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, 30, 32, 47, 51, 54, 56, and 58-62 are examined on the merits.
Claim Objections
Claim 2 is objected to as a typo for “endogenous PUFF gene” should be “PUF gene”.
Claim 3 is objected to as grammatical ambiguity for “The corn plant or part thereof claim 1….” Should be “…of claim 1”.
Claim 54 is objected to as minor form defect for “The method claim 47” should be “The method of claim 47”.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim 1 is rejected under 35 U.S.C. 102(a)(1) as being anticipated by US20040034888A1 (Jingdong Liu et. al., Application filed 2003-04-28, Published 2004-02-19).
Claim 1 recites a corn plant or plant part comprising at least one mutation in an endogenous PUF gene encoding a Pumilio-domain containing (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.
The claim does not specify how the “at least one mutation” is generated; therefore, the term encompasses any sequence alteration in the endogenous PUF gene (including naturally occurring polymorphisms/allelic variants), and is not limited to mutations intentionally induced by laboratory technique.
Maize has undergone thousands of years of intensive human domestication and modern breeding, resulting in extensive naturally occurring allelic diversity across the genome. Due to continuous artificial selection, introgression, and recombination among divers germplasm pools, numerous allelic variants exist at most endogenous loci. In such a genetic background, it is difficult to define any particular sequence variant as a “mutation” attributable to any specific human interventions, as opposed to naturally occurring or historically selected alleles. Accordingly, sequence differences relative to a reference genome represent ordinary allelic variation commonly present in cultivated maize populations rather than necessarily reflecting a distinct, human-engineered mutation.
US20040034888A1 discloses a maize protein sequence (SEQ ID NO: 71078) corresponding to the claimed PUF locus and meeting the claim’s protein-identity requirement (≥80% identity to SEQ ID NO:71) (alignment below). US20040034888A1 further discloses at least one amino-acid difference (8 amino acid insertion with two locations, one location has 3 amino acids insertion, one location has 5 amino acids insertion) among maize PUF protein variants/alleles. Eight amino acid insertions in the encoded PUF polypeptide necessarily reflects alterations in the underlying endogenous PUF coding sequence. Therefore, US20040034888A1 discloses a corn plant comprising at least one mutation in an endogenous PUF gene encoding a Pulimio-domain protein as recited.
Accordingly claim 1 is anticipated with US20040034888A1.
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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, 9, 14, 47, 51, 54, and 60-62 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).
Claim 1 is drawn to a corn plant or plant part comprising at least one mutation in an endogenous PUF gene encoding a pumilio-domain containing (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.
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 that are yield-related components (page 1240, abstract). Huang further teaches that this finding is significant because it “lays the foundation for developing promising new approaches to improve crop yield” (page 1251). 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 both a technical basis that PUF/Pumilio-domain genes influence yield-related seed traits, and an explicit suggestion to examine and utilize homologs in maize for yield improvement.
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 Pumio proteins (19 ZmPUM genes) identified through database/domain searches (e. g., InterPro/PLAZA-based identification) (page 1 and 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 (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. 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 at least one mutation in ZmPUM4 protein.
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). Thus, Char teaches producing a corn plant comprising at least one mutation in an endogenous gene encoding a protein of interest.
Claims 3, 5, and 9 are drawn to a corn plant or part of claim1, wherein at least one mutation is a base substitution, a base deletion, and/or a base insertion (claim 3); at least one mutation is a deletion or an insertion of at least one base (claim 5); at least one mutation is a non-natural mutation (claim 9).
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).
Claim 14 is drawn to the corn plant or part of claim 1, and 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.
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).
Claim 47 is drawn to a method for producing a corn plant or part thereof comprising at least one cell having a mutated endogenous PUF gene, the method comprising contacting a target site within an endogenous PUF gene with a nuclease comprising a cleavage domain and a nucleic acid binding domain, 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, thereby producing the corn plant or part thereof comprising at least one cell having a mutation in the endogenous PUF gene.
Char 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).
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.
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.
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).
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 seed development/yield-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 targets), and Char teaches routine, predictable generation of targeted mutations in endogenous maize genes using CRISPR-based nucleases and regeneration of edited plants. Given the established feasibility of producing targeted indel mutations in maize and the availability of endogenous maize PUF/Pumilio gene targets, a POSITA would have had a reasonable expectation of success in generating a corn plant comprising at least one mutation in an endogenous PUF gene encoding a PUF polypeptides as claimed.
Once a specific endogenous PUM/PUF gene and target region are identified, selection and design of an appropriate guide RNA spacer complementary to that target region would have been a routine and recitable step, performed in accordance with well-established CRISPR design principles, such as sequence complementarity, PAM compatibility, and avoidance of off-target effects. Under these routine design considerations, spacer sequences corresponding to disclosed target regions, including a spacer targeting the sequence of SEQ ID NO:94, would have been selected as a matter of ordinary skill.
A person of ordinary skill in the art also would have had a reasonable expectation of success that the resulting edited PUF allele would remain highly homologous to the endogenous PUF locus sequence because the art-taught edits are minor, localized modifications within a defined target fragment, rather than replacement or extensive rewriting of the gene. Thus, once the target fragment within the endogenous PUF locus is determined and edited according to Char, it would have been an expected and predicable outcome that the resulting mutated PUF gene sequence would retain at least 90% nucleotide identity across the edited region, which correspondence to the claimed ≥90% identity requirement relative to representative mutated PUF sequence.
The claimed invention in claims 1, 3, 5, 9, 14, 47, 51, 54, and 60-62 as a whole is prima facie obvious over the combined teachings of the prior arts above.
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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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Claims 2, 8, 10, 11, 30, 32, 56 and 58-59 are rejected under 35 U.S.C. §103 as being unpatentable over Huang (2021), in view of Huh (2021) and Char (2017) as applied to claim 1 and 47, and further in view of Shi (Jinrui Shi et.al., Plant Biotechnology Journal (2017) 15, pp. 207–216).
Claims 1 and 47 as the teachings of Huang, Huh and Char are discussed above.
Claim 2 is drawn to the corn plant or part of claim 1, wherein the at least one mutation is located upstream of the start codon of the endogenous PUF gene.
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 207).
Claim 10 is drawn to the corn plant or part 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. 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), 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).
Claim 8 is drawn to the corn plant or part thereof of claim1, wherein the at least one mutation in an endogenous PUF gene encoding a PUF polypeptide results in 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, such as increase yield (paragraph 0073).
Claim 11 is drawn to the corn plant or part of claim10, and Claim 32 is drawn to the corn plant of claim 30, wherein the improved yield trait comprises increased yield (bu/acre), increased kernel row number (KRN), and/or increased branch number, as compared to a corn plant that is devoid of the at least one mutation.
Shi teaches compared to the WT, the edited variants increased grain yield by five bushels per acre (Page 207).
Claim 30 is drawn to 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 56 is drawn to the method of claim 47, wherein the mutation in an endogenous PUF gene encoding a PUF polypeptide results in a hypermorphic mutation.
Claim 58 is drawn to the method of claim 47, Claim 59 is drawn to the method of claim 58, wherein the corn plant comprising the mutation exhibits an improved yield trait (claim 58), 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 (claim 59).
A POSITQA would have been motivated to apply the maize genome-editing approach of Char to an endogenous maize PUF/Pumilio gene identified by Huh, including introducing a mutation upstream of the start cordon (e.g., within the promoter/5’UTR regulatory region) teaches by Shi, because at the time of the invention it was well understood that mutations in 5’regulatory regions provide a predictable means to modulate gene expression, and CRISPR-based nuclease editing in maize provide a routine, site-detectable approach for introducing such upstream mutations at a chosen target site. Huang teaches that PUF/Pumlio-domain proteins are a conserved family of RNA-binding post-transcriptional regulators that influence seed development and seed-related traits contributing to yield, thereby identifying PUF genes as a credible class of targets for yield improvement. A POSITA would have appreciated that PUF proteins act through conserved RNA-binding motifs and conserved post-transcriptional regulatory mechanisms, such that differences in primary sequence or precise downstream targets among family members would not dissuade testing maize PUF family members as candidate targets in a crop species. Further, Shi demonstrated that targeted CRISPR-based modification of 5’UTR/regulatory sequences in endogenous maize genes can modulate gene output and translate into increased grain yield under field conditions, reinforcing that upstream regulatory edits can yield inheritable alleles with measurable agronomic effect.
Accordingly, a POSITA would have had a reasonable expectation of success that introducing a targeted upstream (promoter/5’UTR/uORF) mutation in an endogenous maize PUF gene, as taught by Char and exemplified by Shi, would produce maize plants exhibiting altered PUF expression and associated yield-related trait improvements.
The claimed invention in claims 2, 8, 10, 11, 30, 32, 56 and 58-59 as a whole is prima facie obvious over the combined teachings of the prior arts above.
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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/YANXIN SHEN/Examiner, Art Unit 1663
/WEIHUA FAN/Primary Examiner, Art Unit 1663