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 .
Priority
Acknowledgment is made of applicant’s claim for foreign priority of App. No. CN202210423670.9 (04/22/2022) and PCT/CN2022/130494 (11/08/2022) under 35 U.S.C. 119 (a)-(d). The certified copy has been filed in the instant application, filed on November 4th, 2024. However, an English translation has not been filed.
Should applicant desire to obtain the benefit of foreign priority under 35 U.S.C. 119(a)-(d) prior to declaration of an interference, a certified English translation of the foreign application must be submitted in reply to this action. 37 CFR 41.154(b) and 41.202(e).
Failure to provide a certified translation may result in no benefit being accorded for the non-English application. Thus, the earliest possible priority for the instant application is currently October 11th, 2024.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on October 11th, 2024 was considered, initialed, and attached hereto. A signed copy of the list of references cited is included with this Office Action.
Examiner notes that a copy of Ware, D. "Accession number AQK42656: Yucca 2 [Zea mays]" GenBank Database, 16 February 2017 (2017-02-16), as cited in the IDS was not provided and thus was not considered.
Status of Claims
Claims 1-11 filed October 11th, 2024 are pending and examined herein.
Drawings
The drawings are objected to because they lack reference to the SEQ ID NOs. Figure 4 additionally shows the ZmYUC4 single mutant under the double mutant section (as shown below). It appears that the ZmYUC2/ZmYUC4 double mutant with the ZmYUC4 mutation should read Zmyuc2/4#1 and Zmyuc2/4#2) as in the ZmYUC2 mutation above it (instead of Zmyuc4#1 and Zmyuc4#2). Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
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Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code (see pg. 15, ¶87 and pg. 16, ¶90, for example). Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Claim Objections
Claims 1, 4, 6, and 10 are objected to because of the following informalities: The claims recite a polynucleotide sequence as set forth in SEQ ID NO: 2, 4, or 6, as an encoded amino acid sequence of said gene. The sequence listing reflects SEQ ID NO. 4, however, the specification (pg. 15, ¶88) denotes SEQ ID NO. 2 as the amino acid sequence for both the ZmYUC2-T001 and the ZmYUC2-T002 gene. Based on the sequence listing, it is clear that there is intent for SEQ ID NO. 4 to be an amino acid sequence correlated with the ZmYUC2-T002 gene. In the interest of compact prosecution, it appears that this is simply a typographical error.
Claim 5 is objected to because of the following informalities: The claim currently reads “the gene mutant sequences of claims 1.” Examiner suggests amending this to read, “the gene mutant sequences of claim 1.”
Claims 11 and 2 are objected to because of the following informalities: The claims are lacking a space between claim and the claim number (i.e. claim2).
Appropriate correction is required.
Claim Interpretation
Claim 1, 4, 6, and 10 recite a polynucleotide sequence as set forth in SEQ ID NO: 2, 4, or 6, as an encoded amino acid sequence of said gene. The sequence listing reflects SEQ ID NO. 4, however, the specification (pg. 15, ¶88) denotes SEQ ID NO. 2 as the amino acid sequence for both the ZmYUC2-T001 and the ZmYUC2-T002 gene. It appears that SEQ ID NO. 4 is the amino acid sequence correlated with the ZmYUC2-T002 gene and will be interpreted as such unless further clarification provides alternative interpretation.
Claim 1, and 2-5 depending therefrom, and claim 6, and 7-11 depending therefrom, recite “a polynucleotide sequence as set forth in SEQ ID NO…” A polynucleotide sequence set forth in a sequence may be anything from a dimer, trimer, or full length of the referenced sequence. Based on the specification, “a polynucleotide sequence as set forth in SEQ ID NO…” is taken to mean a sequence with 100% identity to the SEQ ID NO. of the claim.
The term “mutation” as recited in the claims is not defined in the instant specification; however, Merriam Webster defines mutation as a relatively permanent change in hereditary material that involves either a change in chromosome structure or number (as in translocation, deletion, duplication, or polyploidy) or a change in the nucleotide sequence of a gene's codons. Thus, the mutation of a gene nucleotide sequence from which the gene mutant sequence is derived is taken to mean any possible mutation.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claim 6 is rejected under 35 U.S.C. 101 because the claimed invention is directed to a natural phenomenon without significantly more. The claim recites a method for enhancing lodging resistance in maize by decreasing expression or function of a lodging-related gene. This judicial exception is not integrated into a practical application because lodging-related genes are found in nature and are shown to fluctuate in function throughout the growth of a plant.
SEQ ID NO. 2 of the instant application, an encoded amino acid sequence of the lodging-related gene claimed, is disclosed in Li, W. et al. (2015, “Genome-Wide Analysis and Expression Patterns of the YUCCA Genes in Maize,” Journal of Genetics and Genomics, 42:707-710). Li discloses Probable indole-3-pyruvate monooxygenase YUCCA5 [Zea mays] with NCBI Reference SEQ ID NO. NM_001368031.1 (as cited in IDS filed 10/11/2024, see alignment below).
Score
Identities
Positives
Gaps
861 bits(2224)
422/422(100%)
422/422(100%)
0/422(0%)
Query 1 MGLLPTDRMDSLFSPRCQWVNGPIIVGAGPSGLAVAACLREQGVPYVMLERADCIASLWQ 60
MGLLPTDRMDSLFSPRCQWVNGPIIVGAGPSGLAVAACLREQGVPYVMLERADCIASLWQ
Sbjct 1 MGLLPTDRMDSLFSPRCQWVNGPIIVGAGPSGLAVAACLREQGVPYVMLERADCIASLWQ 60
Query 61 RRTYDRLKLHLPKQFCELPRMPFPDHYPEYPTRRQFIDYLEDYAARFEIRPEFSTTVVLA 120
RRTYDRLKLHLPKQFCELPRMPFPDHYPEYPTRRQFIDYLEDYAARFEIRPEFSTTVVLA
Sbjct 61 RRTYDRLKLHLPKQFCELPRMPFPDHYPEYPTRRQFIDYLEDYAARFEIRPEFSTTVVLA 120
Query 121 RYDETSGLWRVTTSAPANGGDVEYIGRWLVVATGENAEAVVPDIPGLGGFHGKVTHVSDY 180
RYDETSGLWRVTTSAPANGGDVEYIGRWLVVATGENAEAVVPDIPGLGGFHGKVTHVSDY
Sbjct 121 RYDETSGLWRVTTSAPANGGDVEYIGRWLVVATGENAEAVVPDIPGLGGFHGKVTHVSDY 180
Query 181 KSGEAYAGKRVLVVGCGNSGMEVSLDLSDHGARPAMVVRDAVHVLPREVLGKSTFQLAVL 240
KSGEAYAGKRVLVVGCGNSGMEVSLDLSDHGARPAMVVRDAVHVLPREVLGKSTFQLAVL
Sbjct 181 KSGEAYAGKRVLVVGCGNSGMEVSLDLSDHGARPAMVVRDAVHVLPREVLGKSTFQLAVL 240
Query 241 LRRWLPLWLVDKIMVILAWLVLGNLAKLGLRRPAAGPLELKETHGRTPVLDTGALARIRA 300
LRRWLPLWLVDKIMVILAWLVLGNLAKLGLRRPAAGPLELKETHGRTPVLDTGALARIRA
Sbjct 241 LRRWLPLWLVDKIMVILAWLVLGNLAKLGLRRPAAGPLELKETHGRTPVLDTGALARIRA 300
Query 301 GDIAVVPAVTRFGKGGQVELADGRTLDFDAVILATGYRSNVPQWLESSNDSFNKEGYPKT 360
GDIAVVPAVTRFGKGGQVELADGRTLDFDAVILATGYRSNVPQWLESSNDSFNKEGYPKT
Sbjct 301 GDIAVVPAVTRFGKGGQVELADGRTLDFDAVILATGYRSNVPQWLESSNDSFNKEGYPKT 360
Query 361 AFPHGWKGQSGLYAVGFTRRGLLGASTDAVRIAKDLGNVWREETKPTKRAGACHRRCISV 420
AFPHGWKGQSGLYAVGFTRRGLLGASTDAVRIAKDLGNVWREETKPTKRAGACHRRCISV
Sbjct 361 AFPHGWKGQSGLYAVGFTRRGLLGASTDAVRIAKDLGNVWREETKPTKRAGACHRRCISV 420
Query 421 VF 422
VF
Sbjct 421 VF 422
Li discloses fourteen candidate YUC genes in maize using the amino acid sequences of Arabidopsis YUC genes as queries, resulting in previously reported ZmYUC1, ZmSPI1, and 12 novel members, ZmYUC2-ZmYUC13 [pg. 707, col. 1, ¶2]. Expression patterns of the ZmYUC genes at various stages of female inflorescence development showed that five genes, ZmYUC2, -4, -5, -6, and -10, were highly expressed at the spikelet-pair meristem stage in maize (specialized meristem that initiates the formation of spikelet branches) [pg. 709, col. 1, ¶3]. A naturally occurring decrease in expression of said ZmYUC genes was observed at the next stage, the early stage of spikelet meristem [Fig. S4]. As Li teaches a sequence (NM_001368031.1, predicted ZmYUC5) with 100% identity to SEQ ID NO. 2 of the instant application, which is inherently a polynucleotide sequence of a lodging-related gene as claimed in claim 6, and teaches that there is a natural decrease in expression of ZmYUC2, -4, -5, -6, and -10 over spikelet meristem growth, this is a natural process or natural phenomenon.
The claim does not include additional elements that are sufficient to amount to significantly more than the judicial exception because there are no method steps other than decreasing expression of a lodging-related gene wherein the polynucleotide sequence of said gene is selected from a group of sequence including SEQ ID NO. 2. Examiner notes that the claims depending from claim 6, do recite additional elements that amount to significantly more than the judicial exception because they specify how the decrease or inhibition in the normal expression or protein function of the lodging-related gene is achieved. For example, through RNA interference or mutation as in claim 7.
Claim Rejections - 35 USC § 112(b)
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 6-11 are 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 7-11 are rejected insofar as they depend from claim 6.
Claim 6 recites a method for enhancing lodging resistance in maize by decreasing or inhibiting “a normal expression or protein function” of a lodging-related gene. It is unclear what a normal expression means or to what the normal expression is in comparison. Is a normal expression the baseline for a control plant; and of what kind? Is normal expression a specific value? Would normal expression change over time or change depending on the lodging-related gene? It is also unclear if it is the expression or protein function that is intended to be “normal.” Claims 7-11 depend from claim 6 and claims 7 and 9 additionally recite normal expression or protein function.
Further, claim 11 recites the limitation "said gene mutation" yet does not recite a gene mutation within the claim. Claim 6, from which 11 depends, also does not recite a gene mutation. There is insufficient antecedent basis for this limitation in the claim.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-11 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.
Regarding claim 1, and claims 2-5 depending therefrom, the claims are broadly drawn to a gene mutant sequence that generates a phenotype of increased brace root growth angle and lodging resistance that is derived from any mutation of a polynucleotide sequence (a) set forth in SEQ ID NO. 1, 3, 5, 9, or 10, (b) an amino acid encoded sequence of said gene, set forth in SEQ ID NO. 2, 4, or 6, (c) a polynucleotide sequence of SEQ ID NO. 1-6 and 9-10 that hybridizes with the polynucleotide sequence under stringent hybridization conditions, (d) a polynucleotide sequence that has at least 95% or greater identity to the SEQ ID NOs. set above (a-c), or (e) a poly nucleotide sequence that is complementary to the sequences above (a-d). The claims state that the mutation may be generated through physical mutagenesis, chemical mutagenesis, ZFN, TALEN, and/or CRISPR/Cas gene editing.
Claim 6 and claims 7-11 depending therefrom are more broadly drawn to a method for enhancing lodging resistance in maize by decreasing or inhibiting a normal expression or protein function of a lodging-related gene. Similarly to claim 1, claim 6 specifies that the gene is (a) set forth in SEQ ID NO. 1, 3, 5, 9, or 10, (b) an amino acid encoded sequence of said gene, set forth in SEQ ID NO. 2, 4, or 6, (c) a polynucleotide sequence of SEQ ID NO. 1-6 and 9-10 that hybridizes with the polynucleotide sequence under stringent hybridization conditions, (d) a polynucleotide sequence that has at least 95% or greater identity to the SEQ ID NOs. set above (a-c), or (e) a poly nucleotide sequence that is complementary to the sequences above (a-d). The claims state that mutation or RNA interference decreases or inhibits normal expression or protein function or the lodging related gene and that the mutation may be generated through physical mutagenesis, chemical mutagenesis, ZFN, TALEN, and/or CRISPR/Cas gene editing.
The instant specification reduces to practice the mutation of polynucleotide sequences of SEQ ID NO. 1-6 and 9-10 to generate either single or double mutant maize plants. The ZmYUC4 gene (genomic nucleotide sequence as set forth in SEQ ID No: 10) had only one transcript (nucleotide sequence as set forth in SEQ ID NO. 5, amino acid sequence as set forth in SEQ ID NO. 6), whereas the ZmYUC2 gene (genomic DNA sequence as set forth in SEQ ID NO. 9) had two transcripts: ZmYUC2-T001 (nucleotide sequence as set forth in SEQ ID NO. 1,amino acid sequence as set forth in SEQ ID NO. 2) and ZmYUC2-T002 (nucleotide sequence as set forth in SEQ ID NO. 3, amino acid sequence as set forth in SEQ ID NO. 2 (interpreted to mean SEQ ID NO. 4 according to claim interpretation above)) [pg. 15, ¶88]. The instant specification reduces to practice the mutation of the gene nucleotide sequences using CRISPR/Cas9 resulting in the four gene mutant sequences with SEQ ID NOs. 11-14. The specification reduces to practice the ZmYUC4 single mutants (with gene mutant sequence of SEQ ID NO. 13 or 14 derived from polynucleotide sequences NO. 10 or 5 or encoding amino acid sequence NO. 6) or a ZmYUC2/4 double mutant to increase brace root growth angle and lodging resistance in maize plants over the wild-type (WT) control. Examiner notes that the ZmYUC2/4 double mutants are not claimed in the instant claims.
The instant specification does not reduce to practice any other gene nucleotide sequences, fragments thereof, a polynucleotide sequence that hybridize with the polynucleotide sequences under stringent hybridization conditions, or a polynucleotide sequence that is complementary to the sequence. The instant specification does not reduce to practice any method of creating mutations other than the CRISPR/Cas9 gene editing vectors. The instant specification does not reduce to practice the use of the single ZmYUC2 mutants with only the mutation of SEQ ID NO. 11 or 12 in increasing brace root growth angle and lodging resistance, showing that the single ZmYUC2 mutants were not significantly different than the WT in influencing lodging resistance or brace root growth angle [Fig. 7]. Thus, the use of polynucleotide sequences with SEQ ID NO. 9, 1, and 3, with encoding amino acids with SEQ ID NO. 2 and 4 are not reduced to practice with the function as claimed.
The Applicant concedes that the use the ZmYUC2 single mutants (with SEQ ID NO. 9, 1, and 3, with encoding amino acids of SEQ ID NO. 2 and 4, and with the gene mutation sequences of SEQ ID NO. 11 or 12) did not generate a phenotype of increased brace root growth angle and lodging resistance in maize plant containing the gene mutant sequence. Thus, a complementary polynucleotide sequence or a polynucleotide sequence that hybridizes with the polynucleotide sequence of SEQ ID NO. 9, 1, and 3, or encoding amino acids of SEQ ID NO. 2 and 4, are not guaranteed to generate a phenotype of increased resistance. The Applicant additionally concedes that “stringent hybridization conditions” are sequence dependent and vary under different environmental conditions [pg. 10, ¶64], indicating that undue experimentation would be required to create polynucleotide sequences that would be able to hybridize and maintain the function as claimed.
Yali et al. (2022, “Mutation Breeding and Its Importance in Modern Plant Breeding”, Journal of Plant Sciences, 10(2): 65-70) teaches that mutations may be spontaneous or induced and are the result of any change in an organism's DNA that is not caused by normal recombination and segregation [Abstract]. Mutations can be artificially generated through numerous methods that may have variable results, varying from site specific single nucleotide substitutions through site directed mutagenesis to extensive chromosomal abnormalities caused by ionizing radiation [pg. 67, col. 1, ¶1-6]. Mutations may additionally may have a strong negative effect on other traits, such as low yield [Table 2]. Zischewski et al. (2017, “Detection of on-target and off-target mutations generated by CRISPR/ Cas9 and other sequence-specific nucleases”, Biotechnology Advances, 35:95-104) discloses that the mutation ability of CRISPR/Cas9 to create double-strand breaks at any desired position in the genome is dependent on the target sequence, which may be altered by changing the 20-nt sequence of the single guide RNA (gRNA). [pg. 96, col. 1, ¶2]. Thus, any mutation in the claimed gene nucleotide sequences may not result in the desired effect of generating a phenotype of an increased brace root growth angle and lodging resistance in maize plants, and may result in no change or even deleterious effects.
The Applicant only reduces to practice the use of CRISPR/Cas9 to create the mutant gene sequences of SEQ ID NO. 11-14. Undue experimentation would be required to investigate the type of mutation required to create the gene mutant sequence that can perform this function.
Further regarding claim 4 and claim 9, the claim is broadly drawn to the gene mutant sequence of claim 1 derived through mutation, wherein said mutation involves CRISPR/Cas9, in which a target sequence is selected from a fragment of a nucleotide sequence as set forth in SEQ ID NO. 1, 3, 5, 9, or 10 that conforms to a sequence arrangement rule of 5’-Nx-NGG-3’, wherein N represents any of A, G, C, and T, X is between 14-30, and Nx represents X consecutive nucleotides, or a nucleotide sequence that is complementary to the polynucleotide sequence.
The instant specification reduces to practice the target sequences shown in Fig. 4A. Fig. 4A shows the design of the ZmYUC2 and ZmYUC4 gene with three target sites, two found in SEQ ID NO. 10, the genomic nucleotide sequence of the ZmYUC4 gene, and one found in SEQ ID NO. 9, the genomic nucleotide sequence of the ZmYUC2 gene [Fig. 4B]. Two types of mutations were found in the ZmYUC2 gene sequence, a deletion of 1288bp-1292bp (ATTGC) downstream of the start codon (ATG) (SEQ ID NO. 11) in the genomic DNA sequence and an insertion of an A base between 1285bp and 1286bp (SEQ ID NO. 12); and the two types of mutations in the ZmYUC4 gene were a deletion of the base (A) at 255bp downstream as well as a deletion of the base (G) at 938bp downstream (SEQ ID NO. 13), and a deletion of 253bp-267bp (GAAGACTACCCGGAG) and a deletion of 936bp-937bp (CG) downstream of the start codon (ATG)（SEQ ID NO. 14). Amino acid sequence analysis indicated that these insertions and deletions of bases caused premature termination or frame shift mutations in the ZmYUC2 and/or ZmYUC4 genes in the mutants [pg. 17, ¶97].
The instant specification does not reduce to practice any other target sequences or specify alterations of a single guide RNA to allow for alterative targeting. The instant specification does not reduce to practice any CRISPR/Cas9 target sequence with the sequence arrangement of any 14-30 consecutive nucleotides, followed by any single nucleotide and GG, in the 5’ to 3’ direction. The instant specification additionally does not reduce to practice any other mutations than those of the gene mutant sequences set forth in SEQ ID NOs. 11-14, and does not reduce to practice the generation of the phenotype of increased brace root growth angle and lodging resistance in maize plants using the gene mutant sequences of SEQ ID NO. 11 and 12 in single mutants alone.
As above, Zischewski et al. teaches that the ability of CRISPR/Cas9 to create double-strand breaks at any desired position in the genome is dependent on the target sequence, which may be altered by changing the sequence of the gRNA. [pg. 96, col. 1, ¶2]. Zischewski teaches that the target region needs to be sequenced to confirm and ultimately determine the outcome of genome editing [pg. 97, col. 1, ¶1]. Undue experimentation would be required alter the gRNA and to confirm that any target sequence conforming to the sequence arrangement recited in claim 4 provides a gene mutant sequence that performs the necessitated function of generating a phenotype of increased brace root growth angle and lodging resistance.
Thus, claims 1-11 do not meet the written description requirement.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
Claims 1 and 2 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Alexandrov, N. et al. (2008), “Insights into corn genes derived from large-scale cDNA sequencing,” Plant Mol. Biol. 69(1-2):179-194, (Zea mays clone 1583668 disulfide oxidoreductase/ monooxygenase/ oxidoreductase mRNA, complete cds (GenBank Acc. No. EU957130.1)).
Claim 1 recites a gene mutant sequence that is derived from mutation of a gene nucleotide sequence that has at least 95% or greater identity to the polynucleotide sequence of SEQ ID NO. 9.
Claim 2 recites the gene mutant sequence of claim 1, wherein said gene mutant sequence is derived through the mutation comprising substitution, deletion, and/or addition of one or more nucleotides in SEQ ID NO. 9.
Regarding claim 1, Alexandrov explicitly discloses a Zea mays clone 1583668 disulfide oxidoreductase/ monooxygenase/ oxidoreductase mRNA, complete cds (GenBank: EU957130.1) with 96% identity to SEQ ID NO. 9 of the instant application (see alignment below). This sequence has at least 95% or greater identity to the polynucleotide of the claimed invention, meeting requirement (e) above of claim 1.
Regarding claim 2, it additionally could be the gene mutant sequence derived from a mutation of SEQ ID NO. 9, with 46 nucleotide bases that could have been mutated from the gene nucleotide sequence of SEQ ID NO. 9 by any mutation (see above claim interpretation). Thus, any mutation of any number of nucleotides may have occurred to a sequence derived from SEQ ID NO. 9 to obtain a gene mutant sequence 95% identity to SEQ ID NO. 9. Alexandrov additionally teaches that sequences disclosed may have insertions or deletions (i.e. mutation comprising deletion) [pg. 183, col. 1, ¶2; pg. 181, col. 1, ¶4] (cf. instant claim 2).
Score
Expect
Identities
Gaps
2012 bits(1089)
0.0
1198/1244(96%)
33/1244(2%)
Query 1 ACCAACACAACAACATCTCTTCCAGCTCCCACCAGCAACTACAAGAGAACATCACATCCT 60
|||||||||||||||||||||||||||||||||||||||||||| |||||||||||||||
Sbjct 2 ACCAACACAACAACATCTCTTCCAGCTCCCACCAGCAACTACAATAGAACATCACATCCT 61
Query 61 AGCTAGCTACTAGCTCGAGCTCTCATCAGAAGA-ACATACTG---ATC-AT---C-G--- 108
||||||||||||||||||||||||||||||||| |||||||| ||| || | |
Sbjct 62 AGCTAGCTACTAGCTCGAGCTCTCATCAGAAGATACATACTGATCATCGATCACCAGTCA 121
Query 109 --------A--TCA--C-CAGTCACCACCACCACCTAAGGCTCTCAGACGACCTCAGAGA 155
| | | | |||||||||||||||||||||| ||||||||| ||||||||
Sbjct 122 CCACCACCACCTAAGGCTCAGTCACCACCACCACCTAAGG--CTCAGACGATCTCAGAGA 179
Query 156 TTGAAAGCCACACATCAGCCATGGGGCTCCTTCCTACCGACCGCATGGACAGCCTCTTCT 215
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 180 TTGAAAGCCACACATCAGCCATGGGGCTCCTTCCTACCGACCGCATGGACAGCCTCTTCT 239
Query 216 CCCCGCGCTGCCAGTGGGTGAACGGCCCCATCATCGTGGGCGCGGGGCCCTCGGGTCTCG 275
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 240 CCCCGCGCTGCCAGTGGGTGAACGGCCCCATCATCGTGGGCGCGGGGCCCTCGGGTCTCG 299
Query 276 CCGTGGCGGCGTGCCTGCGCGAGCAGGGCGTCCCCTACGTCATGCTGGAGCGCGCCGACT 335
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 300 CCGTGGCGGCGTGCCTGCGCGAGCAGGGCGTCCCCTACGTCATGCTGGAGCGCGCCGACT 359
Query 336 GCATCGCGTCGCTGTGGCAGAGGCGCACCTACGACCGCCTCAAGCTGCACCTGCCCAAGC 395
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 360 GCATCGCGTCGCTGTGGCAGAGGCGCACCTACGACCGCCTCAAGCTGCACCTGCCCAAGC 419
Query 396 AGTTCTGCGAGCTGCCGCGCATGCCCTTCCCCGACCACTACCCGGAGTACCCCACCCGCC 455
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 420 AGTTCTGCGAGCTGCCGCGCATGCCCTTCCCCGACCACTACCCGGAGTACCCCACCCGCC 479
Query 456 GCCAGTTCATCGACTACCTCGAGGACTACGCCGCCAGGTTCGAGATCAGGCCGGAGTTCA 515
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 480 GCCAGTTCATCGACTACCTCGAGGACTACGCCGCCAGGTTCGAGATCAGGCCGGAGTTCA 539
Query 516 GCACCACGGTGGTGTTGGCGCGCTACGACGAGACGTCGGGCCTCTGGCGCGTCACCACCT 575
||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 540 GCACCACCGTGGTGTTGGCGCGCTACGACGAGACGTCGGGCCTCTGGCGCGTCACCACCT 599
Query 576 CGGCGCC------CGCCAACGGTGGCGACGTGGAGTACATCGGCCGCTGGCTCGTCGTCG 629
||||||| |||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 600 CGGCGCCCGCCGGCGCCAACGGTGGCGACGTGGAGTACATCGGCCGCTGGCTCGTCGTCG 659
Query 630 CCACCGGCGAGAACGCCGAGGCCGTCGTGCCCGACATCCCGGGTCTCGGCGGCTTCCACG 689
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 660 CCACCGGCGAGAACGCCGAGGCCGTCGTGCCCGACATCCCGGGTCTCGGCGGCTTCCACG 719
Query 690 GCAAGGTCACCCACGTCAGCGACTACAAGTCAGGGGAGGCCTACGCCGGGAAGCGCGTGC 749
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 720 GCAAGGTCACCCACGTCAGCGACTACAAGTCAGGGGAGGCCTACGCCGGGAAGCGCGTGC 779
Query 750 TCGTGGTCGGCTGCGGCAACTCCGGGATGGAGGTGTCGCTCGACCTCTCCGACCACGGGG 809
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 780 TCGTGGTCGGCTGCGGCAACTCCGGGATGGAGGTGTCGCTCGACCTCTCCGACCACGGGG 839
Query 810 CCCGCCCGGCCATGGTCGTGCGCGACGCCGTGCACGTCCTCCCCCGTGAGGTGCTCGGCA 869
|||||||||||||||||||||||||||| |||||||||||||||||||||||||||||||
Sbjct 840 CCCGCCCGGCCATGGTCGTGCGCGACGCTGTGCACGTCCTCCCCCGTGAGGTGCTCGGCA 899
Query 870 AGTCCACCTTCCAGCTCGCCGTGCTCCTCAGGCGCTGGCTCCCGCTCTGGCTCGTCGACA 929
||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 900 AGTCCACCTTCGAGCTCGCCGTGCTCCTCAGGCGCTGGCTCCCGCTCTGGCTCGTCGACA 959
Query 930 AGATCATGGTCATCCTCGCCTGGCTCGTCCTGGGCAACCTCGCCAAGCTCGGCCTCCGAC 989
||||||||||||||||||||||||||||||||||||||||||||| |||||||||||| |
Sbjct 960 AGATCATGGTCATCCTCGCCTGGCTCGTCCTGGGCAACCTCGCCAGGCTCGGCCTCCGCC 1019
Query 990 GCCCCGCCGCCGGCCCGCTCGAGCTCAAGGAGACACACGGCCGCACACCCGTCCTCGACA 1049
||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||
Sbjct 1020 GCCCCGCCGCCGGCCCGCTTGAGCTCAAGGAGACACACGGCCGCACACCCGTCCTCGACA 1079
Query 1050 CCGGCGCGCTCGCGCGCATCCGTGCCGGAGATATCGCCGTCGTCCCTGCCGTGACACGTT 1109
||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||
Sbjct 1080 CCGGCGCGCTCGCGCGCATCCGTGCCGGAGATATCGCCGTCGTCCCTGCCGTGGCACGTT 1139
Query 1110 TCGGAAAGGGCGGCCAGGTTGAGCTCGCCGACGGACGCACTCTTGACTTCGACGCCGTCA 1169
|| |||||||||| ||||||||||||||||||||||||||||| ||||||||||||||||
Sbjct 1140 TCCGAAAGGGCGGTCAGGTTGAGCTCGCCGACGGACGCACTCTCGACTTCGACGCCGTCA 1199
Query 1170 TCCTCGCCACCGGATACCGCAGCAACGTTCCCCAGTGGCTCGAG 1213
||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1200 TCCTCGCCACCGGATACCGCAGCAACGTTCCCCAGTGGCTCGAG 1243
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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 and 6-8 are rejected under 35 U.S.C. 103 as being unpatentable over Li, W. et al. (2015), “Genome-Wide Analysis and Expression Patterns of the YUCCA Genes in Maize,” Journal of Genetics and Genomics, 42:707-710, Zea mays probable indole-3-pyruvate monooxygenase YUCCA5 (NCBI Reference SEQ ID NO. NM_001368031.1, as cited in the IDS filed 10/11/2024), in view of Chen, Q. et al. (2014), “Auxin Overproduction in Shoots Cannot Rescue Auxin Deficiencies in Arabidopsis Roots,” Plant & Cell Physiology, 55(6):1072-1079, and Hostetler, A.N. et al. (2022) “Multiple brace root phenotypes promote anchorage and limit root lodging in maize.” Plant, Cell & Environment, 45, 1573–1583 (Accepted 1/15/2022, available online 4/7/2022).
Claim 1 recites a gene mutant sequence that is derived from a mutation of a gene nucleotide sequence, and generates a phenotype of an increased brace root growth angle and lodging resistance in maize plants, wherein said gene nucleotide sequence is a polynucleotide SEQ ID NO. 2 as an encoded amino acid sequence of said gene.
Claim 2 recites the gene mutant sequence of claim 1, wherein said gene mutant sequence is derived through the mutation comprising substitution, deletion, and/or addition of one or more nucleotides in the nucleotide sequence of said gene.
Claim 6 recites a method for enhancing lodging resistance in maize by decreasing or inhibiting a normal expression or protein function of a lodging-related gene, wherein the polynucleotide sequence of said gene is selected from one of the following sequences: (a) a polynucleotide sequence as set forth in SEQ ID No: 1, 3, 5, 9, or 10; (b) a polynucleotide sequence as set forth in SEQ ID No: 2, 4, or 6, as an encoded amino acid sequence of said gene; (c) a polynucleotide sequence that hybridizes with the polynucleotide sequence in (a) or (b) under stringent hybridization conditions, inhibiting the expression of said polynucleotide sequence endowing the maize with an increased brace root growth angle and lodging resistance; (d) a polynucleotide sequence that has at least 95% or greater identity to the polynucleotide sequence as set forth in any of (a)-(c), inhibiting the expression of said polynucleotide sequence endowing the maize with the increased brace root growth angle and lodging resistance; or (e) a polynucleotide sequence that is complementary to the sequence as set forth in any of (a)-(d).
Claim 7 recites the method of claim 6, wherein decreasing or inhibiting the normal expression or protein function of the lodging-related gene is achieved by RNA interference and/or mutation.
Claim 8 recites the method of claim 7, wherein decreasing or inhibiting gene expression or gene mutation comprises substitution, deletion, and/or addition of one or more nucleotides in a nucleotide sequence of said gene or a gene promoter.
Regarding claims 1-2 and 6-8, Li discloses Zea mays probable indole-3-pyruvate monooxygenase YUCCA5 (NCBI Reference SEQ ID NO. NM_001368031.1) with 100% identity to SEQ ID NO. 2 (see alignment below). Li discloses that, in Arabidopsis, YUCCA (YUC) genes, which are involved in auxin biosynthesis, encode flavin monooxygenases (FMOs) that convert indole-3-pyruvate (IPA) to indole-3-acetic acid (IAA) [pg. 707, col. 1, ¶1]. Li teaches that overexpression of Arabidopsis YUC1 in tobacco or its orthologs in petunia and rice leads to auxin overproduction. This suggestion that the functions of YUC genes are conserved among plant species led to the identification of ZmYUC1, ZMSPI1, and 12 novel members of the YUC genes in maize, ZmYUC2-ZmYUC13 [pg. 707, col. 1, ¶1]. In a phylogenetic analysis, the protein sequences from Arabidopsis thaliana, Oryza sativa, and several other plant species were aligned, revealing that ZmYUC2, ZmYUC4, ZmYUC5, and ZmYUC7 were closely related to OsYUC7 and OsYUC6, and in the same clade as AtYUC3, AtYUC7, AtYUC5, and AtYUC9, indicating that they might have similar functions [Fig. 1A].
Li does not explicitly teach that these YUC genes or the disclosed Zea mays probable indole-3-pyruvate monooxygenase YUCCA5 sequence may be mutated to result in a gene mutant sequence to generate a phenotype of increased brace root growth angle and lodging resistance in maize plants or that decreased or inhibited expression or protein function would result in a method for enhancing lodging resistance. However, Chen teaches mutant YUC genes in Arabidopsis, wherein the inactivation of five YUC genes lead to the development of agravitropic roots (i.e. decreasing or inhibiting a normal expression or protein function) [Abstract]. Chen teaches that an auxin gradient is a key step for several processes of root development and is synthesized locally in the roots [pg. 1072, col. 2, ¶2]. Chen teaches that IAA is synthesized from IPA by the YUC family of flavin-containing monooxygenases and that there are 11 YUC genes in Arabidopsis [pg. 1073, col. 1, ¶2], five of which (YUC3, YUC5, YUC7, YUC8, and YUC9) have detectable expression in Arabidopsis roots at seedling stages [pg. 1073, col. 1, ¶3]. Chen teaches single mutants of the five YUC genes and simultaneous inactivation of the five YUC genes (i.e. gene mutant sequence; decreasing or inhibiting gene expression) [pg. 1073, col. 2, ¶4]. The mutants were obtained by T-DNA insertion or transposon insertion from publicly available sources and are as follows: yuc3, (GABI_376G12), yuc5 (CSHL_GT6160), yuc7 (SALK_059832), yuc8 (SM_3.23299), yuc9 (SAIL_762_D07) (i.e. derived from mutation of a gene nucleotide sequence; a mutation comprising the addition/insertion of one or more nucleotides in the sequence of a gene) [pg. 1077, col. 2, ¶3].
Score
Identities
Positives
Gaps
861 bits(2224)
422/422(100%)
422/422(100%)
0/422(0%)
Query 1 MGLLPTDRMDSLFSPRCQWVNGPIIVGAGPSGLAVAACLREQGVPYVMLERADCIASLWQ 60
MGLLPTDRMDSLFSPRCQWVNGPIIVGAGPSGLAVAACLREQGVPYVMLERADCIASLWQ
Sbjct 1 MGLLPTDRMDSLFSPRCQWVNGPIIVGAGPSGLAVAACLREQGVPYVMLERADCIASLWQ 60
Query 61 RRTYDRLKLHLPKQFCELPRMPFPDHYPEYPTRRQFIDYLEDYAARFEIRPEFSTTVVLA 120
RRTYDRLKLHLPKQFCELPRMPFPDHYPEYPTRRQFIDYLEDYAARFEIRPEFSTTVVLA
Sbjct 61 RRTYDRLKLHLPKQFCELPRMPFPDHYPEYPTRRQFIDYLEDYAARFEIRPEFSTTVVLA 120
Query 121 RYDETSGLWRVTTSAPANGGDVEYIGRWLVVATGENAEAVVPDIPGLGGFHGKVTHVSDY 180
RYDETSGLWRVTTSAPANGGDVEYIGRWLVVATGENAEAVVPDIPGLGGFHGKVTHVSDY
Sbjct 121 RYDETSGLWRVTTSAPANGGDVEYIGRWLVVATGENAEAVVPDIPGLGGFHGKVTHVSDY 180
Query 181 KSGEAYAGKRVLVVGCGNSGMEVSLDLSDHGARPAMVVRDAVHVLPREVLGKSTFQLAVL 240
KSGEAYAGKRVLVVGCGNSGMEVSLDLSDHGARPAMVVRDAVHVLPREVLGKSTFQLAVL
Sbjct 181 KSGEAYAGKRVLVVGCGNSGMEVSLDLSDHGARPAMVVRDAVHVLPREVLGKSTFQLAVL 240
Query 241 LRRWLPLWLVDKIMVILAWLVLGNLAKLGLRRPAAGPLELKETHGRTPVLDTGALARIRA 300
LRRWLPLWLVDKIMVILAWLVLGNLAKLGLRRPAAGPLELKETHGRTPVLDTGALARIRA
Sbjct 241 LRRWLPLWLVDKIMVILAWLVLGNLAKLGLRRPAAGPLELKETHGRTPVLDTGALARIRA 300
Query 301 GDIAVVPAVTRFGKGGQVELADGRTLDFDAVILATGYRSNVPQWLESSNDSFNKEGYPKT 360
GDIAVVPAVTRFGKGGQVELADGRTLDFDAVILATGYRSNVPQWLESSNDSFNKEGYPKT
Sbjct 301 GDIAVVPAVTRFGKGGQVELADGRTLDFDAVILATGYRSNVPQWLESSNDSFNKEGYPKT 360
Query 361 AFPHGWKGQSGLYAVGFTRRGLLGASTDAVRIAKDLGNVWREETKPTKRAGACHRRCISV 420
AFPHGWKGQSGLYAVGFTRRGLLGASTDAVRIAKDLGNVWREETKPTKRAGACHRRCISV
Sbjct 361 AFPHGWKGQSGLYAVGFTRRGLLGASTDAVRIAKDLGNVWREETKPTKRAGACHRRCISV 420
Query 421 VF 422
VF
Sbjct 421 VF 422
Li and Chen do not explicitly teach that mutation resulting in a decrease or inhibition of expression or protein function of this gene effecting root growth in maize would alter lodging resistance or brace root growth angle, however, Hostetler teaches that brace roots (and brace root angle) directly contribute to anchorage and lodging resistance by establishing a ridged base (i.e. lodging-related gene) [Abstract; pg. 1577, col. 2, ¶1]. Hostetler teaches that genotypes with higher brace root contribution to anchorage are more likely to resist root lodging [pg. 1582, col. 1, ¶3]. Hostetler indicates that future studies must define the genetic basis for plant anchorage and that the above ground root system may reflect the below ground root system [pg. 1582, col. 2, ¶2].
Given that Li teaches a probable YUCCA5 amino acid sequence, teaches that the function of YUC genes are likely conserved among plant species, and reveals in a phylogenetic analysis that ZmYUC2, ZmYUC4, ZmYUC5, and ZmYUC7 are in the same clade as AtYUC3, AtYUC7, AtYUC5, and AtYUC9, indicating that they may have the same function in development; given that Chen teaches that mutants of AtYUC3, AtYUC5, AtYUC7, AtYUC8, and AtYUC9 in Arabidopsis alter the development and gravitropism of roots by inhibiting gene expression; and given that Hostetler teaches that brace root lodging susceptibility varies among maize genotypes, is dependent on plant root architecture, and may reflect below ground root development, it would have been prima facie obvious to one of ordinary skill in the art at the time of filing to mutate a YUC gene polynucleotide sequence in maize, particularly ZmYUC2, ZmYUC4, ZmYUC5, and ZmYUC7, or encoded amino acid sequence of said gene, such as the Zea mays probable indole-3-pyruvate monooxygenase YUCCA5 (NCBI Reference SEQ ID NO. NM_001368031.1) to decrease expression/protein function and alter root growth, brace root angle, and lodging resistance in maize. One of ordinary skill in the art would be motivated to use NM_001368031.1 as a lodging-related gene due to its indicated functional similarities to the Arabidopsis YUC genes. One of ordinary skill in the art would have reasonable expectation of success in the method of mutating the ZmYUC2, ZmYUC4, ZmYUC5, and ZmYUC7 to alter root growth, as the Arabidopsis genes in the same clade that were predicted to have the same or similar functionality did alter root growth when mutated. Additionally, the change in root gravitropism taught by Chen may directly influence the root angle, which would be beneficial to lodging resistance, as taught by Hostetler, providing further motivation to modify the mutation of Chen in Arabidopsis to maize.
Claims 3 and 9 are rejected under 35 U.S.C. 103 as being unpatentable over Li in view of Chen and Hostetler as applied to claims 2 and 6-8 above, and further in view of Lee, K. et al. (2019), “CRISPR/Cas9-mediated targeted T-DNA integration in rice,” Plant Molecular Biology, 99:317-328.
Claim 3 recites the gene mutant sequence of claim2, wherein said mutation is generated by techniques comprising physical mutagenesis, chemical mutagenesis, ZFN, TALEN, and/or CRISPR/Cas gene editing.
Claim 9 recites the method of claim 6, wherein inhibiting the normal expression of the lodging-resistant genes is achieved by means comprising RNAi, physical mutagenesis, chemical mutagenesis, ZFN, TALEN, and/or CRISPR/Cas gene editing.
Regarding claims 3 and 9, Li, Chen, and Hostetler teach a sequence with 100% identity to SEQ ID NO. 2 that may be mutated to produce a gene mutant sequence to generate a phenotype of lodging resistance in maize plants/a method for enhancing lodging resistance in maize by decreasing or inhibiting expression or protein function of this lodging-related gene. Chen teaches Arabidopsis YUC gene mutants created through T-DNA or transposon insertion. Li, Chen, and Hostetler do not teach that the mutation is generated by techniques comprising physical mutagenesis, chemical mutagenesis, ZFN, TALEN, or CRISPR/Cas9.
However, Lee teaches that T-DNA integration into the rice genome may be precisely targeted with a combined CRISPR/Cas9 system and Agrobacterium-mediated transformation. Lee teaches the use of a standard Agrobacterium binary vector to construct a T-DNA that contains a CRISPR/Cas9 system using SpCas9 and a gRNA targeting the exon of the rice AP2 domain-containing protein gene Os01g04020 to allow for efficiently generated targeted T-DNA insertions [Abstract]. Lee teaches that this system can be easily applied to other plants, ensuring optimal expression of transgenes without unwanted insertional mutagenesis [pg. 326, col. 1, ¶2; col. 2, ¶2].
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing that using T-DNA insertion could be used in tandem with CRISPR/Cas9. One would have been motivated to do so based on the higher accuracy and lack of unwanted insertional mutagenesis. Additionally, CRISPR/Cas9 relies on a gRNA to reach a targeted sequence within the genome. Using such a system for to create mutations is well known in the prior art, as are alternatives such as RNAi, ZFN and TALEN.
Claims 4 and 10 are rejected under 35 U.S.C. 103 as being unpatentable over Li in view of Chen, Hostetler, and Lee, as applied to claims 3 and 9 above, and further in view of Li, B. et al. (US Patent Application No. US 20170114356 A1, published 04/27/2017).
Claim 4 recites the gene mutant sequence of claim 3, wherein said CRISPR/Cas gene editing involves CRISPR/Cas9, in which a target sequence is selected from one of the following sequences: (a) a sequence being a fragment of a nucleotide sequence as set forth in SEQ ID No: 1, 3, 5, 9, or 10 that conforms to a sequence arrangement rule of 5’-Nx-NGG-3’, wherein N represents any of A, G, C, and T, 14 < X < 30, X is an integer, and Nx represents X consecutive nucleotides; or (b) a nucleotide sequence that is complementary to the polynucleotide sequence as set forth in (a).
Claim 10 recites the method of claim 9, wherein said CRISPR/Cas is the CRISPR/Cas9 gene editing method, with a target sequence selected from one of the following sequences: (a) a sequence being a fragment of a nucleotide sequence as set forth in SEQ ID No: 1, 3, 5, 7, 8, 9, or 10 that conforms to a sequence arrangement rule of 5’-Nx-NGG-3’, wherein N represents any of A, G, C, and T, 14 < X < 30, X is an integer, and Nx represents X consecutive nucleotides; or (b) a nucleotide sequence that is complementary to the polynucleotide sequence as set forth in (a).
Regarding claims 4 and 10, Li, Chen, Hostetler, and Lee do not explicitly teach that the target sequence a sequence being a fragment of a nucleotide sequence as set forth in SEQ ID No: 1, 3, 5, 9, or 10 that conforms to a sequence arrangement rule of 5’-Nx-NGG-3’, wherein N represents any of A, G, C, and T, 14 < X < 30, X is an integer, and Nx represents X consecutive nucleotides; or a nucleotide sequence that is complementary to the polynucleotide sequence of SEQ ID No: 1, 3, 5, 9, or 10.
However, Li, B. teaches novel alternatively spliced transcripts and uses thereof for improvement of agronomic characteristics in crop plants. Li, B. teaches novel transcripts in maize, recombinant DNA constructs, transgenic plants or cells thereof, and methods for generating transgenic seed and plants with improved agronomic characteristics [Abstract]. Li, B. teaches that the agronomic characteristic may be improved root or stalk lodging [¶153]. Li, B. teaches SEQ ID NO. 97954 that is 98.5% complementary to SEQ ID NO. 1 of the instant application (i.e. a nucleotide sequence that is complementary to the polynucleotide sequence as set forth in SEQ ID No: 1, 3, 5, 9, or 10) (alignment shown below). Li, B. teaches that the CRISPR guide RNA may comprise a variable targeting domain of 12 to 30 nucleotide sequences and an RNA fragment that can interact with a Cas endonuclease [¶79].
Query Match 98.5%; Score 1250; Length 1281;
Best Local Similarity 99.8%;
Matches 1263; Conservative 0; Mismatches 0; Indels 3; Gaps 1;
Qy 1 ATGGGGCTCCTTCCTACCGACCGCATGGACAGCCTCTTCTCCCCGCGCTGCCAGTGGGTG 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1263 ATGGGGCTCCTTCCTACCGACCGCATGGACAGCCTCTTCTCCCCGCGCTGCCAGTGGGTG 1204
Qy 61 AACGGCCCCATCATCGTGGGCGCGGGGCCCTCGGGTCTCGCCGTGGCGGCGTGCCTGCGC 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1203 AACGGCCCCATCATCGTGGGCGCGGGGCCCTCGGGTCTCGCCGTGGCGGCGTGCCTGCGC 1144
Qy 121 GAGCAGGGCGTCCCCTACGTCATGCTGGAGCGCGCCGACTGCATCGCGTCGCTGTGGCAG 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1143 GAGCAGGGCGTCCCCTACGTCATGCTGGAGCGCGCCGACTGCATCGCGTCGCTGTGGCAG 1084
Qy 181 AGGCGCACCTACGACCGCCTCAAGCTGCACCTGCCCAAGCAGTTCTGCGAGCTGCCGCGC 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1083 AGGCGCACCTACGACCGCCTCAAGCTGCACCTGCCCAAGCAGTTCTGCGAGCTGCCGCGC 1024
Qy 241 ATGCCCTTCCCCGACCACTACCCGGAGTACCCCACCCGCCGCCAGTTCATCGACTACCTC 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1023 ATGCCCTTCCCCGACCACTACCCGGAGTACCCCACCCGCCGCCAGTTCATCGACTACCTC 964
Qy 301 GAGGACTACGCCGCCAGGTTCGAGATCAGGCCGGAGTTCAGCACCACGGTGGTGTTGGCG 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 963 GAGGACTACGCCGCCAGGTTCGAGATCAGGCCGGAGTTCAGCACCACGGTGGTGTTGGCG 904
Qy 361 CGCTACGACGAGACGTCGGGCCTCTGGCGCGTCACCACCTCGGCGCCCGCCAACGGTGGC 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 903 CGCTACGACGAGACGTCGGGCCTCTGGCGCGTCACCACCTCGGCGCCCGCCAACGGTGGC 844
Qy 421 GACGTGGAGTACATCGGCCGCTGGCTCGTCGTCGCCACCGGCGAGAACGCCGAGGCCGTC 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 843 GACGTGGAGTACATCGGCCGCTGGCTCGTCGTCGCCACCGGCGAGAACGCCGAGGCCGTC 784
Qy 481 GTGCCCGACATCCCGGGTCTCGGCGGCTTCCACGGCAAGGTCACCCACGTCAGCGACTAC 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 783 GTGCCCGACATCCCGGGTCTCGGCGGCTTCCACGGCAAGGTCACCCACGTCAGCGACTAC 724
Qy 541 AAGTCAGGGGAGGCCTACGCCGGGAAGCGCGTGCTCGTGGTCGGCTGCGGCAACTCCGGG 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 723 AAGTCAGGGGAGGCCTACGCCGGGAAGCGCGTGCTCGTGGTCGGCTGCGGCAACTCCGGG 664
Qy 601 ATGGAGGTGTCGCTCGACCTCTCCGACCACGGGGCCCGCCCGGCCATGGTCGTGCGCGAC 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 663 ATGGAGGTGTCGCTCGACCTCTCCGACCACGGGGCCCGCCCGGCCATGGTCGTGCGCGAC 604
Qy 661 GCCGTGCACGTCCTCCCCCGTGAGGTGCTCGGCAAGTCCACCTTCCAGCTCGCCGTGCTC 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 603 GCCGTGCACGTCCTCCCCCGTGAGGTGCTCGGCAAGTCCACCTTCCAGCTCGCCGTGCTC 544
Qy 721 CTCAGGCGCTGGCTCCCGCTCTGGCTCGTCGACAAGATCATGGTCATCCTCGCCTGGCTC 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 543 CTCAGGCGCTGGCTCCCGCTCTGGCTCGTCGACAAGATCATGGTCATCCTCGCCTGGCTC 484
Qy 781 GTCCTGGGCAACCTCGCCAAGCTCGGCCTCCGACGCCCCGCCGCCGGCCCGCTCGAGCTC 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 483 GTCCTGGGCAACCTCGCCAAGCTCGGCCTCCGACGCCCCGCCGCCGGCCCGCTCGAGCTC 424
Qy 841 AAGGAGACACACGGCCGCACACCCGTCCTCGACACCGGCGCGCTCGCGCGCATCCGTGCC 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 423 AAGGAGACACACGGCCGCACACCCGTCCTCGACACCGGCGCGCTCGCGCGCATCCGTGCC 364
Qy 901 GGAGATATCGCCGTCGTCCCTGCCGTGACACGTTTCGGAAAGGGCGGCCAGGTTGAGCTC 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 363 GGAGATATCGCCGTCGTCCCTGCCGTGACACGTTTCGGAAAGGGCGGCCAGGTTGAGCTC 304
Qy 961 GCCGACGGACGCACTCTTGACTTCGACGCCGTCATCCTCGCCACCGGATACCGCAGCAAC 1020
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 303 GCCGACGGACGCACTCTTGACTTCGACGCCGTCATCCTCGCCACCGGATACCGCAGCAAC 244
Qy 1021 GTTCCCCAGTGGCTCGAGAGCAGCAACGATTCCTTCAACAAGGAAGGATACCCCAAGACG 1080
||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||
Db 243 GTTCCCCAGTGGCTC---AGCAGCAACGATTCCTTCAACAAGGAAGGATACCCCAAGACG 187
Qy 1081 GCCTTCCCTCACGGGTGGAAGGGTCAATCTGGGCTCTACGCCGTGGGTTTCACCCGGCGT 1140
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 186 GCCTTCCCTCACGGGTGGAAGGGTCAATCTGGGCTCTACGCCGTGGGTTTCACCCGGCGT 127
Qy 1141 GGTCTCTTGGGCGCCTCCACCGACGCCGTGCGCATTGCCAAGGACCTCGGCAACGTCTGG 1200
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 126 GGTCTCTTGGGCGCCTCCACCGACGCCGTGCGCATTGCCAAGGACCTCGGCAACGTCTGG 67
Qy 1201 AGGGAGGAGACCAAGCCCACGAAGAGGGCCGGCGCCTGCCACAGGCGCTGCATCTCCGTC 1260
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 66 AGGGAGGAGACCAAGCCCACGAAGAGGGCCGGCGCCTGCCACAGGCGCTGCATCTCCGTC 7
Qy 1261 GTCTTC 1266
||||||
Db 6 GTCTTC 1
Given that Li, Chen, Hostetler, and Lee teach a method for enhancing lodging resistance using a gene mutant sequence derived from a mutation of a gene nucleotide sequence generating a phenotype of an increased brace root growth angle and lodging resistance; given that Lee teaches CRISPR/Cas9 implementation of such a mutation; and given that Li, B. teaches a nucleotide sequence that is complementary to the polynucleotide sequence of SEQ ID NO. 1 that can be used for agronomic improvement with CRISPR/Cas, it would have been prima facie obvious to one of ordinary skill at the time of filing to use the nucleotide sequence of Li, B. as the target sequence for CRISPR/Cas0 gene editing. One would have been motivated to do so as Li, B. discloses that the novel transcripts in maize may be used for the creation of transgenic plants with improved agronomic characteristics, such as root lodging.
Subject Matter Free of Art
The gene mutated sequences of claims 5 and 11 (with 100% identity to SEQ ID NOs. 11-14) appear to be free of prior art, pending the above rejections. To the extent that claim 5 and 11 are directed to the single mutants of ZmYUC4 and the double mutants of ZmYUC2/4 with the mutations of SEQ ID NOs. 11-14, which appear to function in lodging resistance and increased brace root angle, the subject matter of claims 5 and 11 may be found free of prior art.
Contact Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to EMILY K. JOHNSON whose telephone number is (571)272-5761. The examiner can normally be reached Monday - Friday 7:30 am - 5:00 pm.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Bratislav Stankovic can be reached at 571-270-0305. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/EMILY K JOHNSON/Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Units 1661 & 1662