Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status
Claims 1, 4-5, 8-10, 12-15 and 53-54 are pending and being examined.
Claim 7 is cancelled by the Applicant.
All previous objections and rejections in previous Office action and not set forth below have been withdrawn in view of applicant’s amendments to the claims (dated 03//20/2026).
Claim Rejections - 35 USC § 103
Claims 1, 4-5, 8-10, 12-15 and 53-54 are rejected under 35 U.S.C. 103 being unpatentable over Dhanasekar et al. (A novel mutation in TFL1 homolog affecting determinacy in cowpea (Vigna unguiculata), 2015, Mol. Genet. Genomics, 290:55–65) in view of Lemmon et al. ((Rapid improvement of domestication traits in an orphan crop by genome editing, 2018, Nature Plants, 4:766-770 (U) and Lemmon et al. Supplemental Information, Suppl. Pages 1-7 (V)) and Che et al. (Developing a rapid and highly efficient cowpea regeneration transformation and genome editing system using embryonic axis explants, 2021, The Plant Journal, 106:817–830; published online on 17th Feb. 2021).
Claim 1 is drawn to a method for producing a modified cowpea (Vigna unguiculata) plant exhibiting determinant plant architecture and synchronous and/or early flowering, wherein said method comprises the step of introducing (by targeted genome editing) a gene disruption mutation in Vigna unguiculata SELF PRUNING 2 (VuSP2) gene, comprising the nucleic acid sequence SEQ IDNO:113.
Dhanasekar et al. describes a method to transform indeterminate cowpea plants (page 58, left column, last para, line 1) into determinate one (page 58, right column, first para, line 1-2) by mutating the TFL1 gene in cowpea (VuTLF1) via gamma radiation (abstract). Dhanasekar et al. identifies several point mutations including a cytosine base (C) in the indeterminate plant type being substituted with adenine (A) base in mutants resulting in a change of a single amino acid at position 136 from proline (Pro) in wild types to histidine (His) (P136H) in the mutants (page 59, Fig. 3; page 60, left column, last para, last 3 lines and right column, first para, first 2 lines).
Dhanasekar et al. identified the TFL1 gene in cowpea (VuTLF1) and describes a method to produce determinate cowpea plants (page 58, right column, first para, line 1-2). Sequence data taught by Dhanasekar et al. has been submitted to NCBI/GenBank (p. 58, left col., 2nd paragraph). The TFL1 gene sequence as well as the translated amino acid sequence are described by Dhanasekar et al. and deposited in GenBank (Accession No. KJ569524, published on 09/02/2014). The TFL1 protein sequence has 100% sequence identity to instant SEQ ID NO: 114 (which is encoded by instant SEQ ID NO: 113), as shown below.
Title: US-18-279-761-114
Perfect score: 912
Sequence: 1 MARMPLEPLIVGRVIGEVLD..........GLPVAAVYFNAQRETAARRR 173
Searched: 1 seqs, 173 residues
Total number of hits satisfying chosen parameters: 1
Database : AASEQ2_07282025_122435.pep:*
RESULT 1
AASEQ2_07282025_122435
Query Match 100.0%; Score 912; DB 1; Length 173; Best Local Similarity 100.0%;
Matches 173; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MARMPLEPLIVGRVIGEVLDSFTTSTKMTVSYNKKQVYNGHEFFPSSINIKPKVEIEGGD 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MARMPLEPLIVGRVIGEVLDSFTTSTKMTVSYNKKQVYNGHEFFPSSINIKPKVEIEGGD 60
Qy 61 MRSFFTLIMTDPDVPGPSDPYLREHLHWIVTDIPGTTDATFGKELVSYEIPKPNIGIHRF 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 MRSFFTLIMTDPDVPGPSDPYLREHLHWIVTDIPGTTDATFGKELVSYEIPKPNIGIHRF 120
Qy 121 VFVLFKQKRRQCVTPPSSRDHFNTRNFAAQNELGLPVAAVYFNAQRETAARRR 173
|||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 VFVLFKQKRRQCVTPPSSRDHFNTRNFAAQNELGLPVAAVYFNAQRETAARRR 173
The name of the cowpea gene (VuSP2) in the instant invention is different than VuTFL1, as taught by Dhanasekar et al. However, as the two genes encode the same polypeptide as described above, these two genes are essentially the same. Instant SEQ ID NO: 123 comprises 100% sequence identity to the VuTFL1/VuSP2 coding region, starting at position 25, as described by Dhanasekar et al., as described below.
Lemmon et al. describes targeted genome editing (abstract) in the (CENTRORADIALIS, TERMINAL FLOWER 1, SELF-PRUNING) CETS gene family comprising SELF PRUNING gene(s), in tomato and groundcherry (page 767, Fig. 1; page 768, Fig. 2). Lemmon et al. asserted that the method would be valid for crops including cowpea (page 769, left column, last para, line 12-16).
Che et al. specifically describes CRISPR/Cas based targeted genome editing, efficient plant transformation and regeneration of transgenic plants in cowpea (abstract).
Before the effective filing date of the invention, it would have been obvious to one ordinarily skilled artisan to down-regulate the expression of the VuTFL1/VuSP2 gene, as identified by Dhanasekar et al., to produce a cowpea plant exhibiting determinate plant architecture and early flowering. Use of CRISPR/Cas based targeted gene editing to downregulate a specific gene is now a routine and standard process in the art, as described by Lemmon et al., and genome edited cowpea plant can be regenerated, as described by Che et al. Downregulating a specific gene can be done in many way including deleting the gene itself (knockout) or to get out-of-frame indel mutation(s) that either introduce frame-shift mutation producing a very different protein or by creating a nonsense mutation(s) in the coding region, preferably in the first exon and after the start ATG codon so that the translation terminated early. Targeted genome editing using CRISPR/Cas followed by regeneration of the mutated plants (as described by Lemmon et al. and Che et al.) is faster and avoids off-target mutations compared to random mutagenesis or T-DNA insertion or RNAi based gene silencing.
Given that cowpea is a commercially important legume crop, one of ordinary skill would have been motivated to downregulate the TFL1/SP2 gene in other cowpea varieties, given that early flowering and determinate growth habit exhibit several advantages including optimizing photosynthesis allocation between vegetative and reproductive growth, averting inter-twining of adjacent plants, facilitating easy execution of weeding, spraying agro-chemicals and harvesting, as well as enhancing its adaptability and climate change resilience potential, as asserted by Dhanasekar et al. (paragraph bridging p. 55-56). It is known in the art that determinate growth habit includes early flowering and synchronous pod maturity, besides ease in manual harvesting and short crop duration1.
Cowpea transformation and regeneration is described by Che et al. (abstract). It is a standard and well-known practice to delete any gene, its regulatory elements including the immediate upstream promoter sequence, or part of the gene to generate loss of function mutants using CRISPR-Cas technique, as we often observe in nonsense mutations arising due to premature stop codon.
Before the effective filing date, an ordinarily skilled artisan would have been motivated to use CRISPR/Cas9 gene editing technique using a suitable gRNA including SEQ ID NO:123 with a realistic goal to silence or downregulate expression of the functional VuTFL1/VuSP2 protein encoded by the VuTFL1/VuSP2 gene for developing determinate cowpea plants in elite and indeterminate or semi-determinate cowpea plants. Using any specific gRNA sequence in the first exon and/or the promoter sequence ((the gRNAs including SEQ ID NO: 123 span the first exon/coding region of the VuSP2 gene which is from position 92 to 292 of SEQ ID NO: 113 (data not shown)), to downregulate/silence the expression of a functional VuTFL1/VuSP2 protein is within the experimental design choice of the ordinary skilled artisan.
Regarding claim 4, Lemmon et al. (U) describes expressing Cas9 polypeptide in a plant (page 3, Fig. 2). Lemmon et al. (V) describes cloning polynucleotide encoding Cas9 and two guide RNAs (gRNAs) in a vector (supplementary information, page 2, last para; page 3, para 1). The vectors are introduced in plants using Agrobacterium infiltration method (supplementary information, page 2, para 2). Expressed endonuclease (Cas9) protein and the gRNA(s) interact to produce a ribonucleoprotein (RNP) complex(es) comprising the Cas9 protein and the gRNA(s).
It is a well-known and standard practice to undertake screening the genome of genome edited plant cells to identify the cells/plants having the targeted mutation(s) followed by regenerating plants carrying the mutation(s); and then screening regenerated plants for desired trait(s) which are determinate plant architecture and early flowering in this case.
Regarding claim 5, an ordinarily skilled artisan would have screened the genome of the T0 transformants developed from the targeted genome editing leading to insertion and/or deletion in cowpea, as described above, using well-known nucleic acid amplification or PCR technique, as also described by Lemmon et al. (V) (Supplementary information, page 3, para 2), to confirm presence of the mutation(s) in the transformants.
Regarding claims 8-9, Being an important agricultural crop, an ordinarily skilled artisan would have introduced mutations in cowpea SELF-PRUNING (SP) gene using CRISPR/Cas9 system of gene editing in the plants for several reasons, as described above. All these mutations are stably integrated in the host genome and, thus, are heritable.
Regarding claim 10, Lemmon et al. (U) describes multiple chimeric mutant T0 plants comprising several insertion and deletion mutant alleles (page 767, left column, para 1, line 2-3).
Regarding Claims 12-13, Dhanasekar et al. describes a cowpea (Vigna unguiculata) Terminal Flower 1 (VuTFL1) gene (GenBank Accession No. KJ569524) (page 57, right column, para 3, line 22). Instant SEQ ID NO: 123 comprises 100% sequence identity to the VuTFL1/VuSP2 coding region, starting at position 25, as shown below.
Title: US-18-279-761-123
Perfect score: 20
Sequence: 1 cttatagtggggagagtcat 20
Searched: 1 seqs, 1291 residues
Database : NASEQ2_07282025_190514.seq:*
RESULT 1
NASEQ2_07282025_190514
Query Match 100.0%; Score 20; DB 1; Length 1291; Best Local Similarity 100.0%;
Matches 20; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 CTTATAGTGGGGAGAGTCAT 20
||||||||||||||||||||
Db 25 CTTATAGTGGGGAGAGTCAT 44
The SEQ ID NO: 123 (blue highlight) is in the coding region of the VuTFL1/VuSp2 gene (within the first exon), as shown below (brown highlighted areas are different exons).
However, Dhanasekar et al. does not describe any Cas9 protein or any gRNA.
Lemmon et al. (V) describes targeted genome editing using a polynucleotide encoding Cas9 protein and two guide RNAs (gRNAs) that bind to the coding sequence of the target gene (supplementary information, page 2, last para; page 3, para 1) to mutate the SELF PRUNNING gene in groundcherry. Lemmon et al. also asserted that the method would be valid for legume crops including cowpea (page 769, left column, last para, line 12-16). Moreover, designing gRNAs to mutate specific target gene(s) and using it in conjunction with Cas9 for targeted genome editing in plants, either by introducing the polynucleotide sequences encoding the gRNA(s) and/or the Cas endonuclease into the target plant host OR introducing the ribonucleoprotein complex(es) (RNPs) consisting of gRNA(s) and the Cas endonuclease, is a standard practice in the art.
Before the effective filing date of the claimed invention, it would have been obvious to one of ordinary skill in the art to downregulate the expression of functional cowpea VuTFL1/VuSP2 protein encoded by the coding region of VuTFL1/VuSP2 gene to develop determinate plants, as described by Dhanasekar et al., in different (than the ones used by Dhanasekar et al.)
PNG
media_image1.png
380
567
media_image1.png
Greyscale
elite cowpea varieties using CRISPR/Cas9 gene editing technique using a polynucleotide encoding a Cas9 protein and suitable gRNA(s), as described by Lemmon et al. and regeneration of genome edited cowpea plants, as described by Che et al., in shorter time while avoiding off-target mutations.
Before the effective filing date, an ordinarily skilled artisan would have been motivated to use CRISPR/Cas9 gene editing technique using a suitable gRNA including SEQ ID NO:123 with a realistic goal to silence or downregulate expression of the functional VuTFL1/VuSP2 protein encoded by the VuTFL1/VuSP2 gene for developing determinate cowpea plants in elite and non-determinate cowpea genotypes. Using any specific gRNA sequence preferably in the first exon and/or the promoter sequence ((the gRNAs including SEQ ID NO: 123 span the first exon/coding region of the VuSP2 gene which is from position 92 to 292 of SEQ ID NO: 113 (data not shown)) to mutate VuTFL1/VuSP2 gene is within the experimental design choice of the ordinary skilled artisan.
Regarding claims 14 and 53, Lemmon et al. (U), teaches using Cas9 protein and the NGG Protospacer Adjacent Motif (PAM) (page 768, Fig. 2b and 2h). It is well known in the art that Cas9 protein recognizes the 3’-NGG-5’ Protospacer Adjacent Motif (PAM)2. The method for targeted genome editing, as described above, would include a 3’ NGG PAM sequence in the gRNA, as taught by Lemmon et al. (U) (page 768, Fig. 2b and 2h) and Lemmon et al. (V) (page 6, Supplementary Fig. 1a), while using Cas9 endonuclease.
Regarding claims 15 and 54, Targeted genome editing to silence/downregulate specific VuTFL1/VuSP2 gene, requires plant transformation to introduce the polynucleotide sequence encoding Cas9 protein and the gRNA(s). Plant transformation using agrobacterium-mediated infiltration is a well-known method which is also described by Lemmon et al. (U) (page 766, right column, para 2, line 2-4) and Che et al. (abstract).
Response to Applicant’s arguments
The Office acknowledges the Declaration under 37 C.F.R 1.132, submitted on 3/20/2026.
The argument set forth in the Applicant’s reply on 03/20/2026 along with the Declaration under 37 C.F.R 1.132 has been considered but is not found persuasive.
The Applicant argues that the cited prior art “overlooks the critical qualitative difference in the nature of the mutation taught by Dhanasekar versus the specific type and location of mutation explicitly claimed by Applicant in claim 1” (response, page 7, para 2, line 9-11). The Applicant continues to argue that “the amended claims specifically define the gene as the VuSP2 gene comprising a sequence as denoted by SEQ ID NO: 113, and specifies that the insertion and/or deletion (indel) mutation is generated by a gRNA with a sequence selected from SEQ ID NO:115-137 (targeted to exon 1 region of the gene). This is a critical distinction from Dhanasekar, which identifies a successful substitution mutation in exon 4” (p. 10, para 3, line 1-6). The applicant continues to argue that “The reference focuses on how this substitution affects protein function and stability while maintaining the overall structural integrity of the protein” (page 8, para 2, line 2-4).
The Examiner disagrees after fully considering all the arguments by the Applicant. As discussed above, cited prior art references including Dhanasekar et al., describe that downregulating/silencing of the functional VuSP2/VuTFL1 protein encoded by the VuSP2/VuTFL1 coding region results in producing determinant plant architecture and early flowering. Mutating the first exon (first coding region) of the VuSP2/VuTFL1 gene would delete and/or alter the amino acid sequence of the functional VuSP2/VuTFL1 protein and making it ineffective or nonfunctional.
It is not clear to the Examiner what the Applicant implies by “overall structural integrity of the protein”. The cited references describe specific substitution mutation in the VuSP2/VuTFL1 resulting in a different protein structure and, thus, does not maintain the “the overall structural integrity of the protein”. Such mutation(s) resulting in a changed structure of the mRNA transcript (and encoding a mutated protein) do(es) compromise translation of the non-mutated protein.
Regarding Applicant’s argument that Dhanasekar teaches a substitution mutation in exon 4 produced by site-directed mutagenesis, whereas the claims require an indel produced by using CRISPR/Cas: the teachings of Dhanasekar show that a cowpea plant will have determinate architecture when the wild-type VuSP2 protein is not expressed. It was obvious to the ordinary artisan that any other manipulation of the VuSP2 gene that disrupts expression of the wild type VuSP2 protein would result in determinate plant architecture. Other methods of introducing alterations to plant genes were of course known in the prior art, including CRISPR/Cas, as discussed in the rejection. One of ordinary skill would have been motivated to use the CRISPR/Cas to avoid off-target mutations, also as discussed in the rejection.
The declaration under 37 C.F.R 1.132 mentions that “genome editing outcomes in plants are highly species dependent and often require significant optimization of transformation methods, regeneration systems, and editing constructs” (response, page 4, para 14, line 3-5), implying that genome editing in cowpea would not have been obvious to POSITA based on the cited references. The Applicant is reminded that Che et al. explicitly describes CRISPR/Cas based genome editing in cowpea (abstract).
The Declaration also argues that the results demonstrating use of gRNA sequence SEQ ID NO: 123 to edit the VuSP2 gene in cowpea, was surprising and unexpected, and produces edits that are different from that of Dhanasekar (p. 4-10). However, the results presented in the declaration are not unexpected. As discussed in the rejection above, while Dhanasekar teaches an amino acid substitution to the VuSP2 protein, this structural change resulted in a detrimental effect on protein function (page 55, left column, para 1, line 19-21) and led to determinant plant architecture. Therefore, it is obvious, and not unexpected, that other edits to the VuSP2 gene that also negatively affect protein function would also cause the phenotype. Use of CRISPR/Cas to cause the gene editing is also not surprising or unexpected, as the cited prior art teaches its use in cowpea, as discussed in the rejection.
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). 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.
Contact Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JAY CHATTERJEE whose telephone number is (703)756-1329. The examiner can normally be reached (Mon - Fri) 8.30 am to 5.30 pm..
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
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.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/Jay Chatterjee/ Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/ Supervisory Patent Examiner, Art Units 1661 & 1662
1 Kaldate et al. (Allelic characterization and protein structure analysis reveals the involvement of splice site mutation for growth habit differences in Lablab purpureus (L.) Sweet, 2021, Journal of Genetic Engineering and Biotechnology, 19:34, published online on 22 February 2021) provides the evidence that determinate growth habit includes early flowering and synchronous pod maturity, besides ease in manual harvesting and short crop duration.
2 Hirano et al. (Structural Basis for the Altered PAM Specificities of Engineered CRISPR-Cas9, 2016, Molecular Cell, 61:886–894) provides the evidence that Cas9 protein recognizes and needs the 3’-NGG-5’ Protospacer Adjacent Motif (PAM) (summary; page 866, right column, para 1, line 13-15).
Prosecution Insights