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 .
Second Action Non-Final
It is noted that this Office action is a Second Action Non-Final. The Office is re-opening prosecution to make a new rejection over the position that any mutation introduced using any guide system in any gene location that disrupts the binding of DNA is rendered obvious.
Status of Claims
The amendments received on 04/01/2026 have been entered. Claims 37, 42, 44, 47, 72, 81, 83, 85-86, 99, and 128-130 are pending.
Claims 37, 42, 44, 47, 72, 81, 83, and 85-86 remain withdrawn for being directed to a non-elected invention(s).
Claim 130 has been newly added.
Claims 99 and 128-130 are examined in this Office Action.
Objections/Rejections that are Withdrawn
The 35 USC 112(a) Written Description rejection of claim 128 has been withdrawn in
light of Applicant’s addition of claim 130 and accompanying arguments.
The text of those sections of Title 35, U.S. Code, not included in this action, can be found in the prior Non-Final Office action dated 01/02/2026.
Claim Rejections - 35 USC § 103
Claims 99 and 128-129 remain rejected and claim 130 is newly rejected under 35 U.S.C. 103 as being unpatentable over Gerasimova (Gerasimova et al., 2019, Vavilov Journal of Genetics and Breeding, Vol. 22(8), pp. 1033-1039) (included in IDS) in view of Guo (Guo et al., Pub. No.: US 2012/0278947 A1, Pub. Date: Nov. 1, 2012) (included in IDS) and in further view of Harris (Harris et al., 2011, New Phytologist, Vol. 190, pp. 823-837).
The claims recite “[a] guide nucleic acid that binds to a target site in an endogenous HD-Zip I transcription factor gene, the target site encoding a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 83; wherein the target site comprises a sequence having at least 80% identity to nucleotide sequences SEQ ID NOs: 75 or 76.”
Gerasimova teaches targeted genome modification using an RNA-guided Cas9 endonuclease (entire document; see Title, Abstract, for example). Gerasimova teaches that targeted genome modification is a modern, particularly versatile tool to genetically engineer plants, and there are many reports of successful crop improvement using different applications of RNA-guided endonucleases (page 1034, left column, first paragraph). The targeted sites, which were mutated, exhibited deletions of various sizes, resulting in site-directed genome modification (Abstract).
Gerasimova teaches selecting the Vrs1 gene of barley (the barley Vrs1 gene encodes a Homeodomain-Leucine Zipper (HD-Zip) Class I transcription factor) for targeted genome modification using CRISPR/Cas technology (page 1034, left column). Gerasimova further teaches the target-specific 20bp region of the guide RNA inserted into pSH121 vector (i.e., a guide nucleic acid that binds to a target site in an endogenous HD-Zip I transcription factor gene) (page 1035, right column, first full paragraph).
Gerasimova does not explicitly teach a target site in an endogenous HD-Zip I transcription factor gene encoding a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 83.
However, Guo teaches the down-regulation of a homeodomain-leucine zipper I-class homeobox (i.e., transcription factor) gene for improved plant performance (entire document; see Title, Abstract, for example).
Guo teaches ZmME293, a maize homeodomain-leucine zipper I-class homeobox gene (page 1, paragraph 0004). Guo teaches and claims a method of reducing the expression of ZmME293 or the activity of ZmME293 in a plant, the method comprising modulating the expression levels of ZmME293 mRNA or the protein level of ZmME293 or the activity of ZmME293 polypeptide, wherein the modulation results in an improved agronomic performance of the plant (Guo, claim 33). Guo teaches that the activity of a ZmME293 polypeptide may be reduced or eliminated by disrupting or excising at least a part of the gene encoding the ZmME293 polypeptide. Mutagenized plants that carry mutations in ZmME293 genes also result in reduced expression of the ZmME293 gene and/or reduced activity of the encoded ZmME293 polypeptide (page 14, paragraph 0142).
Guo teaches SEQ ID NO: 2, a polypeptide from Zea mays encoded by ZmME293, a maize homeodomain-leucine zipper I-class homeobox gene. A GenBank BLAST of Guo SEQ ID NO: 2 discloses the following sequence and its conserved domain:
Homeobox-leucine zipper protein HOX14-like [Zea mays] Sequence ID: NP_001140916.1
Query 1 MEQYDGLFPPAYVDSSSSLLLVPNANGTAQEERPRARRRRRRAARCGGGGGELDGGGDHK 60
MEQYDGLFPPAYVDSSSSLLLVPNANGTAQEERPRARRRRRRAARCGGGGGELDGGGDHK
Sbjct 1 MEQYDGLFPPAYVDSSSSLLLVPNANGTAQEERPRARRRRRRAARCGGGGGELDGGGDHK 60
Query 61 KRRLTDEQVEMLELSFREERKLETGRKVHLAAELGLDPKQVAVWFQNRRARHKSKLLEEE 120
KRRLTDEQVEMLELSFREERKLETGRKVHLAAELGLDPKQVAVWFQNRRARHKSKLLEEE
Sbjct 61 KRRLTDEQVEMLELSFREERKLETGRKVHLAAELGLDPKQVAVWFQNRRARHKSKLLEEE 120
Query 121 FAKLKQAHDAAILHKCHLENEVMRLKDKLVLAEEELTRFRSAGNHAVSGDGGDVMARAVC 180
FAKLKQAHDAAILHKCHLENEVMRLKDKLVLAEEELTRFRSAGNHAVSGDGGDVMARAVC
Sbjct 121 FAKLKQAHDAAILHKCHLENEVMRLKDKLVLAEEELTRFRSAGNHAVSGDGGDVMARAVC 180
Query 181 SGSPSSSFSTGTCQQPGGGGGGGDHLGDDDLLYVPDYAYADSSVVEWFSLYGLM 234
SGSPSSSFSTGTCQQPGGGGGGGDHLGDDDLLYVPDYAYADSSVVEWFSLYGLM
Sbjct 181 SGSPSSSFSTGTCQQPGGGGGGGDHLGDDDLLYVPDYAYADSSVVEWFSLYGLM 234
PNG
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310
950
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Greyscale
The conserved domain of Guo sequence SEQ ID NO: 2 shares 100% sequence identity (i.e., at least 80% sequence identity) with instant sequence SEQ ID NO: 83 (see alignment below). Thus, the sequence taught by Guo would be encoded by an endogenous HD-Zip I transcription factor gene encompassing a target site which could bind a guide nucleic acid.
ALIGNMENT OF GUO SEQUENCE SEQ ID NO: 2 AND INSTANT SEQUENCE SEQ ID NO: 83
Qy 1 EMLELSFREERKLETGRKVHLAAELGLDPKQVAVWFQNRRARHKSK 46
||||||||||||||||||||||||||||||||||||||||||||||
Db 70 EMLELSFREERKLETGRKVHLAAELGLDPKQVAVWFQNRRARHKSK 115
Furthermore, Harris teaches that a common region of DNA named the homeobox (HB) encodes a DNA-binding domain termed the homeodomain (HD). The HB is a 180-nucleotide region of DNA encoding a 60-amino-acid-long HD that consists of three characteristic α-helices. α-helix 3, considered the recognition helix, is the most conserved across HD proteins, and is responsible for the specificity of protein–DNA interactions. The leucine zipper is an α-helix comprised of seven amino acid (heptad) repeats. The residues of the heptad are designated an, bn....gn (n being the number of the heptad), where residue d is a leucine. During dimerization through the monomeric α-helices, the structure forms a coiled coil. Residues a and d form the hydrophobic interface of the coiled coil, while residues at positions e and g form complementary charge interactions that permit or inhibit dimerization between two monomers. The leucine zipper of HD-Zip proteins is immediately downstream of the HD and enables dimerization of HD-Zip proteins, which is a prerequisite for DNA binding. Many HD proteins bind strongly as monomers to DNA, but HD-Zip proteins possess a very weak affinity for DNA as monomers and require dimerization for increased DNA-binding efficiency. If the spacing between the HD and leucine zipper components of HD-Zip I proteins is modified, DNA binding is disrupted, indicating that dimerization is essential for the correct spacing of the HDs with regard to their position on the DNA (page 825, Section 3).
It is noted that it is the position of this Office that any mutation introduced using any guide system in any gene location that disrupts the binding of DNA is rendered obvious by the teachings of Gerasimova, Guo, and Harris.
At the time the instant application was filed, it would have been obvious and within the scope of one of ordinary skill in the art to utilize the CRISPR/Cas genome editing system comprising a guide RNA as taught by Gerasimova to bind to a target sequence in an endogenous HD-Zip I transcription factor gene encoding a sequence having at least 80% sequence identity to instant sequence SEQ ID NO: 83 as taught by Guo, to disrupt the DNA binding in the homeodomain (HD) as taught by Harris. One of ordinary skill in the art would have anticipated that targeting the HD of the HD-Zip I transcription factor gene as taught by Guo would disrupt DNA binding as taught by Harris. One would have been motivated to combine the teachings of Gerasimova, Guo, and Harris knowing that the modification of crop genomes employing functional components of CRISPR/Cas (i.e., guide nucleic acids) has been successful in crop improvement as taught by Gerasimova, and the modification of a homeodomain-leucine zipper I-class homeobox gene (disruption of DNA binding as taught by Harris) leads to transgenic plants displaying increased seed and/or biomass yield as taught by Guo. Thus, one of ordinary skill in the art would have a high expectation of success by combining the teachings of Gerasimova, Guo, and Harris.
The use of a guide nucleic acid to introduce a mutation via targeted genome editing in plants is a technique that was routine in the art at the time the application was filed, as taught by the cited references and the state of the art in general.
In regard to claim 128, Guo teaches SEQ ID NO: 1, a maize homeodomain-leucine zipper I-class homeobox gene (Guo, Specification, page 57, lines 33-34), which shares 99.0% sequence identity with instant sequence SEQ ID NO: 75, and 96.9% sequence identity with instant sequence SEQ ID NO: 76 (i.e., wherein the target site comprises a sequence having at least 80% identity to nucleotide sequences SEQ ID NOs: 75 or 76) (see alignments below).
ALIGNMENT OF GUO SEQUENCE SEQ ID NO: 1 AND INSTANT SEQUENCE SEQ ID NO: 75
Query Match 99.0%; Score 151.4; Length 705;
Best Local Similarity 99.3%;
Matches 152; Conservative 0; Mismatches 1; Indels 0; Gaps 0;
Qy 1 ACCGACGAGCAGGTAGAGATGCTGGAGCTGAGCTTCCGGGAGGAGCGGAAGCTGGAGACC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 193 ACCGACGAGCAGGTAGAGATGCTGGAGCTGAGCTTCCGGGAGGAGCGGAAGCTGGAGACC 252
Qy 61 GGCCGGAAGGTGCACCTGGCCGCCGAGCTCGGGCTCGACCCCAAGCAGGTCGCCGTCTGG 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 253 GGCCGGAAGGTGCACCTGGCCGCCGAGCTCGGGCTCGACCCCAAGCAGGTCGCCGTCTGG 312
Qy 121 TTCCAGAACCGCCGCGCTCGCCACAAGAGCAAG 153
||||||||||||||||| |||||||||||||||
Db 313 TTCCAGAACCGCCGCGCCCGCCACAAGAGCAAG 345
ALIGNMENT OF GUO SEQUENCE SEQ ID NO: 1 AND INSTANT SEQUENCE SEQ ID NO: 76
Query Match 96.9%; Score 148.2; Length 705;
Best Local Similarity 98.0%;
Matches 150; Conservative 0; Mismatches 3; Indels 0; Gaps 0;
Qy 1 AGCGACGAGCAGGCAGAGATGCTGGAGCTGAGCTTCCGGGAGGAGCGGAAGCTGGAGACG 60
| ||||||||||| |||||||||||||||||||||||||||||||||||||||||||||
Db 193 ACCGACGAGCAGGTAGAGATGCTGGAGCTGAGCTTCCGGGAGGAGCGGAAGCTGGAGACC 252
Qy 61 GGCCGGAAGGTGCACCTGGCCGCCGAGCTCGGGCTCGACCCCAAGCAGGTCGCCGTCTGG 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 253 GGCCGGAAGGTGCACCTGGCCGCCGAGCTCGGGCTCGACCCCAAGCAGGTCGCCGTCTGG 312
Qy 121 TTCCAGAACCGCCGCGCCCGCCACAAGAGCAAG 153
|||||||||||||||||||||||||||||||||
Db 313 TTCCAGAACCGCCGCGCCCGCCACAAGAGCAAG 345
In regard to claims 129 and 130, even though Gerasimova and Guo do not explicitly teach instant SEQ ID NOs: 85-90 as spacer sequences needed for binding to the DNA, there is only a finite number of possible targets for this role in the homeodomain region of an endogenous HD-Zip transcription factor gene. Therefore, it would be obvious to a person of ordinary skill in the art to try any one of these finite number of sequences for the identification of spacers to bind to DNA; and that would result in instant SEQ ID NOs:85-90, without any surprising result. One of ordinary skill in the art would have been motivated to find spacer sequences that bind to the homeodomain region of the endogenous HD-Zip transcription factor gene to disrupt gene function knowing that mutagenized plants that carry mutations in ZmME293 genes resulted in reduced expression of the ZmME293 gene and/or reduced activity of the encoded ZmME293 polypeptide as taught by Guo. It is well known in the art that spacers are indeed necessary components of the guide nucleic acid(s).
Response to Applicant’s Arguments
Applicant's arguments filed 04/01/2026 have been fully considered but they are not persuasive.
Applicant argues that Gerasimova, Guo, and Harris fail to provide one of ordinary skill in the art with a finite number of target sites.
The Examiner respectfully disagrees. Gerasimova demonstrated that the VRS1 gene of a Siberian elite barley cultivar can readily be modified at predefined target motifs by Cas endonuclease technology (Conclusion, page 1038). Site-directed plant genome modification by this technology involves the construction of Cas endonuclease- and guide-RNA-encoding vectors, delivery of the plasmid DNA into plant cells, processing of the chosen genomic target site by the corresponding gene products, and regeneration of plants from modified cells (Abstract, page 1033).
Guo teaches compositions and methods for down-regulating the level and/or activity of ZmME293 in plants, including the development of specific RNAi constructs for creation of plants with improved yield and/or improved abiotic stress tolerance (page 1, 0007). Guo teaches ZmME293 (Guo SEQ ID NO:1), a maize homeodomain-leucine zipper I-class homeobox gene, which shares 99.0% sequence identity with instant sequence SEQ ID NO: 75, and 96.9% sequence identity with instant sequence SEQ ID NO: 76; ZmME293 (Guo SEQ ID NO:1) encodes (Guo) SEQ ID NO: 2 which shares 100% identity with instant sequence SEQ ID NO: 83.
Guo further teaches that plant cells and plants can be modified by introduction of a ZmME293 polynucleotide sequence configured for RNA silencing or interference. For example, hairpin RNA expression cassettes may be used for modifying drought tolerance, NUE, seed or biomass yield, density tolerance or other phenotypes in maize. Down-regulation of ZmME293(s) by hairpin RNA (hpRNA) expression can result in plants or plant cells having reduced expression (up to and including no detectable expression) of one or more ZmME293s (page 23, 0209). Additionally, Guo teaches the sense strand of the hairpin (Guo SEQ ID NO: 40) and the anti-sense strand of the hairpin (Guo SEQ ID NO: 41) (page 23, 0205), which both share 99.0% sequence identity with instant sequence SEQ ID NO: 75, and 98% sequence identity with instant sequence SEQ ID NO: 76 (see alignments below). Thus, although Guo does not reference a “homeodomain” or a “conserved domain”, Guo does directly point to the region of interest for mutation by the RNAi hairpin sense and anti-sense sequences.
GUO SEQUENCE SEQ ID NO: 40 ALIGNED WITH INSTANT SEQUENCE SEQ ID NO: 75
Qy 1 ACCGACGAGCAGGTAGAGATGCTGGAGCTGAGCTTCCGGGAGGAGCGGAAGCTGGAGACC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 194 ACCGACGAGCAGGTAGAGATGCTGGAGCTGAGCTTCCGGGAGGAGCGGAAGCTGGAGACC 253
Qy 61 GGCCGGAAGGTGCACCTGGCCGCCGAGCTCGGGCTCGACCCCAAGCAGGTCGCCGTCTGG 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 254 GGCCGGAAGGTGCACCTGGCCGCCGAGCTCGGGCTCGACCCCAAGCAGGTCGCCGTCTGG 313
Qy 121 TTCCAGAACCGCCGCGCTCGCCACAAGAGCAAG 153
||||||||||||||||| |||||||||||||||
Db 314 TTCCAGAACCGCCGCGCCCGCCACAAGAGCAAG 346
GUO SEQUENCE SEQ ID NO: 41 ALIGNED WITH INSTANT SEQUENCE SEQ ID NO: 75
Qy 1 ACCGACGAGCAGGTAGAGATGCTGGAGCTGAGCTTCCGGGAGGAGCGGAAGCTGGAGACC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 350 ACCGACGAGCAGGTAGAGATGCTGGAGCTGAGCTTCCGGGAGGAGCGGAAGCTGGAGACC 291
Qy 61 GGCCGGAAGGTGCACCTGGCCGCCGAGCTCGGGCTCGACCCCAAGCAGGTCGCCGTCTGG 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 290 GGCCGGAAGGTGCACCTGGCCGCCGAGCTCGGGCTCGACCCCAAGCAGGTCGCCGTCTGG 231
Qy 121 TTCCAGAACCGCCGCGCTCGCCACAAGAGCAAG 153
||||||||||||||||| |||||||||||||||
Db 230 TTCCAGAACCGCCGCGCCCGCCACAAGAGCAAG 198
GUO SEQUENCE SEQ ID NO: 40 ALIGNED WITH INSTANT SEQUENCE SEQ ID NO: 76
Qy 1 AGCGACGAGCAGGCAGAGATGCTGGAGCTGAGCTTCCGGGAGGAGCGGAAGCTGGAGACG 60
| ||||||||||| |||||||||||||||||||||||||||||||||||||||||||||
Db 194 ACCGACGAGCAGGTAGAGATGCTGGAGCTGAGCTTCCGGGAGGAGCGGAAGCTGGAGACC 253
Qy 61 GGCCGGAAGGTGCACCTGGCCGCCGAGCTCGGGCTCGACCCCAAGCAGGTCGCCGTCTGG 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 254 GGCCGGAAGGTGCACCTGGCCGCCGAGCTCGGGCTCGACCCCAAGCAGGTCGCCGTCTGG 313
Qy 121 TTCCAGAACCGCCGCGCCCGCCACAAGAGCAAG 153
|||||||||||||||||||||||||||||||||
Db 314 TTCCAGAACCGCCGCGCCCGCCACAAGAGCAAG 346
GUO SEQUENCE SEQ ID NO: 41 ALIGNED WITH INSTANT SEQUENCE SEQ ID NO: 76
Qy 1 AGCGACGAGCAGGCAGAGATGCTGGAGCTGAGCTTCCGGGAGGAGCGGAAGCTGGAGACG 60
| ||||||||||| |||||||||||||||||||||||||||||||||||||||||||||
Db 350 ACCGACGAGCAGGTAGAGATGCTGGAGCTGAGCTTCCGGGAGGAGCGGAAGCTGGAGACC 291
Qy 61 GGCCGGAAGGTGCACCTGGCCGCCGAGCTCGGGCTCGACCCCAAGCAGGTCGCCGTCTGG 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 290 GGCCGGAAGGTGCACCTGGCCGCCGAGCTCGGGCTCGACCCCAAGCAGGTCGCCGTCTGG 231
Qy 121 TTCCAGAACCGCCGCGCCCGCCACAAGAGCAAG 153
|||||||||||||||||||||||||||||||||
Db 230 TTCCAGAACCGCCGCGCCCGCCACAAGAGCAAG 198
As stated in the instant Specification, the objective of the instant invention is to prevent or reduce (disrupt) binding of the encoded HD-Zip I transcription factor to DNA (Summary of the Invention, page 2).
To that point, Guo teaches the maize homeodomain-leucine zipper I-class homeobox gene (Guo SEQ ID NO: 1), its encoded protein (Guo SEQ ID NO: 2), the region of interest for mutation (the RNAi hairpin sense and anti-sense sequences), and the effect of RNA silencing, interference, and/or down-regulation of the gene; Harris teaches the commonality and importance of the region of DNA named the homeobox (HB), which encodes a DNA-binding domain termed the homeodomain (HD); and Gerasimova teaches the method of using a guide nucleic acid that binds to a target site in an endogenous homeodomain-leucine zipper I-class homeobox gene. Taken together, the teachings of Gerasimova, Guo, and Harris provide one of ordinary skill in the art with the target gene comprising a finite number of target sites and the method to precisely target a specific site in the required region. One having ordinary skill in the art would have a high expectation of success by following the teachings of Gerasimova, Guo, and Harris.
It is reiterated that it is the position of this Office that any mutation introduced using any guide system in any gene location that disrupts the binding of DNA is rendered obvious by the teachings of Gerasimova, Guo, and Harris.
Summary
No claim is allowed.
Correspondence
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CHRISTINA MEADOWS whose telephone number is (703)756-1430. The examiner can normally be reached Monday - Friday 9:00 am - 5:00 pm.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Amjad Abraham can be reached on 571-270-7058. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/CHRISTINA L MEADOWS/Examiner, Art Unit 1663
CHRISTINA MEADOWS
Examiner
Art Unit 1663
/Amjad Abraham/SPE, Art Unit 1663
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