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
Applicant’s claim for the benefit of a prior-filed application 63/489,708 filed March 10th, 2023, and PCT/US2024/018921 filed March 7th, 2024, under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged.
Thus, the earliest possible priority for the instant application is March 10th, 2023.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on December 19th, 2025, was considered, initialed, and attached hereto. A signed copy of the list of references cited is included with this Office Action.
Status of Claims
Claims 1-3, 5, 9, 11, 16-23, 26-28, 30-31, 35, 37-38, 41, 45, 47, 50-51, and 54 filed September 8th, 2025 are pending and examined herein.
Claim Objection
Claim 1 is objected to for the recitation of “wherein the plant comprises nucleic acid encoding the…” in line 5. As nucleic acid is a singular term, claim 1 should be amended to recited “wherein the plant comprises a nucleic acid sequence encoding the…”
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.
Claim 22 is 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.
Claim 22 recites the limitation "the rootstock and/or scion" in line 2. Claim 1 does not recite a rootstock and/or scion for this limitation to depend from. 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, 2, 3, 5, 9, 11, 16-23, 26-28, 30-31, 35, 37, 38, 41, 45, 47, 50-51, and 54 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 claims contain 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 inventors, at the time the application was filed, had possession of the claimed invention.
The instant disclosure describes:
Transgenic expression of mobile genome editing reagents by producing hairy rootstocks of soybean cultivar susceptible to A. rhizogenes [Example 2].
Application to leaves by gRNA spray [Example 4] and by wounding [Example 6].
Application of gRNA by injection to the stem of seedlings grown from “suitable Editor lines” [Example 5].
Application of gRNA by bathing of whole seedlings in a humid chamber [Example 7].
Exemplary description of heritable editing in soybean, corn and wheat [Example 8].
The instant disclosure does not describe:
A method of editing a genomic target in a plant meristem comprising delivering a guide RNA for a Cas nuclease to a plant root by any means other than the production of transgenic hairy roots.
A method of editing a genomic target in any plant meristem.
The claims are broadly drawn to a method of editing a genomic target in a plant meristem comprising delivering a guide RNA to a plant root, wherein a genomic target within a cell in the meristem is edited. The Applicant limits the claim to incubating the root with a composition comprising the guide RNA, an A. rhizogenes transformation, or injecting a composition comprising the guide RNA into the root. However, the only methodology reduced to practice for delivering the gRNA to a plant root was A. rhizogenes [Example 2]. The other described methodology included introducing gRNA by application of a gRNA spray to the leaves, injection to the stem, or setting the whole plant in a humid chamber. The Applicant does not provide data or reduce to practice plants with the edited cell in the meristem other than stating that soybean was transformed, and that corn, wheat, and other plant species could be as well.
Even hairy root transformation, which was the only method provided for actual delivery to the soybean plant root, has challenges in induction. The choice of plant species and explant type has profound effects on the transformation success rate. For example, Dicotyledonous plants are more responsive to transformation, due to their compatible architecture and higher levels of phenolic compounds, which act as chemical signals activating the virulence gene of A. rhizogenes (Liu, C. et al. 2025. “Reprogramming Hairy Root Cultures: A Synthetic Biology Framework for Precision Metabolite Biosynthesis.” Plants (Basel). 14(13):1928. doi: 10.3390/plants14131928) [pg. 6, para 2]. The choice of plant species and explant type has profound effects on the transformation success rate. The Applicant has only reduced to practice illustrative species transformed by A. rhizogenes, and not the broad genus of any method of delivery of a guide RNA to any plant root. The variability within the genus is such that a sufficient number of species has not been provided.
Further, the instant claims recite wherein the plant comprises a rootstock and a scion grafted onto the rootstock, and wherein the Cas nuclease is expressed in the rootstock. The art demonstrates that the stock and scion must be compatible (Rothenberger, R. et al. 2022. “Grafting.” Extension. University of Missouri. Pub. No. G6971). Generally, only plants closely related botanically form a good graft union [pg. 1, col. 2, para 3]. Plants of different families cannot be grafted successfully [pg. 2, col. 1, para 4]. Although it has been reported that relatively short-lived grafts of herbaceous plants of different families have been made, there is no successful practice for commercial or home grafting of woody plants of different families. The instant disclosure provides no working examples of grafted rootstock to scion combinations that were effective in editing a genomic target within a cell in the meristem, providing prophetic examples of the guide RNA and simply stating that “Editor lines” were grown to the first trifoliate stage.
Thus, given the large scope of claim 3, wherein the plant comprises any rootstock and any scion, and the unpredictability in the success of the rootstock to scion union, the Applicant has not reduced to practice a method of editing a genomic target in a plant meristem further comprising a rootstock and a scion grafted onto the rootstock. Although claims 16-22 limit the rootstock and the scion material, they do not provide specific enough language as to overcome the written description requirement. For example, claim 22 recites the method of claim 1 wherein the rootstock and/or scion or plant is soy, canola, alfalfa, corn, oat, sugarcane or banana etc. These plants represent three different families and would not necessarily result in a successful union and thus the structure would not lead to the required function of a genetic edit in a meristematic cell.
Therefore, claims 1, 2, 3, 5, 9, 11, 16-23, 26-28, 30-31, 35, 37, 38, 41, 45, 47, 50-51, and 54 are rejected for failing to meet the written description requirement.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1, 3, 11, 16-22, 37, 50 and 54 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Yang, L. et al. (January, 2023). "Heritable transgene-free genome editing in plants by grafting of wild-type shoots to transgenic donor rootstocks." Nat Biotechnol 41, 958–967. https://doi.org/10.1038/s41587-022-01585-8 (see IDS filed 12/19/2025).
Claim 1 recites a method of editing a genomic target in a plant meristem comprising delivering a guide RNA for a Cas nuclease to a plant root, wherein the guide RNA is fused to a meristem transport segment (MTS), wherein the plant comprises nucleic acid encoding the Cas nuclease, and wherein a genomic target within a cell in the meristem is edited.
Claim 3 recites the method of claim 1, wherein the plant comprises a rootstock and a scion grafted onto the rootstock, and wherein the Cas nuclease is expressed in the rootstock.
Claim 11 recites the method of claim 1, wherein the nucleic acid encoding the Cas nuclease is fused to an MTS or to a nucleic acid encoding an MTS.
Claim 16 recites the method of claim 3, wherein the scion and the rootstock are different plant species.
Claim 17 recites the method of claim 3, wherein the scion and the rootstock are the same plant species.
Claim 18 recites the method of claim 3, wherein the scion and/or rootstock is a dicot.
Claim 19 recites the method of claim 1, wherein the plant is a dicot.
Claim 20 recites the method of claim 3, wherein the scion and/or rootstock is a monocot.
Claim 21 recites the method of claim 1, wherein the plant is a monocot.
Claim 22 recites the method of claim 1, wherein the rootstock and/or scion, or plant is soy, canola, alfalfa, corn, oat, sorghum, sugarcane, banana, or wheat.
Claim 37 recites the method of claim 1, wherein the Cas nuclease is selected from the group consisting of Cas9, Cas12a (Cpf1), Cas12e (CasX), Cas12d (CasY), C2c1, C2c2, C2c3, Cas12h, Cas12i, and Cas12j.
Claim 50 recites the method of claim 1, further comprising retrieving a progeny of the plant, wherein the progeny has an altered genome.
Claim 54 recites an edited plant produced by the method of claim 1.
Regarding claim 1, Yang teaches fusions of Cas9 and guide RNA transcripts to tRNA-like sequence (TLS) motifs that move RNAs from transgenic rootstocks to grafted wild-type scions to achieve heritable gene editing (i.e., a method of editing a genomic target comprising delivering a guide RNA for a Cas nuclease to a plant root, wherein the plant comprises nucleic acid encoding the Cas nuclease) [Abstract]. Yang teaches the generation of Arabidopsis thaliana lines expressing zCas9 from an estradiol-inducible promoter and two NIA1-targeting gRNAs (gNIA1) driven by the constitutive Pol-III promoters U6-26 and U6-29 [pg. 958, col. 2, para 2]. The two targeting gRNAs were designed to create double genome deletions resulting in a 1,000 bp deletion of the NIA1 gene for a nai1 knockout. Yang also teaches gRNAs targeting a transgenic construct to introduce a deletion of the Venus and 35S terminator sequences resulting in BASTA-resistant plants [pg. 959, col. 1, para 1]. The Cas9 and the two gNIA1 transcripts were fused to TLS1 or TLS2 sequences.
Yang teaches that, with rootstocks expressing Cas9-TLS1 and gNAI1-TLS1 or Cas9-TLS2 and gNAI1-TLS2, transcripts were detected in grafted transgene-free wild-type shoots, confirming long-distance mobility of the Cas9 and gRNA transcripts with the TLS motifs added [pg. 959, col. 2, para 2]. Yang teaches that these mobile transfusions could be transported to meristems that would then give rise to a genome-edited lineage resulting in edited germline progenitor cells producing the next generation [pg. 961, col. 1, para 2]. Yang teaches that Cas9 and gNAI1 fused to either TLS1 or 2 were detected in samples of grafted plants in scion siliques, flowers, stems, cauline leaves, and rosette leaves.
Yang teaches that the appearance of more gene edits in the seed producing siliques is either due to their clonal origin from flower meristems [pg. 963, col. 2, para 1], suggesting that the target within the plant meristem resulted in edited progenitor cells. The mobile Cas9-TLS and gRNA-TLS fusions relocated to the SAMs in the wild-type grafted scion, demonstrated by the production of heritable gene-edited lines following the production of seed in flowers derived from the transformed meristems (see Fig.1 below) [pg. 963, col. 1, para 2]. Thus, the TLS motifs are taken to read on meristem transport segment (MTS) (i.e., wherein the guide RNA is fused to a meristem transport segment (MTS); wherein a genomic target within a cell in the meristem is edited).
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Regarding claim 3, Yang teaches that the plant comprises rootstocks grafter to scions and fusions of Cas9 and guide RNA transcripts to motifs that move RNAs from the rootstocks (i.e., wherein the plant comprises a rootstock and a scion grafted onto the rootstock) [Abstract]. Yang specifically teaches that the Cas9 and gRNA were both detected in the rootstock samples (i.e., wherein the Cas nuclease is expressed in the rootstock) [pg. 959, col. 2, para 1].
Regarding claim 11, Yang teaches that the Cas9 and the two gNIA1 transcripts were fused to TLS1 or TLS2 sequences (i.e., wherein the nucleic acid encoding the Cas nuclease is fused to an MTS or to a nucleic acid encoding an MTS) [pg. 959, col. 1, para 1].
Regarding claim 16, Yang teaches hypocotyl grafting of Brassica rapa with Arabidopsis (i.e., wherein the scion and the rootstock are different plant species) [pg. 968, col. 2, para 2].
Regarding claim 17, Yang teaches hypocotyl grafting of Arabidopsis with Arabidopsis (i.e., wherein the scion and the rootstock are the same plant species) [pg. 968, col. 1, para 5].
Regarding claim 18-19, as above, Yang teaches specific grafting examples in Brassica rapa and Arabidopsis (i.e., wherein the plant is a dicot; wherein the scion and/or rootstock is a dicot).
Regarding claims 20-22, Yang teaches that in light of the recent studies that demonstrates grafting in monocotyledonous species, that this technique based on mobile Cas9-TLS and gRNA-TLS fusions will find use in main crops such as maize, wheat, and rice (i.e., wherein the scion and/or rootstock is a monocot; wherein the plant is a monocot; wherein the plant is wheat) [pg. 966, col. 1, para 3].
Regarding claim 37, Yang teaches the use of a Cas9 in the method of generating plants from the transgenic rootstocks grafted to wild-type scions (i.e., wherein the Cas nuclease is a Cas9) [Abstract].
Regarding claim 50, Yang teaches that it took approximately 3 months to generate edited plantlets, then an additional 3 to 4 months to produce and select the next generation of seeds, ultimately producing transgene free progeny (i.e., retrieving a progeny of the plant, wherein the progeny has an altered genome) [pg. 966, col. 1, para 1].
Regarding claim 54, Yang teaches the production of mutant plants by the method disclosed (i.e., an edited plant produced by the method of claim 1) [pg. 966, col. 1, para 2].
Thus, Yang anticipates claims 1, 3, 11, 16-22, 37, 50 and 54.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1, 3, 9, 11, 16-22, 23, 26, 35, 37, 45, 50-51, and 54 are rejected under 35 U.S.C. 103 as being unpatentable over Nuccio, M. et al. WO 2021/081200 A1. "Genomic Alteration of Plant Germline." Published 04/29/2021 (see IDS filed 12/19/2025) in view of Yang, L. et al. (January, 2023). "Heritable transgene-free genome editing in plants by grafting of wild-type shoots to transgenic donor rootstocks." Nat Biotechnol 41, 958–967. https://doi.org/10.1038/s41587-022-01585-8 (see IDS filed 12/19/2025).
Examiner notes that the applied reference, Nuccio, has common inventors and applicant with the instant application. Based on the earlier publication date, it constitutes prior art under 35 USC § 102(a)(1) and does not qualify for an exception under 35 USC § 102(b)(1)(A).
Claim 1 recites a method of editing a genomic target in a plant meristem comprising delivering a guide RNA for a Cas nuclease to a plant root, wherein the guide RNA is fused to a meristem transport segment (MTS), wherein the plant comprises nucleic acid encoding the Cas nuclease, and wherein a genomic target within a cell in the meristem is edited.
Claim 3 recites the method of claim 1, wherein the plant comprises a rootstock and a scion grafted onto the rootstock, and wherein the Cas nuclease is expressed in the rootstock.
Claim 9 recites the method of claim 5, wherein the composition comprising the guide RNA comprises a nuclease inhibitor
Claim 11 recites the method of claim 1, wherein the nucleic acid encoding the Cas nuclease is fused to an MTS or to a nucleic acid encoding an MTS.
Claim 16 recites the method of claim 3, wherein the scion and the rootstock are different plant species.
Claim 17 recites the method of claim 3, wherein the scion and the rootstock are the same plant species.
Claim 18 recites the method of claim 3, wherein the scion and/or rootstock is a dicot.
Claim 19 recites the method of claim 1, wherein the plant is a dicot.
Claim 20 recites the method of claim 3, wherein the scion and/or rootstock is a monocot.
Claim 21 recites the method of claim 1, wherein the plant is a monocot.
Claim 22 recites the method of claim 1, wherein the rootstock and/or scion, or plant is soy, canola, alfalfa, corn, oat, sorghum, sugarcane, banana, or wheat.
Claim 23 recites the method of claim 1, wherein the MTS comprises (iv) a tRNA-like sequence (TLS), wherein the TLS comprises the nucleotide sequence set forth in SEQ ID NO: 29.
Claim 26 recites the method of claim 1, wherein the nucleic acid encoding the MTS is located 3' of the nucleic acid encoding the Cas nuclease and/or 3' of the guide RNA.
Claim 35 recites the method of claim 1, wherein the method comprises applying two, three, four, five, or more than five guide RNAs to the root.
Claim 37 recites the method of claim 1, wherein the Cas nuclease is selected from the group consisting of Cas9, Cas12a (Cpf1), Cas12e (CasX), Cas12d (CasY), C2c1, C2c2, C2c3, Cas12h, Cas12i, and Cas12j.
Claim 45 recites the method of claim 1, wherein the nucleic acid encoding the guide RNA and the MTS is located between two ribozyme sequences.
Claim 50 recites the method of claim 1, further comprising retrieving a progeny of the plant, wherein the progeny has an altered genome.
Claim 51 recites the method of claim 1, wherein the guide RNA further comprises:(a) one or more modified nucleotides within five nucleotides from the 5' end of the guide RNA; (b) one or more modified nucleotides within five nucleotides from the 3' end of the guide RNA; and/or (c) a 5-methylcytosine group; wherein the one or more modified nucleotides has a modification to a phosphodiester linkage, a sugar, or both a phosphodiester linkage and a sugar.
Claim 54 recites an edited plant produced by the method of claim 1.
Regarding claim 1, Nuccio teaches compositions containing RNA molecules comprising meristem targeting sequences that are fused to RNA cargo sequences which include gene editing molecules [Abstract]. The methods may be used without intervening tissue culture steps to produce plants with altered genomes. Nuccio teaches a method of producing a plant with an altered genome comprising contacting the plant with a composition comprising at least one RNA molecule comprising a cargo segment fused to a meristem transport segment and retrieving a progeny of the plant, wherein the progeny has an altered genome (i.e., a method of editing a genomic target in a plant meristem) [claims 1 and 24]. Nuccio further teaches that the cargo segment has one or more DNA-modifying components, including at least one guide RNA and a coding sequence for a nuclease effector, such as Cas9 (i.e., comprising delivering a guide RNA for a Cas nuclease; wherein the guide RNA is fused to a meristem transport segment; wherein the plant comprises nucleic acid encoding the Cas nuclease) [para 9].
Nuccio teaches that a number of configurations for the RNA molecules can be used, teaching that the meristem transport-competence (MTC) can be assayed by introducing RNAs by grafting, meaning that the RNA molecules can be expressed in the rootstock of the grafted plant, and their effects observed in the scion (i.e., delivering a guide RNA for a Cas nuclease to a plant root [para 36]. Nuccio teaches that the Cas nuclease is intended to be translated inside a plant meristem cell (i.e., wherein a genomic target within a cell in the meristem is edited) [para 44]. Nuccio teaches that viral and cellular-derived RNA molecules that are useful as part of a transport segment include the mRNAs of FT, GAI, CmNACP, LeT6 a tomato KNOX gene, BEL5, or tRNA-like sequences [para 34].
Although Nuccio suggests that the RNA molecules can be expressed in the rootstock of a grafted plant and their effects observed in the scion, Nuccio does not explicitly teach this in practice. However, Yang teaches fusions of Cas9 and guide RNA transcripts to tRNA-like sequence (TLS) motifs that move RNAs from transgenic rootstocks to grafted wild-type scions to achieve heritable gene editing (i.e., a method of editing a genomic target comprising delivering a guide RNA for a Cas nuclease to a plant root, wherein the plant comprises nucleic acid encoding the Cas nuclease) [Abstract].
Yang teaches that in rootstocks expressing Cas9-TLS1 and gNAI1-TLS1 or Cas9-TLS2 and gNAI1-TLS2, transcripts were detected in grafted transgene-free wild-type shoots, confirming long-distance mobility of the Cas9 and gRNA transcripts with the TLS motifs added [pg. 959, col. 2, para 2].
Yang teaches that these mobile transfusions could be transported to meristems that would then give rise to a genome-edited lineage resulting in edited germline progenitor cells producing the next generation [pg. 961, col. 1, para 2]. Yang teaches that Cas9 and gNAI1 fused to either TLS1 or 2 were detected in samples of grafted plants in scion siliques, flowers, stems, cauline leaves, and rosette leaves. Yang teaches that the appearance of more gene edits in the seed producing siliques is either due to their clonal origin from flower meristems [pg. 963, col. 2, para 1], suggesting that the target within the plant meristem resulted in edited progenitor cells. The mobile Cas9-TLS and gRNA-TLS fusions relocated to the SAMs in the wild-type grafted scion, demonstrated by the production of heritable gene-edited lines following the production of seed in flowers derived from the transformed meristems [pg. 963, col. 1, para 2].
Given that Nuccio teaches guide RNA for a Cas nuclease, wherein the guide RNA is fused to a MTS, wherein the plant comprises nucleic acid encoding the Cas nuclease, and suggest application in rootstocks for expression in meristematic tissue of scions; and given that Yang teaches fusions of Cas nucleases with TLS resulting in meristematic expression, it would have been prima facie obvious to one of ordinary skill in the art at the time of filing to combine the inventions to achieve a method of editing a genomic target in a plant meristem. Nuccio explicitly provides reasonable expectation of success by suggesting the use of the RNA molecules in transgenic grafted plants and teaches that TLSs like those of Yang are useful as part of the transport segment. Yang further implements the TLSs with success to create transgene free genetically modified plant. Yang provides explicit motivation, stating hat the graft-mobile gene editing system enables the production of transgene-free offspring in one generation without the need for transgene elimination, culture recovery and selection, or use of viral editing vectors, anticipating
that using graft-mobile editing systems for transgene-free plant production may be applied to a wide range of breeding programs and crop plants [Abstract]. Thus, Nuccio and Yang render obvious the method of claim 1.
Regarding claim 3, Yang teaches that the plant comprises rootstocks grafter to scions and fusions of Cas9 and guide RNA transcripts to motifs that move RNAs from the rootstocks (i.e., wherein the plant comprises a rootstock and a scion grafted onto the rootstock) [Abstract]. Yang specifically teaches that the Cas9 and gRNA were both detected in the rootstock samples (i.e., wherein the Cas nuclease is expressed in the rootstock) [pg. 959, col. 2, para 1].
Regarding claim 9, Nuccio teaches that the composition further comprises an RNase inhibitor (i.e., wherein the composition comprising the guide RNA comprises a nuclease inhibitor) [claim 19].
Regarding claim 11, Nuccio teaches a meristem-delivery vector comprising a cargo segment fused to a meristem transport segment (MTS), wherein the cargo segment comprises one or more guide RNAs for an RNA guided nuclease (Cas nuclease) [claim 22]. Yang teaches that the Cas9 and the two gNIA1 transcripts were fused to TLS1 or TLS2 sequences (i.e., wherein the nucleic acid encoding the Cas nuclease is fused to an MTS or to a nucleic acid encoding an MTS) [pg. 959, col. 1, para 1].
Regarding claims 16-22, Yang teaches hypocotyl grafting of Brassica rapa with Arabidopsis (i.e., wherein the scion and the rootstock are different plant species) [pg. 968, col. 2, para 2]. Yang teaches hypocotyl grafting of Arabidopsis with Arabidopsis (i.e., wherein the scion and the rootstock are the same plant species) [pg. 968, col. 1, para 5]. Yang teaches specific grafting examples in Brassica rapa and Arabidopsis (i.e., wherein the plant is a dicot; wherein the scion and/or rootstock is a dicot). Yang teaches that in light of the recent studies that demonstrates grafting in monocotyledonous species, that this technique based on mobile Cas9-TLS and gRNA-TLS fusions will find use in main crops such as maize, wheat, and rice (i.e., wherein the scion and/or rootstock is a monocot; wherein the plant is a monocot; wherein the plant is wheat) [pg. 966, col. 1, para 3]. Nuccio further teaches that the RNA molecules and provided methods may be used for genomic editing of any plant species, including, but not limited to, monocots and dicots (i.e., monocotyledonous and dicotyledonous, respectively) [para 67]. Nuccio states that examples of plant species of interest include wheat (i.e., wherein the rootstock and/or scion, or plant is wheat).
Regarding claim 23, Nuccio teaches a tRNA-like sequence sharing 100% identity with SEQ ID NO: 29 of the instant application (i.e., wherein the TLS comprises the nucleotide sequence set forth in SEQ ID NO. 29). It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to use the TLS of Nuccio in the invention of Yang as an obvious alternative as Nuccio teaches that the TLS sequences are suitable for RNA transport [para 34]. Nuccio teaches that the composition and plant produced by the method comprise the TLS sequences, indicating that one would have reasonable expectation of success implementing these sequences in a similar methodology.
Regarding claim 35, Nuccio teaches that the composition comprises both a first and a second molecule, each comprising a cargo segment fused to an MTS, wherein the guide RNAs are distinct [claim11]. Nuccio additionally teaches that, in some embodiments, the MTS is operably linked to a cargo segment comprising a plurality of guide RNAs (e.g. 2, 3, 4, or more guide RNAs) [para 46].
Regarding claim 37, Yang teaches the use of a Cas9 in the method of generating plants from the transgenic rootstocks grafted to wild-type scions (i.e., wherein the Cas nuclease is a Cas9) [Abstract].
Regarding claim 45, Nuccio teaches that guide RNA suitable for matching expressed effector polypeptide can be flanked by processing elements, so that functional guide RNAs are excised inside the cells [para 45]. Exemplary processing elements include hammerhead ribozymes, Csy4, and tRNAs. Nuccio teaches that an MTS is operably linked to a cargo segment comprising an array of guide RNAs separated by processing elements [para 46]. As Nuccio teaches that the ribozyme sequences flank the sequences, this is taken to read on claim 45, wherein the nucleic acid encoding the guide RNA and the MTS is located between the two ribozyme sequences.
Regarding claim 50, Yang teaches that it took approximately 3 months to generate edited plantlets, then an additional 3 to 4 months to produce and select the next generation of seeds, ultimately producing transgene free progeny (i.e., retrieving a progeny of the plant, wherein the progeny has an altered genome) [pg. 966, col. 1, para 1].
Regarding claim 51, Nuccio teaches that, in one embodiment, one or more guide RN As are flanked by direct repeats (DR) of the CRISPR array from which the Cas effector polypeptide was first isolated (i.e., wherein the guide RNA further comprises one or more modified nucleotides within 5 nucleotides from the 5' end or 3' end of the guide RNA) [para 45]. Nuccio does not teach the distance, but given that Nuccio specifies that these nucleotides are flanking the guide RNAs, this is taken to read on "within 5 nucleotides".
Regarding claim 54, Yang teaches the production of mutant plants by the method disclosed (i.e., an edited plant produced by the method of claim 1) [pg. 966, col. 1, para 2].
Claims 2, 5, 28, 38 and 41 are rejected under 35 U.S.C. 103 as being unpatentable over Nuccio and Yang as applied to claims 1, 3, 9, 11, 16-22, 26, 35, 37, 45, 50-51, and 54 above, and further in view of Kiryushkin, A. et al. (2021). “Hairy CRISPR: Genome Editing in Plants Using Hairy Root Transformation.” Plants (Basel). (1):51. doi: 10.3390/plants11010051. PMID: 35009056; PMCID: PMC8747350.
Claim 2 recites the method of claim 1, wherein the Cas nuclease is constitutively expressed in the plant.
Claim 5 recites the method of claim 1, wherein the guide RNA is delivered to the plant root by; (i incubating the root with a composition comprising the guide RNA; (ii) an Agrobacterium rhizogenes transformation, wherein the Agrobacterium rhizogenes transformation produces transgenic hairy roots; or (iii) injecting a composition comprising the guide RNA into the root.
Claim 28 recites the method of claim 1, wherein the nucleic acid encoding the Cas nuclease is operably linked to a promoter, wherein the promoter is active in roots and/or phloem companion cells.
Claim 38 recites the method of claim 1, wherein the Cas nuclease is associated with a reverse transcriptase or fused with a reverse transcriptase, and wherein the guide RNA comprises at its 3' end a priming site and an edit to be incorporated into the genomic target.
Claim 41 recites the method of claim 1, wherein the Cas nuclease is associated with a reverse transcriptase or fused with a reverse transcriptase, and wherein the guide RNA comprises at its 3' end a priming site and an edit to be incorporated into the genomic target.
Regarding claim 2 and 5, Nuccio teaches guide RNA for a Cas nuclease, wherein the guide RNA is fused to a MTS, wherein the plant comprises nucleic acid encoding the Cas nuclease, and suggest application in rootstocks for expression in meristematic tissue of scions; and Yang teaches fusions of Cas nucleases with TLS resulting in meristematic expression, rendering obvious the method of claim 1, as detailed above. Nuccio and Yang do not explicitly teach that the Cas nuclease is constitutively expressed in the plant or that the guide RNA is delivered to the root by Agrobacterium rhizogenes transformation, however, Kiryushkin teaches that strong constitutive promoters are typically used to control Cas expression in plants. The Cauliflower Mosaic Virus (CaMV) 35S promoter (p35S) is often (in 53 of the 78 studies that have employed CRISPR/Cas in hairy roots) used to drive expression of Cas9 [pg. 2, para 3]. Ubiquitin promoters (pUbi) from different species, including Arabidopsis, parsley (Petroselinum crispum), maize (Zea mays), rice (Oryza sativa), and soybean (G. max), are also popular choices.
Kiryushkin additionally teaches that hairy root transformation by Agrobacterium rhizogenes strains is a rapid and convenient approach for obtaining transgenic roots, disclosing A. rhizogenes strains that are widely used for hairy root transformation [Abstract]. Kiryushkin additionally teaches CRISPR/Cas vectors, promoters that drive Cas or gRNA expression, the types of Cas nuclease, and the application of CRISPR/Cas genome editing in hairy roots.
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to use a constitutive promoter with the combined teachings of Nuccio and Yang, given that Kiryushkin teaches that the use of constitutive promoters is common to control Cas expression and teaches that hairy root transformation is a fast and rapid approach for obtaining transgenic roots. Kiryushkin provides numerous options with constitutive promoters in various species that are used in root transformation and teaches different Agrobacterium rhizogenes strains widely used for hairy root transformation. Given that the objective of the invention is to deliver a guide RNA for a Cas nuclease to a plant root for expression in meristematic tissue, one would have been motivated to use a strong promoter that has been demonstrated to be effective in root-specific promotion in the transformation of hairy roots and use Agrobacterium rhizogenes transformation due to its rapid and successful transformation potential, which is well known in the art. One would have been motivated and have reasonable expectation of success given that Kiryushkin teaches many options for promoters and strains that have been successful. Further,
Regarding claim 28, Nuccio teaches that the RNA-guided nuclease (Cas nuclease) is operably linked to a promoter which is preferentially expressed in target plant cells and optionally wherein the target plant cells are meristem cells [claim 48]. Nuccio teaches that vegetative stage, meristem-preferred or meristem-specific promoters are set forth in US 20190300890, which is incorporated by reference in its entirety. Nuccio does not explicitly teach the use of a promoter that is active in roots and/or phloem companion cells, however Kiryushkin teaches constitutive promoter CaMV 35S, which is known in the art to be observed in many tissue types, including roots1. It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to use the common CaMV 35S promoter in the methods taught by Nuccio and Yang as it has been demonstrated to be effective in promoting the transformations of hairy roots, which would be useful to the grafting system. One would be motivated and have reasonable expectation of success given that CaMV 35S is expressed in all cell and tissue types and has proven successful in a multitude of species, as demonstrated by Kiryushkin.
Regarding claim 38, 41 and 45, Nuccio teaches that the unprocessed RNA molecules can be detected by a variety of techniques that include reverse transcriptase PCR (RT-PCR) assays where oligonucleotide primers and optionally detection probes which specifically amplify and detect the unprocessed RNA molecule comprising the cargo segments that are operably linked to MTS sequences are used (i.e., wherein the Cas nuclease is associated with a reverse transcriptase) [para 41]. Nuccio teaches that guide RNA suitable for matching expressed effector polypeptide can be flanked by processing elements, so that functional guide RNAs are excised inside the cells [para 45]. Exemplary processing elements include hammerhead ribozymes, Csy4, and tRNAs. Nuccio teaches that an MTS is operably linked to a cargo segment comprising an array of guide RNAs separated by processing elements [para 46]. As Nuccio teaches that the ribozyme sequences flank the sequences, this is taken to read on claim 45, wherein the nucleic acid encoding the guide RNA and the MTS is located between the two ribozyme sequences.
Nuccio and Yang do not explicitly teach that the Cas nuclease is fused to a reverse transcriptase, that the guide RNA comprises a priming site at its 3’ end, or that the Cas nuclease is a Cas nickase. However, Kiryushkin teaches the types of CRISPR/Cas9 systems include using nickase Cas9 activity resulting in a single-stand break, pegRNA- prime editing guide RNA comprised of a target specific crRNA, a conservative tracrRNA, a PBS-- primer binding site, an RT template-- RNA template for reverse transcription, as well as reverse transcriptase [Fig. 5, see below]. Kiryushkin teaches that these variable systems lead to the edit being incorporated into the genomic target (i.e., wherein the Cas nuclease is associated with a reverse transcriptase or fused with a reverse transcriptase, and wherein the guide RNA comprises at its 3' end a priming site and an edit to be incorporated into the genomic target).
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It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to use the well-known CRISPR/Cas system using nickase Cas9 for a single strand break as this was a technique known in the art. Kiryushkin teaches that the approach has been widely used in different fields of biology since its invention, consisting of a Cas nickase fused with a modified reverse transcriptase via a linker [pg. 14, para 4]. There are a finite number of CRISPR/Cas approaches available, as detailed by Kiryushkin, there was a documented design need for further improvement of CRISPR-based grafting systems, as detailed by Yang, and the methodology was available to one of ordinary skill in the art at the time of filing as a potential solution that has been widely use.
It further would have been obvious to use flanking processing elements, such as hammerhead ribozymes, which Nuccio recites as an exemplary processing element so that functional gRNAs can be excised inside the cell. One of ordinary skill in the art would have been motivated to use ribozymes in the engineering of the gRNA because the ribozymes can aid in severing the RNA exactly where the gRNA begins and ends for more precise cuts. One would have reasonable expectation of success as Nuccio suggests the use of ribozymes with functional gRNAs and teaches exemplary options for use with the gRNA construct.
Claim 47 is rejected under 35 U.S.C. 103 as being unpatentable over Yang and Nuccio as applied to claims 1, 3, 9, 11, 16-22, 26, 35, 37, 45, 50-51, and 54 above, and further in view of Gao, Y. et al. (2014). “Self‐processing of ribozyme‐flanked RNAs into guide RNAs in vitro and in vivo for CRISPR‐mediated genome editing.” Journal of Int. Pl. Bio. 56(4):343-349 (see IDS filed 12/19/2025).
Claim 47 recites the method of claim 1, wherein the nucleic acid encoding the guide RNA and the MTS further comprises a hammerhead ribozyme sequence 5' to the nucleic acid encoding the guide RNA and the MTS, and a HDV ribozyme 3' to the nucleic acid encoding the guide RNA and the MTS.
Regarding claim 47, Nuccio teaches guide RNA for a Cas nuclease, wherein the guide RNA is fused to a MTS, wherein the plant comprises nucleic acid encoding the Cas nuclease, and suggest application in rootstocks for expression in meristematic tissue of scions; and Yang teaches fusions of Cas nucleases with TLS resulting in meristematic expression, rendering obvious the method of claim 1, as detailed above.
Nuccio teaches that guide RNA suitable for matching expressed effector polypeptide can be flanked by processing elements, so that functional guide RNAs are excised inside the cells [para 45]. Exemplary processing elements include hammerhead ribozymes, Csy4, and tRNAs. Nuccio teaches that an MTS is operably linked to a cargo segment comprising an array of guide RNAs separated by processing elements [para 46].
Nuccio and Yang do not explicitly teach the method of claim 1 wherein the nucleic acid encoding the gRNA and the MTS further comprise a hammerhead ribozyme sequence 5’ to the gRNA and MTS and an HDV ribozyme sequence 3’ to the gRNA and MTS.
However, Gao teaches the design of an RNA molecule with self-processing capacity for gRNA production [pg. 345, col. 1, para 4]. Gao teaches an RNA molecule containing a Hammerhead type ribozyme at the 5’-end, a gRNA that targets a green florescent protein gene in the middle and a hepatitis delta virus (HDV) ribozyme at the 3’-end [pg. 345, col. 2, para 1]. Gao teaches that gRNA with the HDV ribozyme at the 3’‐end still retained sufficient activity to guide Cas9 to cut target DNA [pg. 345, col. 2, para 3]. Gao teaches that this work demonstrates that gRNAs can be efficiently produced in vitro and in vivo from essentially any promoters when the primary transcripts are flanked by self‐cleaving ribozymes, opening the door to more sophisticated CRISPR-mediate genome editing in many organisms [pg. 347, col. 1, para 2]. Gao teaches that gRNAs can now be produced using tissue‐specific promoters, hormone‐responsive promoters, environmental signal regulated promoters, and other well‐characterized promoters; allowing for automation and high-throughput production of gRNAs [pg. 347, col. 2, para 1-4].
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to use known ribozymes in known and previously demonstrated positions in the construction of a gRNA construct for genetic editing. One would be motivated to use such processing elements in the construct as Gao teaches that the ribozymes promote the self-processing capacity for gRNA production and that the self-cleaving ribozymes allow for more advanced genome editing. One could use these ribozymes in the positions taught in the prior art with reasonable expectation of success as Gao teaches that the gRNAs could be efficiently produced in vitro and in vivo using essentially any promoter when flanked with the self-cleaving ribozymes. Thus, Yang, Nuccio, and Gao render obvious claim 47.
Claim 27 is rejected under 35 U.S.C. 103 as being unpatentable over Yang and Nuccio as applied to claims 1, 3, 9, 11, 16-22, 26, 35, 37, 45, 50-51, and 54 above, and further in view of Cohen, R. et al. (2005). “The positional, structural, and sequence requirements of the Drosophila TLS RNA localization element.” RNA. (7):1017-29. doi: 10.1261/rna.7218905.
Claim 27 recites the method of claim 1, wherein the nucleic acid encoding the MTS is located 5' of the nucleic acid encoding the Cas nuclease and/or 5' of the guide RNA.
Regarding claim 27, Nuccio teaches guide RNA for a Cas nuclease, wherein the guide RNA is fused to a MTS, wherein the plant comprises nucleic acid encoding the Cas nuclease, and suggest application in rootstocks for expression in meristematic tissue of scions; and Yang teaches fusions of Cas nucleases with TLS resulting in meristematic expression, rendering obvious the method of claim 1, as detailed above.
Yang and Nuccio do not explicitly teach the method of claim 1, wherein the nucleic acid encoding the MTS is located 5' of the nucleic acid encoding the Cas nuclease and/or 5' of the guide RNA, however, the location of the MTS within the construct would be merely an optional feature of the invention. Additionally, Cohen teaches that the vast majority of known RNA localization elements map to the 3'UTRs of their respective transcripts [pg. 1019, col. 1, para 2]. Cohen teaches that the TLS can mediate RNA localization within a variety of different sequence contexts and regardless of its position within the transcript [pg. 1026, col. 1, para 3]. Specifically, Cohen teaches that the TLS retains its transport/localization activity when placed in the 5'UTR of the transcript and such placement does not greatly interfere with translation [pg. 1020, col. 1, para 1]. As the TLS of Yang is taken to read on the MTS of the instant claims, the placement of the MTS 5' of the nucleic acid encoding the Cas nuclease and/or guide RNA as claimed would be simple substitution of one known element for another to obtain predicted results.
Nuccio teaches that the MTS is located 3' of the cargo segment, reading on the method of claim 1 yet differing to claim 27 in that the MTS is located 3' of the nucleic acid instead of 5'. Cohen teaches that the substituted component, 5'TLS, and its function, transport/localization activity when placed in the 5'UTR of the transcript, were known in the art at the time of filing of the instant application. Given that Cohen teaches that the placement did not greatly interfere with translation, one of ordinary skill in the art could have substituted one known element for another, and the results of the substitution would have been predictable.
Claims 28 and 30 are rejected under 35 U.S.C. 103 as being unpatentable over Yang and Nuccio as applied to claims 1, 3, 9, 11, 16-22, 26, 35, 37, 45, 50-51, and 54 above, and further in view of James, A. et al. (2022). “Assessment of root-specific promoters in banana and tobacco and identification of a banana TIP2 promoter with strong root activity.” Front. Plant Sci. 13:1009487. (see IDS filed 12/19/2025).
Claim 28 recites the method of claim 1, wherein the nucleic acid encoding the Cas nuclease is operably linked to a promoter, wherein the promoter is active in roots and/or phloem companion cells.
Claim 30 recites the method of claim 28, wherein the promoter is the promoter of a gene selected from the group consisting of Arabidopsis WRKY6, chickpea WRKY31, carrot MYB 113, corn GLU1, strawberry RB7-type TIP-2, and banana TIP2-2, or the promoter of an orthologous gene thereof.
Regarding claims 28 and 30, Nuccio teaches guide RNA for a Cas nuclease, wherein the guide RNA is fused to a MTS, wherein the plant comprises nucleic acid encoding the Cas nuclease, and suggest application in rootstocks for expression in meristematic tissue of scions; and Yang teaches fusions of Cas nucleases with TLS resulting in meristematic expression, rendering obvious the method of claim 1, as detailed above.
Nuccio and Yang do not explicitly teach that the Cas nuclease is operably linked to a promoter active in roots and/or phloem companion cells, however, James teaches that root-specific promoters have been reported from a wide range of plants including model species such as Arabidopsis thaliana as well as from agriculturally important crop plants [pg. 2, col. 1, para 3]. James teaches that TIP-type aquaporins have shown high levels of expression in root tissues, or even root-specific expression [pg. 2, col. 2, para. 2]. This includes rice and maize TIP2-2, as well as the promoters of the RB7-type TIP from tobacco and strawberry driving root-specific expression in their respective host plants, a novel Musa TIP2-2 promoter sequence, and a b‐glucosidase 1 (GLU1) promoter from maize [Abstract]. James teaches successful high expression of GUS in banana, with Nicotiana as a reference model species, teaching that the promoters expand the options for the control of gene expression [Abstract]. Although this teaches specifically banana root expression, James teaches numerous homologues that have been previously demonstrated to be effective in root-specific expression [pg. 2, col. 2, para. 2].
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to use known root-specific promoters in the methods of Nuccio and Yang because there were a finite number of reported, root-specific promoters from a range of plant species at the time of filing. James teaches that root-specific promoters allow for targeted transgene expression for improving plant traits, demonstrating a design need in the agricultural field. It would have been obvious to try these root-specific promoters because there were a finite number of potential predictable solutions across many species, including model plant species. James teaches that these promoters, and numerous homologues have been found to be effective in expression in roots, showing that one of ordinary skill in the art would have reasonable expectation of success.
Claim 31 is rejected under 35 U.S.C. 103 as being unpatentable over Yang, Nuccio, and James as applied to claims 28 and 30 above, and further in view of Stein O, Granot D. (2019). “An Overview of Sucrose Synthases in Plants.” Front Plant Sci. 10:95. doi: 10.3389/fpls.2019.00095.
Claim 31 recites the method of claim 28, wherein the promoter is selected from the group consisting of a promoter from a Flowering Locus T (FT) gene, a promoter from a Fabaceaen FOR1 gene, a rice tungro bacilliform virus promoter, an RmlC-like cupins superfamily protein promoter, a Commelina yellow mottle virus promoter, a wheat dwarf virus promoter, a sucrose synthase promoter, a glutamine synthetase promoter, a phloem-specific isoform of plasmamembrane H+-ATPase promoter, a JMJ18 promoter, and a phloem protein 2 (PP2) promoter.
Regarding claim 31, Nuccio teaches guide RNA for a Cas nuclease, wherein the guide RNA is fused to a MTS, wherein the plant comprises nucleic acid encoding the Cas nuclease, and suggest application in rootstocks for expression in meristematic tissue of scions; Yang teaches fusions of Cas nucleases with TLS resulting in meristematic expression, rendering obvious the method of claim 1, as detailed above; James further teaches alternative promoters useful for RNA constructs, rendering obvious the method of claim 28, as detailed above.
Nuccio, Yang, and James do not explicitly teach that the promoter is a sucrose synthase promoter, however, Stein teaches that Sucrose synthase (SuSy) is a glycosyl transferase enzyme that plays a key role in sugar metabolism, primarily in sink tissues [Abstract]. Work with promoter-GUS fusions has revealed SUS promoter activity in the phloem of many plant species, including potato, Arabidopsis, maize, rice, tomato and Craterostigma plantagineum, thus demonstrating the wide applicability of sucrose synthase promoters in genetic engineering [pg. 6, col. 1, para 1]. Stein teaches that sucrose synthase may play another, less studied role in the development of shoot apical meristem (SAM) [pg. 9, col. 1, para 3]. The SAM receives Suc from the phloem and there is evidence that SUS are expressed in the SAM. The SlSUS4 promoter GUS fusion showed activity in young meristematic areas, including the SAM.
It would have been prima facie obvious to use a sucrose synthase promoter in the method of Yang and Nuccio in order to further target meristematic growth. One would have been motivated to use this promoter in a method of CRISPR editing a rootstock for root to shoot transport as James teaches that sucrose synthase plays a role in the development of shoot apical meristem and shows activity in young meristematic areas. One would have reasonable expectation of success as SUS promoter activity has been shown in the phloem of many plants, and would likely be effective in the methodology of Nuccio and Yang.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
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Claims 1, 2, 3, 5, 9, 11, 16-23, 26-28, 30-31, 35, 37, 38, 41, 45, 47, 50-51, and 54 are rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent Application No. 19/163,330. The applications share the same Applicant, Inari Agriculture Technology, Inc, and two inventors, Palak Kathiria and Micheal Lee Nuccio.
Although the claims at issue are not identical, they are not patentably distinct from each other. The subject matter claimed in the instant application is disclosed in the referenced patent, and the referenced patent and the instant application are claiming common subject matter.
The instant claims are drawn to a method of editing a genomic target in a plant meristem comprising delivering a guide RNA for a Cas nuclease to a plant root, wherein the guide RNA is fused to a meristem transport segment (MTS), and wherein a genomic target within a cell in the meristem is edited. Dependent claims further limit the claims, wherein the Cas nuclease is a Cas9 (claim 37); wherein the Cas nuclease is fused with a reverse transcriptase, and wherein the guide RNA comprises at its 3’ end a priming site and an edit to be incorporated into the genomic target (claim 38); and wherein the Cas nuclease is a Cas nickase, wherein the Case nickase is a Cas9 nickase or a Cas12 nickase (claim 41).
Conflicting claim 1 is drawn to a method of editing a genomic target in a scion comprising grafting the scion onto a rootstock comprising nucleic acid encoding a Cas9 nickase or Cas12 nuclease and nucleic acid encoding a guide RNA for the nickase or nuclease, wherein the nucleic acid encoding the guide RNA and the nucleic acid encoding the nickase or nuclease are fused to nucleic acid encoding a meristem transport segment, wherein the genomic of a cell in the meristem of the scion is edited.
The instant and conflicting claims further overlap in scope as the instant application claims wherein the scion or the rootstock are the same or different plant species and a monocot or dicot (instant claims 16-21, conflicting claims 14-19); wherein the rootstock or scion is soy, canola, alfalfa, corn, oat, sorghum, sugarcane, banana, or wheat (instant claim 22, conflicting claim 18); wherein the MTS comprises an RNA hairpin comprising a first stem of 8 to 12 nucleotides, at least one variable bulge, a second stem of 4 to 7 nucleotides and a variable loop; an FT-derived sequences wherein the FT-derived sequence comprises the nucleotide sequence set forth in SEQ ID NO: 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24; or iv. a tRNA-like sequence (TLS), wherein the TLS comprises the nucleotide sequence set forth in SEQ ID NO: 29 or 30 (instant claim 23, conflicting claim 19); wherein the nucleic acid encoding the MTS is located 3’ or 5’ of the nucleic acid encoding the Cas nuclease and/or 3’ or 5’ of the guide RNA (instant claims 26 and 27, conflicting claims 22-23); wherein the promoter Is active in roots and or phloem (instant claim 28, conflicting claim 24); wherein the promoter is the promoter of a gene selected from Arabidopsis WRKY6, chickpea WRKY31, carrot MYB113, corn GLU1, strawberry RB7-type TIP-2, and banana TIP2-2, or the promoter of an orthologous gene thereof (instant claim 30, conflicting claim 26); wherein the promoter is selected from Flowering Locus T (FT) gene, a promoter from a Fabaceaen FOR1 gene, a rice tungro bacilliform virus promoter, an RmlC-like cupins superfamily protein promoter, a Commelina yellow mottle virus promoter, a wheat dwarf virus promoter, a sucrose synthase promoter, a glutamine synthetase promoter, a phloem-specific isoform of plasmamembrane H+-ATPase promoter, a JMJ18 promoter, and a phloem protein 2 (PP2) promoter (instant claim 31, conflicting claim 27); wherein the nucleic acid encoding the guide RNA and the MTS is located between two ribozyme sequences (instant claim 45, conflicting claim 34); wherein the nucleic acid encoding the guide RNA and the MTS further comprises a hammerhead ribozyme sequence 5' to the nucleic acid encoding the guide RNA and the MTS, and an HDV ribozyme 3' to the nucleic acid encoding the guide RNA and the MTS (instant claim 47, conflicting claim 36); wherein the method comprises applying two-five or more guide RNAs to the roots (instant claim 35, conflicting claim 39); wherein the Cas nuclease is selected from the group consisting of Cas12a (Cpf1), Casl2e (CasX), Cas12d (CasY), Cas12h, Cas12i, and Cas12j (instant claim 37, conflicting claim 41); further comprising retrieving a progeny of the plant, wherein the progeny has an altered genome (instant claim 50, conflicting claim 43); an edited plant produced by the method (instant claim 54, conflicting claim 46).
The instant and conflicting claims differ in that instant claim 1 recites delivering a guide RNA for a Cas nuclease to a plant root and claim 3 recites that the method comprises wherein the Cas nuclease is expressed in the rootstock, while conflicting claim 1 recites a rootstock comprising nucleic acid encoding a Cas9 nickase or Cas 12 nuclease and nucleotide encoding a guide RNA. However, given that instant claim 1 results in a genomic target within a cell in the meristem edited and the conflicting claim 1 recites wherein the genome of a cell in a meristem of the scion, the end result and general methodology to obtain that result are the same. Even though conflicting claims narrow the nickase or nuclease, these are limitations provided in the instant claim set.
Therefore, one of ordinary skill in the art, at the time the claimed invention was filed, would have readily recognized that the conflicting claims of the granted United States Patent Application No. 19/163,330 and the claims in the instant application, as recited above, are obvious variants and are not patentably distinct. Furthermore, there is no apparent reason why Applicants would have been prevented from presenting claims corresponding to those of the instant application in the previously granted patent. In re Schneller, 397 F.2d 350, 158 USPQ 210 (CCPA 1968). See also MPEP § 804.
Examiner notes that claims 2, 5, 9, 51 are rejected on the ground of nonstatutory double patenting as being unpatentable over in view of the above 103 rejections and thus do not overcome the nonstatutory rejection.
Conclusion
No claims allowed.
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/EMILY K JOHNSON/Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Units 1661 & 1662
1 CaMV 35S promoter is the mostly commonly used promoter for driving transgene expression in plants (Sunilkumar, G. et al. 2002. “Developmental and tissue-specific expression of CaMV 35S promoter in cotton as revealed by GFP.” Plant Mol Biol. 50(3):463-74. doi: 10.1023/a:1019832123444). Using GFP gene as a reporter system in cotton, Sunilkumar teaches that varying levels of promoter activity were observed in all cell and tissue types in the hypocotyl, cotyledon, stem, leaf, petiole, and root [Abstract].
Prosecution Insights