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 1-5, 9, 12-14, 17, 35-36, 58, 62-63, 68 and 74-76 are pending.
Claims 1-5, 9, 12-14, 17, 35-36, 58, 62-63, 68 are withdrawn from examination as being part of non-elected inventions.
Claims 74-76 are being examined.
All previous objections and rejections not set forth below have been withdrawn in view of applicant’s amendments to the claims.
The claim amendments by the Applicant by adding new issues, which was not present in any of the claims before, necessitated new grounds of rejections and new prior art reference(s), as discussed below.
Information Disclosure Statement
Initialed and dated copy of Applicant’s information disclosure statement (IDS) filed on 12/10/2025 are attached to the instant Office action. The submissions are in compliance with the provisions of 37 C.F.R. § 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Claim Rejections - 35 USC § 103
Claim 74 is rejected under 35 U.S.C. 103 as being unpatentable over Uga, Y. (US20170306421 A1) in view of Uga et al. (Control of root system architecture by DEEPER
ROOTING 1 increases rice yield under drought conditions, 2013, Nature Genetics, 4:1097-1102) and Feng et al. (ABA-inducible DEEPER ROOTING 1 improves adaptation of maize to water deficiency, 2022, Plant Biotechnology Journal, 20:2077–2088).
Claim 74 is drawn to a guide nucleic acid that binds to a target site within an endogenous gene encoding DEEPER ROOTING 1 (DRO1), wherein the endogenous gene encodes a polypeptide having at least 80% identity to SEQ ID NOs: 77. The protein set forth by SEQ ID NO: 77 is encoded by the DRO1 gene in the genome of a corn plant (spec, page 5, line 15-16 and 21-23).
Uga, Y. describes a maize DRO1 gene encoding a cDNA (SEQ ID NO: 14) comprising 99.3% sequence identity to instant SEQ ID NO: 76 (data not shown), which encodes the coding region (cDNA) of the genomic DNA sequence set forth in instant SEQ ID NO: 75. The cDNA encodes a ZmDRO1 polypeptide (SEQ ID NO: 15, as described by Uga, Y) comprising 99.2% sequence identity (i.e., at least 80%) to instant SEQ ID NO: 77, as discussed in the previous Office action (dated 9/11/2025, bridging paragraph between page 5 and 6) and also shown below.
Title: US-18-775-013-77
Perfect score: 1323
Sequence: 1 MKIFSWVANKIGGKQEPKRS..........LDDDGAKWVKTDSDFIVLEM 255
Scoring table: BLOSUM62
Gapop 10.0 , Gapext 0.5
Searched: 1 seqs, 255 residues
Total number of hits satisfying chosen parameters: 1
Database : US-15-586-096-15.pep:*
RESULT 1
US-15-586-096-15
Query Match 99.4%; Score 1315; DB 1; Length 255; Best Local Similarity 99.2%;
Matches 253; Conservative 2; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MKIFSWVANKIGGKQEPKRSAAHYRGNVSECRNDEFSDWPQSLLAIGTFGDRQLEEGVVE 60
||||||||||||||||||||||||||||||||||||||||||||||||||:|||||||||
Db 1 MKIFSWVANKIGGKQEPKRSAAHYRGNVSECRNDEFSDWPQSLLAIGTFGNRQLEEGVVE 60
51
Qy 61 TSSGNVQAAQDPAKFTEEEEADSIRRELEVLLLQGNNNNGGQAEAQGSRGDERRQVAWKE 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 TSSGNVQAAQDPAKFTEEEEADSIRRELEVLLLQGNNNNGGQAEAQGSRGDERRQVAWKE 120
Qy 121 HDGECSKEKQPTSGEMVTSKARAREMVAGKKRSTLKPRSVASLLRLLACKGGFATPVLEP 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 HDGECSKEKQPTSGEMVTSKARAREMVAGKKRSTLKPRSVASLLRLLACKGGFATPVLEP 180
Qy 181 RSPFPQSRMEKLLKAILEKKIHPQNPSTAAARRHQLDWKLDEKEIDECLEDALRDLDDDG 240
|||||||||||||||||||||||||||||||||||||||||||||||||:||||||||||
Db 181 RSPFPQSRMEKLLKAILEKKIHPQNPSTAAARRHQLDWKLDEKEIDECLDDALRDLDDDG 240
230
Qy 241 AKWVKTDSDFIVLEM 255
|||||||||||||||
Db 241 AKWVKTDSDFIVLEM 255
However, Uga, Y. does not describe using CRISPR/Cas based genome editing technique and/or using a guide nucleic acid (or gRNA).
Uga et al. describes that higher expression of DRO1 gene increases the root growth angle (RGA), whereby roots grow deeper (deep rooting) in a more downward direction (abstract, line 9-11), and becomes resistant to drought (abstract, last line).
Feng et al. teaches that modern maize cultivars have different root architectures, particularly the RGA, compared to ancestral species (page 1, right column, para 1, line 18-19) including Balsas teosinte (page 1, left column, para 2, last line). One of the genes affecting drought avoidance (or tolerance/resistance) trait by modifying root architecture in maize, as in many other plants including rice, is DRO1 (page 2078, left column, para 4). Maize DRO1 (ZmDRO1) has a different allele (ZmDRO1mex) in more drought tolerant ancient maize cultivars like Zea mays ssp. Mexicana (ZmDRO1mex) compared to more modern cultivars like B73 containing the ZmDRO1B73 allele (page 2078, right column, para 1, last 2 lines; and page 2078, right column, para 2, line). The genotype of ZmDRO1mex exists in teosinte but has been lost in most cultivated maize during domestication (page 2078, right column, para 2, last 2 lines). Feng et al. also teaches that differential effects between ZmDRO1mex and ZmDRO1B73 alleles is due to the differences in transcription level (page 2078, right column, para 4, line 1-12). ZmDRO1 displays the most dramatic response to ABA in both B73 and mexicana. ZmDRO1 had a lower basal expression but a stronger response to ABA induction in mexicana than in B73 (page 2078, right column, para 4, line 18-21). Recombinant inbred lines harboring ZmDRO1mex have a stronger response to ABA induction, larger downward root angle upon ABA stimulus, and higher grain yield in water-deficient fields (and page 2084, left column, first para, line 2-4; and page 2084, right column, first para, first 3 lines). Moreover, Feng et al. also describes a method to enhance drought avoidance/tolerance by overexpressing the endogenous DRO1 gene (e.g. GenBank accession No. GRMZM2G 700200; page 2086, right column, last para, line 1) encoding a protein having at least 80% (99.2%) sequence identity to instant SEQ ID NO: 77 (as shown below), in modern cultivars like B73 (page 2079, right column, para 3, line 8-17).
RESULT 1
AASEQ2_01232026_132445
Best Local Similarity 99.2%; Query Match 99.3%; Score 610; DB 1; Length 254;
Matches 119; Conservative 1; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MVTSKARAREMVAGKKRSTLKPRSVASLLRLLACKGGFATPVLEPRSPFPQSRMEKLLKA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 135 MVTSKARAREMVAGKKRSTLKPRSVASLLRLLACKGGFATPVLEPRSPFPQSRMEKLLKA 194
Qy 61 ILEKKIHPQNPSTAAARRHQLDWKLDEKEIDECLDDALRDLDDDGAKWVKTDSDFIVLEM 120
||||||||||||||||||||||||||||||||||:|||||||||||||||||||||||||
Db 195 ILEKKIHPQNPSTAAARRHQLDWKLDEKEIDECLEDALRDLDDDGAKWVKTDSDFIVLEM 254
Before the effective filing date of the invention, it would have been obvious to one ordinarily skilled artisan to modify the method described by Uga Y. using the well-known and widely used targeted gene editing CRISPR-Cas technique to substitute the coding region in the DRO1 gene in a cultivated maize variety (e.g., GenBank accession No. GRMZM2G 700200) to overexpress the modified protein as encoded by ZmDRO1mex gene from mexicana,(as taught by Feng et al.; supplementary Table S1). There is only one amino acid difference between the DRO1 protein in a cultivated maize variety, as in GenBank accession No. GRMZM2G 700200, and the protein encoded by the ZmDRO1mex gene from Mexicana, as shown above.
It would have been obvious to an ordinarily skilled artisan that such targeted gene editing in the coding region of DRO1 gene would change root architecture conferring enhanced drought avoidance/tolerance in commercially important cultivated maize varieties including B73. Designing a guide nucleic acid (gRNA) for CRISPR-Cas based gene editing technique is a well-known and standard practice in the art1.
Before the effective filing date, an ordinarily skilled artisan would have been motivated to design and use a guide nucleic acid (gRNA) to replace the endogenous DRO1 gene in a cultivated variety with the ZmDRO1mex gene from Mexicana comprising the coding region that encodes a polypeptide comprising at least 80% sequence identity to instant SEQ ID NO: 77, with the realistic goal to change root architecture to enhance drought avoidance/tolerance in the genome edited plant.
Claims 75-76 are rejected under 35 U.S.C. 103 as being unpatentable over Uga, Y. in view of Uga et al. and Feng et al. as applied to claim 74 above, and further in view of Kitomia et al. (Root angle modifications by the DRO1 homolog improve rice yields in saline paddy fields, 2020, PNAS, 117:21242–21250).
Claim 75 is drawn to a guide nucleic of claim 74, wherein the target site is in a region of the DRO1 gene having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 103.
Uga, Y., in view of Uga et al. and Feng et al., describes making a guide nucleic acid to edit a target site in the DRO1 gene having at least 80% sequence identity to SEQ ID NO: 77 to change root architecture to enhance drought avoidance/tolerance in the genome edited plant, as discussed above.
However, Uga, Y. in view of Uga et al. and Feng et al. does not explicitly describe a guide nucleic targeting a region of the DRO1 gene having at least 80% sequence identity to the nucleotide sequence comprising SEQ ID NO: 103.
Kitomia et al. describes CRISPR-Cas9 assays revealing several DRO1 homologs involved in influencing root system architecture (RSA) in crops. Introgression lines with combinations of gain-of-function and loss-of-function alleles in qSOR1 (a homologue of DRO1) and DRO1 comprise four different RSAs (ultra-shallow, shallow, intermediate, and deep rooting) suggesting that natural alleles of the DRO1 (including DRO1 homologs) can be utilized to control RSA variations (abstract). Different gain-of-function DRO1 alleles conferring deeper rooting as well as the loss-of-function alleles with soil-surface roots (SOR), which enable upland plants to adapt to waterlogging by allowing them to obtain oxygen from the air (page 21242, left column, para 1, line 2-4), are equally useful. Such loss-of-function alleles in isogenic lines enabled rice to avoid the reducing stresses of saline soils, resulting in increased yields (abstract).
Kitomia et al. teaches that the C-terminal region among the DRO1 homologs is indispensable for RGA control (page 21244, right column, para 3, line 1-2). The C-terminal domain includes the WxxTD and EAR-like motifs which are well conserved despite relatively low homology among the DRO1 proteins (page 21244, right column, para 3, line 2-4).
Kitomia et al. describes using CRISPR-Cas9 based technique to edit DRO1 homologues/alleles (page 21244, right column, para 2, line 13-16; Fig. 2; page 21248, left column, para 5, line 6-8). Genome edited deletion lines without the C-terminal region show shallow RGA phenotypes indicating that the conserved regions in the C-terminal of the DRO1 homologs (or alleles) are essential for RGA control in both dicots and monocots (page 21244, right column, para 3, line 22-27).
A gRNA sequence comprising instant SEQ ID NO: 103 (as recited in claim 75) which includes a spacer sequence comprising instant SEQ ID NO: 110 (as recited in claim 76), would target the C-terminal motif of the DRO1 polypeptide comprising instant SEQ ID NO: 77, around the region from position 204 to 223 in 255 amino acid long DRO1 protein (data not shown). Before the effective filing date of the invention, it would have been obvious to one ordinarily skilled artisan to undertake the standard technique of allele mining for DRO1 gene2 in commercially important (modern) cultivars/varieties in maize by editing/mutating the coding region of endogenous DRO1 allele(s) in the plant using suitable guide RNAs to target the C-terminal sequence in the DRO1 protein, as described by Kitomia et al. Even though instant SEQ ID NO:103 is not explicitly taught by the cited references, using any functionally equivalent specific guide RNA (including SEQ ID NO:103) for targeting the C-terminus region of the DRO1 protein with a realistic goal to get loss-of-function DRO1 alleles conferring shallow RGA phenotype(s), as described by Kitomia et al., is within the experimental design choice of any ordinarily skilled artisan as part of the allele mining process using CRISPR-Cas based technique.
Before the effective filing date, an ordinarily skilled artisan would have been motivated to undertake allele mining for DRO1 gene in commercially important maize cultivars by editing the endogenous DRO1 allele(s) using suitable gRNAs including functional equivaents of instant SEQ ID NO: 103 targeting the C-terminal sequence in the DRO1 protein. Using any specific guide RNA to edit the C-terminus region of the DRO1 protein with a realistic objective to get loss-of-function DRO1 alleles conferring shallow RGA phenotype(s) is within the experimental design choice of any ordinarily skilled artisan as part of the allele mining process using CRISPR-Cas based technique.
Conclusion
All the claims are rejected.
Applicant's amendment also necessitated the new grounds 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.
Correspondence
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..
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Jay Chatterjee
Patent Examiner
Art Unit 1662
/Jay Chatterjee/ Examiner, Art Unit 1662
1 Cui et al. (Review of CRISPR/Cas9 sgRNA Design Tools, 2018, Interdisciplinary Sciences: Computational Life Sciences,10:455–465) provides the evidence that designing a guide RNA for targeted gene editing using CRISPR-Cas based technique is a standard and routine practice (abstract).
2 Singh et al. (Allele mining for a drought responsive gene DRO1 determining root growth angle in donors of drought tolerance in rice (Oryza sativa L.), 2021, Physiol Mol Biol Plants, 27:523–534) provides the evidence of allele mining technique, that too specifically for DRO1 gene.