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, 5, 8-11,14, 30, 32, 47, 51, 54, 56, and 58-62 are pending. Claims 1-3, 5, 8-11, 14, 47, 54, and 56 are currently amended. Claims 1-3, 5, 8-11,14, 30, 32, 47, 51, 54, 56, and 58-62 are examined on the merits.
Response to Amendment
The objection of claims 2, 3, and 54 are withdrawn in view of amendment to the claims.
The rejection of Claim 1 rejected under 35 USC § 102(a)(1) is withdrawn in view of amendment to the claims. However, claim 1 is now rejected under 35 U.S.C. §103 for the reasons set forth below.
The rejections under 35 U.S.C. §103 are maintained and modified in view of Applicant’s claim amendments. The prior art applied continues to render the claimed subject matter obvious for the reasons set forth below.
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 1-3, 5, 8-11, 14, 30, 32, 56, 58, and 59 are rejected under 35 U.S.C. §103 as being unpatentable over Huang (Ruihua Huang et. al., Journal of Integrative Plant Biology (2021) Volume63, Issue7, pp1240-1259), in view of Huh (Sung Un Huh, Biomolecules. (2021) 11, 1851, pp,1-11), and further in view of Char (Si Nian Char et. al., Plant Biotechnology Journal (2017) 15, pp. 257–268), and Shi (Jinrui Shi et.al., Plant Biotechnology Journal (2017) 15, pp. 207–216).
Claim 1 recites a corn plant or plant part thereof comprising at least one mutation in an endogenous PUF gene encoding a Pumilio domain containing protein (PUF) polypeptide, wherein the endogenous PUF gene comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:69, comprises a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs:72, 73 or 74-92, and/or encodes an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:71, wherein the at least one mutation is located upstream of the start codon of the endogenous PUF gene.
Huang teaches that Pumilio RNA-binding proteins (PUM/PUF family) regulate developmentally important processes, and specifically teaches Arabidopsis APUM24 as a PUF/Pumilio-domain protein involved in seed maturation/seed development traits (page 1240, abstract). Huang expressly directs a person of ordinary skill in the art to investigate the functional conservation and potential application of APUM24 homologs in crop plants such as rice, maize, and soybean for agricultural improvement (page 1251, last paragraph).
Accordingly, Huang provides a technical basis that PUF/Pumilio-domain genes are involved in plant developmental processes and suggested examination of homologous genes in crop plants, including maize.
Huang does not teaches PUF/Pumilio gene family in maize.
Huh teaches the endogenous maize PUF/Pumilio gene family. Huh discloses that plant genome encode Pumilio (PUM/PUF) RNA-binding proteins characterized by the Pumilio homology domain and reports that Zea mays contains a set of Pumilio proteins (19 ZmPUM genes) identified through database/domain searches (e. g., InterPro/PLAZA-based identification) (page 1 and 2, Evolutionally Conserved Pumilio RNA Binding Proteins; page 2 fig 1). Huh therefore teaches the existence in maize of endogenous PUF/Pumilio genes encoding PUF polypeptides.
Further, as shown by sequence comparison of a disclosed maize PUF/Pumilio protein Zm00001eb035700 (pum4 - pumilio4) obtained from the publicly available maize sequence resources, including MaizeGDB (https://www.maizegdb.org, the database genome build/version (RefGen_v5) availability date is Jan 2020). The ZmPUM4 polypeptides includes a 241 amino-acid region that aligns with SEQ ID NO:71 at approximately 96% identity with 8 gaps across the full 241-amino-acid length (see below alignment). Accordingly, under the broadest reasonable interpretation, Huh’s disclosure of maize PUF/Pumilio proteins, together with the demonstrated high sequence identity of a maize PUF/Pumilio polypeptide region to SEQ ID NO:71, teaches an endogenous maize gene encoding an amino acid sequence having at least 80% identity to SEQ ID NO:71, as recited.
Huh does not teach producing a corn plant or plant part comprising at least one cell having a mutation in the endogenous PUF gene using a nuclease comprising a cleavage domain and a nucleic acid binding domain.
Char teaches introducing sequence-specific nuclease (e. g., CRISPR/Cas9 systems with guide nucleic acids) into maize cells/embryos(page 258-259, Targeted mutagenesis strategy) to generate targeted mutations (e.g., insertions/deletions/substitutions) in endogenous maize genes (page 260, fig 3), followed by regeneration of maize plants carrying the mutations (page 262, Inherited Cas9 and gRNA expression induces new mutations in progeny plants). Char further teaches use of a CRISPR/Cas9 genomic editing system, wherein a guide RNA (gRNA) is a chimeric molecules of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) that includes a spacer sequence of approximately 18-20 nucleotides complementary to a target DNA sequence withing an endogenous maize, thereby providing sequence-specific binding to the target site. Cas9 contains both RuvC and HNH DNA cleavage domains that cause DNA double-strand breaks (DSB) (page 257, Introduction).
A person of ordinary skill in the art would have been motivated to apply the maize genome-editing approach of Char to an endogenous maize PUF/Pumilio gene taught by Huh because Huang teaches that PUF/Pumilio-domain proteins regulate plant developmental processes and identifies APUM24 homologs in crop plants, including maize, should be explored for agricultural improvement. Huh identifies maize PUF/Pumilio proteins as a defined gene family in corn, thereby providing concrete endogenous maize PUF targets. Char teaches routine and predictable generation of targeted mutations in endogenous maize genes using CRISPR-based nuclease and regeneration of edited maize plants. Given the established feasibility of producing targeted mutations in maize and the availability of endogenous maize PUF/Pumilio gene targets, a person of ordinary skill in the art would have had a reasonable expectation of success in producing a corn plant or plant part comprising at least one cell having a mutation in an endogenous PUF gene as claimed.
Huh does not teach at least one mutation in ZmPUM4 gene, nor does Huh teach that the mutation is located upstream of the start codon of the endogenous PUF gene.
Char teaches introducing sequence-specific nuclease (e. g., CRISPR/Cas9 systems with guide nucleic acids) into maize cells/embryos(page 258-259, Targeted mutagenesis strategy) to generate targeted mutations (e.g., insertions/deletions/substitutions) in endogenous maize genes (page 260, fig 3), followed by regeneration of maize plants carrying the mutations (page 262, Inherited Cas9 and gRNA expression induces new mutations in progeny plants). Thus, Char teaches producing a corn plant comprising at least one mutation in an endogenous gene encoding a protein of interest.
Char does not specifically teach introducing the mutation upstream of the start codon of an endogenous maize PUF gene.
Shi teaches targeted genome editing of an upstream regulatory region of an endogenous maize gene. In particular, Shi teaches a method of producing an upstream mutation relative to the start codon of an endogenous gene, including “a 7-bp duplication in the 5’-untranslated region (5’-UTR) which produces an in-frame ATG codon upstream of the original translation start codon” (page 208, right column, pa1). Shi further teaches that targeted genome editing of endogenous maize genes in the 5’UTR can modulate gene expression (page 207, Summary; page 212, both left and right column).
Accordingly, Shi teaches targeted editing of regions upstream of the start codon of an endogenous maize gene, including the 5’-UTR and other upstream regulatory sequences.
A person of ordinary skill in the art would have been motivated to apply the maize genome-editing approach of Char to an endogenous maize PUF/Pumilio gene taught by Huh, and to introduce the mutation upstream of the start codon as taught by Shi, because Huang teaches that PUF/Pumilio-domain proteins regulate seed development -related traits and expressly suggests that APUM24 homologs in crop plants including maize should be explored for agricultural improvement. Huh identifies maize PUF/Pumilio proteins as a defined gene family in corn, thereby providing concrete endogenous maize PUF targets. Char teaches routine, predictable generation of targeted mutations in endogenous maize genes using CRISPR-based nucleases and regeneration of edited plants. Shi further teaches the upstream/5’UTR regulatory-region editing of endogenous maize genes can modulate gene expression. Accordingly, a POSITA would have had a reasonable expectation of success in generating a corn plant comprising at least one upstream mutation in an endogenous PUF gene encoding a PUF polypeptide as claimed.
Therefore, claim 1 is prima facie obvious over Huang in view of Huh and Char, and further in view of Shi.
Claim 2 recites the corn plant or plant part thereof of claim 1, wherein the corn plant comprises a mutation in an upstream open reading frame (uORF) of the endogenous PUF gene.
As set forth above with respect to claim 1, Shi teaches the method of producing upstream mutation of the start codon of endogenous gene: “result of a 7-bp duplication in the 5’-untranslated region (5’-UTR) which produces an in-frame ATG codon upstream of the original translation start codon” (page 208, right column, pa1). A uORF is located upstream of the main coding sequence and functions as an upstream translational regulatory element. Therefore, it would have been obvious to introduce a mutation in a uORF of the endogenous maize PUF gene for the same reasons set forth above with respect to claim 1. Accordingly, claim 2 is prima facie obvious.
Claim 3 recites the corn plant or plant part thereof of claim 1, wherein the start codon is at position 11500 with reference to SEQ ID NO:69.
As set forth above with respect to claim 1, Zm00001eb035700 corresponds to the claimed PUF genomic sequence. The Zm00001eb035700 genomic sequence of 11592 nucleotides aligns to nucleotide 1816-1347 of SEQ ID NO: 69 with only one nucleotide mismatch (see below alignment). Further, translation of SEQ ID No: 69 beginning at nucleotide 11500 produces the start codon. Therefore, the position recited in claim 3 identifies the start codon of the claimed PUF gene (see below for sequence and translation). Shi teaches targeted mutation upstream of the original translation start codon in the 5’untranslated/upstream regulatory region of an endogenous maize gene (page 208, right column, pa1). Accordingly, it would have been obvious to introduce a mutation upstream of the start codon located at position 11500 with reference to SEQ ID NO: 69 for the same reasons set forth above with respect to claim 1. Accordingly, claim 3 is prima facie obvious.
Claim 5 recites the corn plant or plant part thereof of claim 1, wherein the at least one mutation is a deletion or an insertion of at least one base.
As set forth above with respect to claim 1, Char teaches at least one mutation is a base deletion, a base insertion, two base deletion, 4 base deletion, 32 base deletion, and non-natural mutation (page 260, fig 3). Accordingly, claim 5 is prima facie obvious.
Claim 8 recites the corn plant or plant part of claim 1, wherein the at least one mutation in the endogenous PUF gene encoding a PUF polypeptide results in a dominant allele, a dominant negative allele, a recessive allele, a hypomorphic allele, or a hypermorphic allele.
In instant application, a “hypermorphic mutation” is a mutation that results in increased expression of the gene product and/or increased activity of the gene product (paragraph 0073). As set forth above with respect to claim 1, claim 8 is prima facie obvious.
Claim 9 recites the corn plant or plant part thereof of claim 1, wherein the at least one mutation is located upstream of a coding sequence encoding SEQ ID NO:71.
As set forth above with respect to claim 1, the ZmPUM4 polypeptide includes a 241 amino-acid region that aligns with SEQ ID NO: 71 at approximately 96% identity (see below for alignment). Shi teaches targeted mutation in the 5’ untranslated /upstream regulatory region of an endogenous maize gene upstream of the original translation start codon (page 208, right column, pa1). Therefore, it would have been obvious to introduce the mutation upstream of the coding sequence encoding the PUF polypeptide corresponding to SEQ ID NO: 71 for the same reasons set for the above with respect to claim 1. Accordingly, claim 9 is prima facie obvious.
Claim 10 recites to the corn plant or plant part thereof of claim 1, wherein the corn plant comprising the at least one mutation exhibits an improved yield trait as compared to a corn plant devoid of the at least one mutation.
As set forth above with respect to calm 1, the upstream mutation in the endogenous PUF gene would have been obvious over Huang, Huh, Char, and Shi. Huang teaches both a technical basis that PUF proteins as components of post-transcriptional regulatory pathways influencing reproductive development, and PUF/Pumilio-domain genes influence yield-related seed traits (page 1250, right column), and an explicit suggestion to examine and utilize homologs in maize for yield improvement.
Huang does not teach mutating maize PUF (Pumilio-domain) protein improves yield.
Shi teaches that targeted genome editing of endogenous maize genes in the 5’untrnslated region (5’UTR) can be used to modulate gene expression and produce plants exhibiting increased maize grain yield, measured in bushels per acre, under field conditions (page 207, summary). Shi teaches that editing regulatory regions upstream of the coding sequence, without altering the encode protein, is an effective and predictable strategy for improving yield traits in maize, thereby providing a reasonable expectation of success for achieving yield improvement through regulatory -region genome editing (page 212, both left and right column).
Claim 11 recites corn plant of claim 10, wherein the improved yield trait comprises increased yield (bu/acre), kernel row number (KRN), and/or branch number. Claim 30 recites a corn plant regenerated from the plant part of claim 1, wherein the corn plant comprising the at least one mutation exhibits an improved yield trait. Claim 32 recites a corn plant of claim 30, wherein the improved yield trait comprises increased yield (bu/acre), kernel row number (KRN), and/or branch number.
As set forth above with respect to claim 1, Shi teaches compared to the WT, the edited variants increased grain yield by five bushels per acre (Page 207, Summary). Char teaches regeneration of maize plants carrying targeted mutations (page 257, Summary).
Accordingly, claim 11, 30, and 32 are prima facie obvious over Huang, Huh, Char, and Shi.
Claim 14 recites the corn plant or plant part thereof of claim 1, wherein the at least one mutation results in a mutated PUF gene comprising a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs:100, 101, 102, 103 or 104.
As set forth above with respect to claim 1, Huh teaches the relevant endogenous maize PUF/PUM (e.g., Zm00001eb035700) gene and its genomic locus as a target for genetic modification (e.g., a 12-nucleotide deletion within a 3177(SEQ ID NO:100) nucleotide aligned region). Char teaches that CRISPR editing in maize results in localized sequence changes confirmed to the target site (e.g., small indels generated by repair of a nuclease-induced double-strand break), such that the remainder of the endogenous genomic sequence remains unchanged (page 265, Discussion).
Accordingly, claim 14 is prima facie obvious over Huang, Huh, Char, and Shi.
Claims 47, 51, 54, and 60-62 are rejected under 35 U.S.C. §103 as being unpatentable over Huang (2021), in view of Huh (2021), and further in view of Char (2017).
Claim 47 recites the method for producing a corn plant comprising at least one cell having a mutated PUF gene, the method comprising contacting a target site within an endogenous PUF gene in the corn plant or plant part thereof with a nuclease comprising a cleavage domain and a nucleic acid binding domain, wherein the nucleic acid binding domain of the nuclease binds to the target site within the endogenous PUF gene, wherein the endogenous PUF gene:(a) comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:69;(b) comprises a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs:72, 73 or 74-92; and/or (c) encodes an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:71, thereby producing the corn plant or plant part thereof comprising at least one cell having a mutation in the endogenous PUF gene.
As discussed above with respect to claim 1, Huang teaches PUF/Pumilio-domain proteins as targets associated with yield-related traits, Huh teaches endogenous maize PUF/Pumilio genes including genes satisfying the claimed sequence identity limitations, and Char teaches CRISPR/Cas-mediated targeted mutagenesis of endogenous maize genes.
Claim 47 recites the method step of contacting the endogenous PUF gene target site with a nuclease comprising a cleavage domain and a nucleic acid binding domain.
Char teaches introducing sequence-specific nuclease, including CRISPR/Cas9 systems, into maize cells to target endogenous maize genes and generate mutations (p258-260), wherein Cas9 contains DNA cleavage domains and a guide RNA provide sequence-specific binding to the target site (p257).
Therefore, it would have been obvious to use the CRISPR/Cas9 editing method taught by Char to generate the claimed mutation in the endogenous maize PUF gene taught by Huh for the reasons discussed above with respect to claim 1.
Accordingly, claim 47 is prima facie obvious over Huan in view of Huh and Char.
Claim 51 is drawn to the method of claim 47, wherein the target site is within a region of the PUF gene having at least 80% sequence identity to a nucleotide sequence of any one of SEQ ID NOs:72, 73 or 74-92.
Huh teaches the ZmPUM4 genomic fragment comprises a 63-nucleotide sequence that is 100% identical to SEQ ID NO:92 (alignment below).
Claim 54 is drawn to the method of claim 47,wherein the mutation is a deletion or an insertion of at least one base.
Char teaches targeted mutations in endogenous maize genes, including insertions/deletions generated at the target site (page 260, fig. 3).
Claim 60 is drawn to the method of claim 47, wherein the mutation results in a mutated PUF gene comprising a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs:100, 101, 102, 103 or 104.
Char teaches that CRISPR editing in maize results in localized sequence changes at the target site, such that the remainder of the endogenous genomic sequence remains unchanged (page 265, Discussion). Therefore, the resulting edited PUF gene would retain at least 90% sequence identity to the representative mutated PUF sequences.
Claim 61 is drawn to a guide nucleic acid that binds to a target site within an endogenous gene encoding a PUF polypeptide, wherein the endogenous PUF gene is defined by sequence identity such that the gene comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:69, and/or comprises a region having at least 80% sequence identity to SEQ ID NOs:72, 73 or 74-92, and/or encodes an amino acid sequence having at least 80% to SEQ ID NO:71.
As set forth above with respect to claim 47, Huh teaches endogenous maize PUF/Pumilio genes, and Char teaches CRISPR guide nucleic acids having spacer sequences complementary to target DNA sequences withing endogenous maize genes (page 257, Introduction)
Claim 62 is drawn to a guide nucleic acid of claim 61, wherein the guide nucleic acid comprises a spacer comprising any one of the nucleotide sequences of SEQ ID NOs:93-95, 105 and 106.
SEQ ID NO:94 is a 23-nucleotide sequence, matching a segment within the genomic DNA of Zm00001eb035700 (ZmPUM4) (below).
Accordingly, the claimed invention in claims 51, 54, and 60-62 as a whole is prima facie obvious over the combined teachings of the prior arts above.
Claims 56, 58 and 59 are rejected under 35 U.S.C. §103 as being unpatentable over Huang (2021), in view of Huh (2021) and Char (2017) as apply to claim 47, and further in view of Shi (2017).
Claim 47 as the teachings of Huang, Huh, and Char are discussed above.
Claims 56 and 58 are interpreted as dependent of claim 47.
Claim 56 recites the method of claim 47, wherein the mutation in the endogenous PUF gene encoding a PUF polypeptide results in a dominant mutation, a dominant negative mutation, a recessive mutation, a hypomorphic mutation, or a hypermorphic mutation.
For the same reasons set forth above with respect to claim 8, the prior art teaches or suggests at least the hypermorphic mutation alternative. Claim 56 is prima facie obvious.
Claim 58 recites the method of claim 47, wherein the corn plant comprising the mutation exhibits an improved yield trait as compared to a plant devoid of the mutation. Claim 59 recites the method of claim 58, wherein the improved yield trait comprises increased yield (bu/acre), increased kernel row number (KRN), and/or increased branch number, as compared to a plant that is devoid of the mutation.
Huang teaches PUF/Pumilio-domain proteins are involved in seed development/yield-related traits and suggests investigating crop homologs for agricultural improvement (page 1251, right column, pa3). Shi further teaches that the edited maize variants increased grain yield by five bushels per acre compared to wild type (page 207, Introduction).
Therefore, claims 58-59 are prima facie obvious.
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CLUSTAL O(1.2.4) multiple sequence alignment
Zm00001eb035700 MATESAFRLIGGTGARDWSKGFGAFGSSAGALSGEDLGFVDNDTGVYGGWNKSVPNRSGS 60
SEQIDNO71 ------------------------------------------------------------ 0
Zm00001eb035700 APPSMEGSLAALGHLIDQQSGSFEASLTTLDNITDSSKSEEQLRADPAYFEYYGSKVNLN 120
SEQIDNO71 ------------------------------------------------------------ 0
Zm00001eb035700 PRLPPPLISRESRRLMNRVGKAKEWRMVSQDNSSKGSIYVPRSMLSTHKEEPEDDKSPRL 180
SEQIDNO71 ------------------------------------------------------------ 0
Zm00001eb035700 DSSSVEDAQIVSSASNFQSQDFMLERFQQSVASSPDSSSSNPSNSNTGDSMPVYSDINLL 240
SEQIDNO71 ------------------------------------------------------------ 0
Zm00001eb035700 KSLSFDALKQSDLNSWTPKGPLKSNVNNDLSSPPLSSSSYPGSKTGTQTFEQEKAAADTK 300
SEQIDNO71 ------------------------------------------------------------ 0
Zm00001eb035700 HGNVVLGSGAAVTEVDNVDSIMKNLKLSLDVHTSSPAKQRWQDNVLQQYGSFLPAQGDPI 360
SEQIDNO71 ------------------------------------------------------------ 0
Zm00001eb035700 QLTTQGPHPPHVPFVDNLSHAQLKLPDIHQNLPQPSMTTPFYTPNSFGNPYYQNLHPANA 420
SEQIDNO71 ------------------------------------------------------------ 0
Zm00001eb035700 FPTSIGTGGYAVSGSILPPFMAGYAPQGPLATPLDSSMTPSFSGRPSGFLPAGNLTGGTD 480
SEQIDNO71 ------------------------------------------------------------ 0
Zm00001eb035700 FMQSCKVYGQFEPVFQGQTLPGVLPPVRRNDSAGFLPPSRNITGSPGIQGQRARQKFDES 540
SEQIDNO71 ------------------------------------------------------------ 0
Zm00001eb035700 KTCSFLEELKSNRARMVELSDITGRVVEYSADQHGSRFIQQKLENCTAEEKTSVFAEILP 600
SEQIDNO71 ------------------------------------------------------------ 0
Zm00001eb035700 HASALMTDVFGNYVIQKFFEHGTREQRRDLATKLVGHVLPLSLQMYGCRVIQKALEVMEL 660
SEQIDNO71 ---------------------------------------------------------MEL 3
***
Zm00001eb035700 DQKIDLVHELDGHIMRCVRDQNGNHVIQKCIECVPTEHIGFVVSAFQGQVTSLSMHPYGC 720
SEQIDNO71 DQKIDLVHELDGHIMRCVRDQNGNHVIQKCIECVPTEHIGFVVSAFQGQVTSLSMHPYGC 63
************************************************************
Zm00001eb035700 RVIQRILEHCGGNSQGQCIIDEILQWVCILAQDQYGNYVTQHVLERGKAHERSQIITKLA 780
SEQIDNO71 RVIQRILEHCGGNSQGQCIIDEILQWVCILAQDQYGNYVTQHVLERGKAHERSQIITKLA 123
************************************************************
Zm00001eb035700 GQVVTMSQNKYASNVIEKCFQHGDIAERDLLIRRIVEQTEGNNNLL---AMMKDQYANYV 837
SEQIDNO71 GQVVTMSQNKYASNVIEKCFQHGDIAERDLLIRRIVEQTEGNNNLLVCLAMMKDQYANYV 183
********************************************** ***********
Zm00001eb035700 VQKILETCNEDQRELLLSRVKDHMQALRKYTYGKHIVSRVEQLCGD-----GTAESGS 890
SEQIDNO71 VQKILETCNEDQRELLLSRVKDHMQALRKYTYGKHIVSRVEQLCGDVVTLSGTAESGS 241
********************************************** *******
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329
975
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191
1373
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362
975
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191
1366
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346
1346
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Sequence close to location near position 11500 of SEQ ID NO: 69
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Translation of the above sequence:
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Response to Applicant’s Remarks:
Applicant’s argument has been considered but are not persuasive.
Argument 1: Huang does not teach ZmPUM4/Zm00001eb035700, nor demonstrate that ZmPUM4 is a homolog of APUM24.
Applicant’s argument is not persuasive. The rejection does not rely on Huang alone to teach the claimed maize PUF gene, Huang is relied upon for teaching that PUF/Pumilio-domain proteins are involved in seed development and yield-related traits and for motivating investigation of crop homologs. Huh teaches endogenous maize PUF/Pumilio genes, and the record demonstrates that ZmPUM4/Zm00001eb035700 encodes a PUF polypeptide region having approximately 96% identity to SEQ ID NO: 71. Therefore, the combination teaches or suggests the claimed endogenous maize PUF gene.
Argument 2: Huh merely identifies 19 putative maize PUM genes and does not specifically disclose ZmPUM4.
Applicant’s argument is not persuasive. Huh teaches that Zea mays contains a family of endogenous maize PUF/Pumilio genes (19 ZmPUM genes) identified through database/domain searches. Further, sequence comparison of the publicly available maize PUF/Pumilio protein ZmPUM4/Zm00001eb035700 demonstrates that the ZmPUM4 polypeptide includes a 241 amino-acid region that aligns with SEQ ID NO:71 at approximately 96% identity. Therefore, Huh’s disclosure of endogenous maize PUF/Pumilio genes, together with the demonstrated sequence identity of ZmPUM4 to SEQ ID NO: 71, teaches or at least suggests an endogenous maize PUF gene within the scope of the claims.
Argument 3: Char does not edit a PUF gene and therefore does not teach the claimed invention.
Applicant’s argument is not persuasive. Char is not relied upon for teaching a PUF target gene. Char is relied upon for teaching CRISPR/Cas-mediated editing of endogenous maize genes and regeneration of edited maize plants. Hur provides the maize PUF target, while Huang provides the motivation to investigate PUF/Pumilio genes for yield-related traits.
Argument 4: there is no motivation to select ZmPUM4 or reasonable expectation that mutating ZmPUM4 would improve yield.
Applicant’s argument is not persuasive. Claim 1 does not require improved yield. Huang teaches that PUF/Pumilio-domain proteins and suggests investigation of homologous genes in crop plants, including maize. Huh identifies endogenous maize PUF/Pumilio genes. Char teaches targeted editing of endogenous maize genes, and Shi teaches upstream/5’UTR editing of endogenous maize genes. Accordingly, the rejection of claim 1 is based on the claimed upstream mutation in a maize PUFG gene and not on any reburied yielded phenotype.
For the phenotype claims 10, 11, 30, 32, 58, and 59, to the extent applicant argues lack of evidence of improved yield, Shi teaches that upstream regulatory-region editing can produce increased maize grain yield. Moreover, the claims broadly encompass numerous PUF genes and mutation types, and applicant’s examples are limited to specific embodiments and are not commensurate in scope with the full breadth of the claims.
Argument 5: The prior art does not teach the target regions recited in claim 51 (SEQ IN NO: 72, 73, or 74-92)
Applicant’s argument is not persuasive. The alignment demonstrates that the ZmPUM4 genomic sequence contains a63-nucleotide region that is 100% identical to SEQ ID NO: 92. Once the endogenous maize PUF gene was identified, selection of a CRISPR target site within the corresponding region would have been routine.
Argument 6: The prior art does not teach the mutated PUF sequences of claim 60 (SEQ ID NO: 100-104).
Applicant’s argument is not persuasive. Claim 60 requires only at least 90% sequence identity to the recited representative mutated sequences. Char teaches localized CRISPR-induced mutations while the remainder of the genomic sequence remains unhinges. Thus, the resulting edited PUF gene would reasonably retain at least 90% sequence identity to the representative mutated sequence.
Argument 7: The prior art does not teach the guide nuclei acids/spacer sequences of claims 61 and 62.
Applicant’s argument is not persuasive. Char teaches CRISPR guide nucleic acids having spacer sequences complementary to endogenous maize target sequences. Once the target region withing the endogenous maize PUF gene was identified, selection of a complementary spacer sequence would have been a routine design choice. The alignment further shows that SEQ ID NO: 94 matches a segment with Zm00001eb035700.
Argument 8 (for amended claims 1-3, 9, etc.): The prior art does not teach mutations upstream of the start codon, in a uORF, or upstream of the coding sequence encoding SEQ ID NO: 71)
Applicant’s argument is not persuasive. Shi teaches targeted editing of the 5’untranslated/upstream regulatory region of an endogenous maize gene, including introduction of an upstream ATG and modification of the region upstream of the original translation start codon (page 207). Shi further teaches that such upstream regulatory-region editing can modulate gene expression and increase maize grain yield. Therefore, it would have been obvious to introduce the claimed upstream/uORF mutations in the endogenous maize PUF gene for the same reasons set forth in the rejection.
Conclusion
No claims are allowed.
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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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/YANXIN SHEN/ Examiner, Art Unit 1663
/WEIHUA FAN/ Primary Examiner, Art Unit 1663
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