2 recited in the claims exhibits 100% sequence identity to a portion of the LOC100527871 gene sequences (see below alignment), indicating that the claimed sequence corresponds to a naturally occurring sequence present within the soybean genome. The miR396a sequence identified in the alignment corresponds to the claimed miR396a, which is represented by SEQ ID NO: 2 in the present application.
It would have been obvious to one of ordinary skill in the art to mutated soybean MIR396 family members, such as miR396a, as taught by Noon in view of Zhang’s teaching that CRISPR mutation/disruption of MIR396 genes improving grain size, grain weight, grain number, and yield. A person of ordinary skill would have been motivated to apply Zhang’s MIR396 disruption strategy to soybean because Noon teaches that soybean contains MIR396 gene family, especially, the claimed soybean miR396a sequence, represented by SEQ ID NO:2, corresponds to a naturally occurring soybean miR396a/pre-mirR396a region and exhibits 100% identity to a corresponding fragment of soybean locus LOC100527871. A person of ordinary skill would have had a reasonable expectation that reducing or disrupting soybean miR396a sequence using Zhang’s CRISPR-based MIR396 disruption approach to obtain soybean plants with altered miR396 regulation and improved yield-related traits.
Claim 2 recites the mutant miR396 of claim 1, wherein the mutation is a base deletion in one or more specified combinations of miR396a, miR396b, miR396c, miR396d, miR396f, and/or miR396i.
For the same reason set forth above with respect to claim 1, Zhang teaches CRISPR-generated MIR396 mutants, including insertion and deletion mutations at sgRNA targets sites (p104, Fig 1B), where Zhang explains that plus and minus numbers represent nucleotides inserted and deleted at sgRNA target sites and that mutations on different mir396 members are separated by slashes. Zhang further teaches that CRISPR lines with mutations in mature miR396 regions were selected because such mutations should disrupt miR396 function, and that mir396ef mutants with different genotypes displayed the same seed phenotypes (p103, right column pa3). Zhang teaches single (mir3963), double (mir396ef), triple (mir396abc), and higher-order (mir396abced) MIR396 mutants (p103, right column, pa2), demonstrating the CRISPR editing of individual and multiple MIR396 family members was known and that selection of particular combinations of MIR396 mutation was within the ordinary skill in the art. Noon teaches the corresponding soybean MIR396 genes. therefore, it would have been obvious to produce deletion mutants in one or more soybean MIR396 family members, including the claimed soybean MIR396 combinations, because Zhang teaches that CRISPR-generated MIR396 mutations, including, deletions at target sites, disrupt MIR396 function and improve yield-related traits, and Noon identifies the soybean MIR396 targets to which Zhang’s strategy would be applied.
Claim 2 is obvious over Zhang and Noon.
Claims 3, and 5-14, 16, 18-19 are rejected under 35 U.S.C. §103 as being unpatentable over Zhang (2019) in view of Noon (2019) as apply to claim 1, and further in view of Xie (Hongtao Xie, CN114107370 A, Application 2021-12-03, Publication 2022-03-01).
Claim 1 as the teachings of Zhang and Noon are discussed above.
Claims 3 and 5 are interpreted as dependent of claims 1.
Claim 3 recites a gene-editing reagent comprising Cas12i and gRNA configured to generated the mutant miR396 of claims 1 in soybean.
For the same reason set forth above with respect to claim 1-2, Xie teaches using Cas12i and a gRNA for gene editing in soybean, wherein the gRNA comprises a backbone region that binds Cas12i and a guide sequence that hybridizes to a target sequence (claim 1). Xie further teaches that the gRNA includes a first segment, also referred to as a framework/backbone region, protein-binding segment, protein-binding sequence, or direct repeat sequence, and a second segment, also referred to as a targeting sequence or guide sequence, wherein the first segment interacts with Cas12i and the guide sequence directs Cas12i to a specific nucleotide sequence within the target nucleic acid (p2, Disclosure of Invention, III). Xie also teaches that the guide sequence may be altered or modified to hybridize to any desired sequence within the target nucleic acid (p2, Disclosure of Invention, III). Xie teaches constructing a soybean gene-editing vector comprising Cas12i protein and gRNA, including gRNA guide sequence, PAMs, and a direct repeat sequence, and using Cas12i for gene editing in soybean (Example 1; claim 5).
Xie does not teach gene editing on soybean MIR396.
Noon teaches soybean MIR396 genes, and Zhang teaches that MIR396 disruption improves yield-related traits. It would have been obvious to substitute the soybean MIR396 target sequences taught by Noon for the target of Xie in order to use Xie’s Cas12i/gRNA soybean editing system to disrupt soybean MIR396 genes for the yield-related purpose taught by Zhang. Accordingly, claim 3 is obvious over Zhang, Noon and Xie.
Claim 5 recites a method for producing soybean with an improved seed size, seed weight, number of seeds per plant, or yield by introducing the mutant miR396 of claim 1 into soybean material and regenerating a soybean plant. Claim 6 recites the method of claim 5, wherein endogenous soybean miR396 is mutated to introduce the mutant miR396, optionally through gene editing.
For the same reasons set forth above with respect to claim 1, Zhang teaches generating MIR396 mutants by CRISPR-mediated mutagenesis and obtaining plants exhibiting improved yield-relate traits. Noon teaches the corresponding soybean MIR396 gene. Xie teaches gene editing, transformation, regeneration, and recovery of edited soybean plants (claim 1 and Example 1). Therefore, it would have been obvious to mutate endogenous soybean MIR396 and regenerate a soybean plant as recited.
Claims 5 and 6 are obvious over Zhang, Noon, and Xie.
Claim 7 recites the method according to claim 6, wherein the gene editing is conducted with Cas12i in the soybean cell, the soybean seed, the soybean tissue, or the soybean part; and preferably, an amino acid sequence of the Cas 12i undergoes mutations of 369th and 433rd amino acids relative to the sequence shown in SEQ ID No. 1; or the amino acid sequence of the Cas12i undergoes mutations of 7th, 233rd, 267th, 369th, and 433rd amino acids relative to the sequence shown in SEQ ID No. 1. Claim 8 recites a method of gene editing soybean using Cas12i and gRNA targeting soybean miR396, followed by regeneration of a soybean plant.
For the same reasons set forth above with respect to claim 6, Xie teaches gene editing in soybean using Cas12i and gRNA and delivering Cas12i/gRNA into soybean plant cells, seeds, tissues, or plant parts, and regenerating edited soybean (claim 1 and Example 1). Xie further teaches a Cas12i nuclease having an amino acid sequence that is 100% identity to SEQ ID NO: 1 (see alignment below). The term “preferably” is permissive rather than mandatory and therefore does not constitute a positive claim limitation. Consequently, the obviousness analysis is based on the required limitations of claim7, and the prior art need not disclose the preferred Cas12i mutation. Therefore, claim 7 and 8 are obvious.
Claim 9 recites the method according to claim 8, wherein the guide sequence for hybridizing with the target sequence in the gRNA is shown in any one of SEQ ID No. 8-11.
For the same reasons set forth above with respect to claim 8, Xie teaches that the guide sequence may be altered or modified to hybridize to any desired sequence within the target nucleic acid, and shows designing gRNA guide sequences according to soybean target genes and PAMs (Example 1). Noon identifies the soybean MIR396 target genes. therefore, it would have been obvious to design guide sequences hybridizing to soybean MIR396 target sequences. The use of particular guide sequences targeting soybean MIR396 would have been an obvious matter of routine guide design once soybean MIR396 genes were selected as the target. Accordingly, claim 9 is obvious.
Claim 10 recites a method for producing soybean with an improved seed size, seed weight, number of seeds per plant, or yield, comprising a step of hybridizing a soybean seed or a soybean plant prepared by the method according to claims 5 with another soybean variety to produce the soybean with the improved seed size, seed weight, number of seeds per plant, or yield.
Xie teaches a method of preparing a soybean plant comprising crossing an edited high-oleic soybean plant with another soybean plant to produce a soybean plant (claim 9). Therefore, once the soybean MIR396-edited plant would have been obvious from Zhang, Noon, and Xie, hybridizing the edited soybean plant with another soybean variety to produce soybean with improved seed size, seed weight, number of seeds per plant, or yield would have been obvious.
Claim 10 is obvious over Zhang, Noon, and Xie.
Claim 11 depends from claim 3 and further specifies particular combinations of soybean miR396 family members undergoing base deletions.
For the same reasons discussed with respect to claims 2 and 3, Zhang teaches generating deletion mutations in MIR396 family members using CRISPR. In particular, Zhang teaches that mir396ef double mutants showed increased grain length, width, thickness, and 100-grain weight compared with wild type, and further teaches that both MIR396e and MIR396f contribute to grain-size development (Fig 1B-D; p103, right column). Zhang also teaches generating higher-order miR396avcef mutants by crossing mir396abc mutants with mir396ef mutants (p103, right column, pa3), thereby suggesting that multiple MIR396 family members may be edited in combination to regulate seed/grain trats. Noon teaches the corresponding soybean MIR396 family members, and Xie teaches Cas12i/gRNA gene editing in soybean. Selection of particular combinations of edited soybean MIR396 genes would have been an obvious design choice in applying the known CRISPR editing strategy to soybean MIR396 targets. Accordingly, claim 11 is obvious.
Claim 12 recites quantitative improvements in seed size, seed weight, seed number, and yield.
Zhang expressly teaches MIR396 mutant plants exhibiting increased grain size, grain weight, grain member, and yield. In particular, Zhang teaches that mir396ef mutants had increased grain length, width, and thickness by 8.46%, 7.95%, and 8.16%, respectively, and increased 1000-grain weight of 31.78 +/- 0.55 g compared with 25.12+/-0.30 g in WT (Fig 1D; Fig 3E-3G; p106). Thus, Zhang teaches quantitative improvements in grain/seed size, grain/seed weight, deed number, and yield. Applying the same MIR396 editing strategy to soybean as suggested by Noon and implemented using the soybean Cas12i editing system of Xie would have rendered the claimed performance limitation obvious. Accordingly, claim 12 is obvious.
Claim 13 recites the method of claim 5, and recites the same deletion combinations discussed from claim 11 in the context of the production method of claim 5.
For the same reasons discussed with respect to claims 5, 6, and 11, claim 13 would have been obvious.
Claim 14 recites the method of claim 13, wherein the introducing the mutant miR396 comprises a step of gene editing of the soybean cell, the soybean seed, the soybean tissue. Claim 16 recites the method of claim 10, wherein the introducing the mutant miR396 comprises a step of gene editing endogenous miR396 into the soybean cell, the soybean seed, the soybean tissue, or the soybean part.
For the same reasons set forth above with respect to claim 10, and 13, Xie expressly teaches gene editing endogenous soybean genes using Cas12i/gRNA in soybean cells, tissues, seeds, regenerated plants (claim 1; Example 1). Claim 14 and 16 are obvious.
Claim 18 recites hybridizing the edited soybean plant with another soybean variety to produce progeny processing the improved agronomic traits.
For the same reasons set forth above with respect to claims 8 and 10, Xie teaches a method of preparing a soybean plant comprising crossing an edited high-oleic soybean plant with another soybean plant to produce a soybean plant (claim 9). A person of ordinary skill would have been motivated to apply conventional plant breeding by crossing an improved plant with another variety to transfer desirable traits. Therefore, claim 18 is obvious.
Claim 19 recites the method according to claim 18, wherein the guide sequence for hybridizing with the target sequence in the gRNA is shown in one of SEQ ID NOS: 8-11.
For the same reasons set forth above with respect to claims 1 and 18, Xie teaches designing guide RNAs for hybridization to desired soybean target sequence, while Noon teaches the soybean MIR396 target loci. Selecting particular guide sequence targeting soybean MIR396 would have been routine optimization once the target loci were chosen. Accordingly, claim 19 is obvious.
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Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to YANXIN SHEN whose telephone number is (571)272-7538. The examiner can normally be reached Monday-Friday.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Amjad A Abraham can be reached at (571)272-7058. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/YANXIN SHEN/Examiner, Art Unit 1663
/WEIHUA FAN/Primary Examiner, Art Unit 1663
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