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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 12/19/2025 has been entered.
Claim Status
Claims 2, 10-11, 15-17, 19-25, 30-32, 34, and 36 are pending.
Claims 15, 17, 30-32, 34, and 36 are withdrawn from examination.
Claims 1, 3-9, 12-14, 26-29, 33, 35, and 37-90 are cancelled by the Applicant.
Claims 2, 10-11, 16, 19-25 are being examined.
The claim amendments (dated 12/19/2025) obviate the claim objections and rejections presented in the Final Office action dated 09/19/2025.
Claim Rejections - 35 USC § 103
Claims 2, 10-11 and 19-23 are rejected under 35 U.S.C. 103 as being unpatentable over Jacobs et al. (Targeted genome modifications in soybean with CRISPR/Cas9, 2015, BMC Biotechnology,15:16) in view of Gaudelli et al. (Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage, 2017, Nature, 551:464-471) and He et al. (Technological breakthroughs in generating transgene-free and genetically stable CRISPR-edited plants, 2020, aBIOTECH, 1:88–96; first published 3 Dec. 2019).
Claim 2 is drawn to an edited transgenic plant genome comprising a signature protospacer adjacent motif (sPAM) site, wherein the sPAM site is introduced and is operably linked to a DNA junction polynucleotide of a modified transgenic locus in the transgenic plant genome and wherein the sPAM site is absent from a transgenic plant genome comprising an original transgenic locus.
Jacobs et al. describes a CRISPR system comprising Cas9 endonuclease and a guide RNA (gRNA) (page 1, left column, last para, line 1-3). It also describes an edited transgenic (soybean) plant genome comprising a protospacer adjacent motif (PAM) site comprising the specific PAM sequence in the transgenic plant expressing a GFP transgene (page 1, abstract; page 2, Fig. 1). It is noted that a PAM site is not structurally distinct from a sPAM site as it both are nucleotide sequences which are recognized by a specific RNA dependent DNA endonuclease, like Cas9. The PAM sites taught by Jacobs et al. are shown to be operably linked to the DNA junction polynucleotide where the modified transgenic locus containing a GFP transgene have been introduced before. Jacobs et al. also describes targeted genetic change in the genome of transgenic plant cells as recited in claims 19-22, by deleting several other genes in soybean comprising Glyma01g38150 and Glyma11g07220 genes (page 3, left column, last para), and several genomic sequences associated with MicroRNAs (miRNAs) in soybean (page 3, right column, last para). Jacobs et al. describes seeds (page 8, para 2) and roots (page 2, Fig. 1; page 8, para 2) of the transgenic plants, as recited in claim 23. Jacobs et al. teaches targeted genomic change in a plant genome wherein the modified transgenic locus, which reads on to “first modified transgenic locus”, uses GFP for selection (page 7, left column, para 3, line 9-11; page 7, right column, para 3, line 1-3) and, thus, lacks a selectable marker transgene conferring resistance to an antibiotic, tolerance to an herbicide, or an ability to grow on mannose, as recited in claim 11.
However, Jacobs et al. does not describe a PAM site which have been absent from the original transgenic locus.
Gaudelli et al. describes introducing nucleotide base conversions using Base Editing (BE) technique and expanding the development of base editors with different PAM compatibilities with enhanced DNA specificity, which further improve editing efficiency and final product purity (page 464, left column last para and right column first para). The new sites (developed due to permanent conversion of C•G to T•A base pair) (page 464, left column, para 3, line 12), and the guide RNAs are recognized by Cas9 (page 465, Fig. 1), which is a class 2 type II RNA dependent DNA endonuclease (RdDe)1, as recited in claim 10.
Before the effective filing date, it would have been obvious to a person with ordinary skill in the art to use the method taught by Gaudelli et al. to introduce new protospacer adjacent motifs (sPAM) by using base editing technique in a transgenic plant as taught by Jacobs et al. to expand the option of available PAM sequences operably linked to any specific genomic sequence while improving editing efficiency and final product purity, as taught by Gaudelli et al. Every Cas endonuclease has its own preferred sPAM sequence(s) to cleave the polynucleotide sequence in a specific way. A specific sPAM sequence(s) may not be present in any specific target sequence in a genome and, thus, the need to use base editing technique for editing any original (pre-edited) plant genome. The newly introduced sites to be used as sPAM sites were absent in the original plant. It is irrelevant to CRISPR-Cas based gene editing technique how the PAM site(s) in the polynucleotide sequence (being targeted) has been introduced into the genome.
It would also have been obvious to an ordinarily skilled artisan to use the well-known and standard genome editing technique to introduce new PAM site(s), as recognized by a specific Cas endonuclease, at a specific locus for the same reason, i.e., to create new PAM site(s) not present in a specific target sequence in a plant genome. The newly created PAM site(s) can be used by the artisan to edit/modify the specific locus in the genome using CRISPR-Cas genome editing technique.
Targeted genome editing at that specific locus containing the newly introduced site(s), which can act as PAM site(s), can also be achieved by crossing the first genome edited plants having the newly introduced PAM site operably linked to a DNA junction at a specific genomic locus with a previously created stable transgenic plant expressing specific Cas9 variant which recognizes the newly introduced PAM site(s).
Before the effective filing date, an ordinarily skilled artisan would have been motivated to introduce new signature protospacer adjacent motifs (sPAM) either by using base editing technique in a plant (transgenic or otherwise) to expand the options of available PAM sequences operably linked to any specific genomic sequence in a transgenic plant for improving editing efficiency and/or final product purity.
Regarding claim 11, CRISPR-Cas based targeted gene editing technique at the time of filing was widely used and a standard process to remove any transgene including marker gene(s) from a plant, as described by He et al. (abstract).
Claim 16 is rejected under 35 U.S.C. 103 as being unpatentable over Jacobs et al. in view of Gaudelli et al., as applied to claims 2, 10-11, 19-23 above, and further in view of Yau et al. (Less is more: strategies to remove marker genes from transgenic plants, 2013, BMC Biotechnology, 13:36) and Official Journal of the European Union (Commission Implementing Decision (EU) 2017/2450, Authorising the placing on the market of products containing, consisting of, or produced from genetically modified soybean DAS-44406-6, pursuant to Regulation (EC) No 1829/2003 of the European Parliament and of the Council on genetically modified food and feed, 12/21/2017).
Claim 16 is drawn to the edited transgenic plant genome of claim 2, wherein the first modified transgenic locus is in soybean variety DAS44406-6 and comprises the modification of original transgenic locus in the transgenic soybean plant.
Jacobs et al. in view of Gaudelli et al. describe deletion of several genes in soybean by introducing new PAM sites using base editing technique to further improve editing efficiency and/or final product purity, as described above.
However, Jacobs et al. in view of Gaudelli et al. does not describe the soybean variety DAS44406-6.
Yau et al. describes that the presence of transgenes including selectable marker genes (SMGs) in genetically modified (GM) plants, and subsequently in food, feed, and in the environment, are of concern and subject to special government regulations in many countries. Presence of SMGs in GM plants might also result in a metabolic burden for the host plants (abstract). Use of SGMs also prevents re-use of the same SMGs when a second transformation scheme is needed to be performed on the transgenic host (page 1, abstract).
Official Journal of the European Union (Commission Implementing Decision (EU) 2017/2450) teaches soybean variety DAS-44406-6 expressing the following proteins: 5-enolpyruvyl shikimate-3-phosphate synthase (2mEPSPS), aryloxyalkanoate dioxygenase-12 (AAD-12), and phosphinothricin acetyl transferase (PAT), which confer tolerance to glyphosate-based herbicides, 2,4-dichlorophenoxy acetic acid (2,4-D) and other related phenoxy herbicides, and glufosinate ammonium-based herbicides, respectively (page 2, para 7).
Before the effective filing date, it would have been obvious to any person with ordinary skill in the art to use CRISPR-Cas technique to remove selectable markers gene(s) from transgenic plants (as described above) including from the soybean variety DAS44406-6 containing several selection marker genes, as taught by the Official Journal of the European Union (Commission Implementing Decision (EU) 2017/2450). The SMGs in the soybean variety DAS44406-6 would have been removed with the goal to minimize and/or eradicate the concerns associated with genetically modified crops containing SGMs, as taught by Yau et al.
Before the effective filing date, an ordinarily skilled artesian would have been motivated to use CRISPR/Cas9 based genome editing technique to remove one or more undesirable selectable markers gene(s) from a transgenic plant including soybean variety DAS44406-6.
Claims 24-25 are rejected under 35 U.S.C. 103 as being unpatentable over Jacobs et al. in view of Gaudelli et al., as applied to reject claims 2, 10-11, 19-23 above, and further in view of Afzal et al. (Seed Production Technologies of Some Major Field Crops. In: Hasanuzzaman, M. (eds) Agronomic Crops. Springer, Singapore. 2019).
Claim 24 depends from claim 21, and is drawn to a method for obtaining a bulked population of inbred seed for commercial seed production comprising selfing the transgenic plant resulting in selfed elite crop pant and harvesting seed from the selfed elite crop plants.
Jacobs et al. in view of Gaudelli et al. describes genome edited transgenic plants modified to contain at least one newly introduced sPAM site, as discussed above.
However, Jacobs et al. in view of Gaudelli et al. do not teach a method for obtaining a bulked population of (selfed) inbred seed comprising selfing the transgenic plant and harvesting seed from the selfed elite crop plants.
Afzal et al. teach seed production techniques in some major field crops including soybean. Afzal et al. teach that soybean is a self-pollinated crop and, thus, breeding methodologies applicable to self-pollinated crops such as development of pure lines through selfing and bulk selection methods are applicable to soybean (page 675, para 1, line 9).
Before the effective filing date, it would have been obvious to any person with ordinary skill in the art to modify the method taught by Afzal et al. to develop pure lines or inbreed lines of the transgenic soybean plant produced by CRISPR-Cas technique (as described above) and then develop selfed bulked population starting with the genome edited plants for commercial seed production of a self-pollinated crop like soybean.
Before the effective filing date, an ordinarily skilled artisan would have been motivated to modify the methods taught by Afzal et al. to produce selfed inbreed lines to develop bulked population starting with genome edited plants, produced by using CRISPR/Cas9 based genome editing technique, for commercial seed production of a self-pollinated crop like soybean.
Regarding claim 25, claim 25 indirectly depends from claim 2. Claim 25 is drawn to a method of obtaining hybrid crop seeds comprising crossing a first transgenic plant (of claim 2) to a second crop plant and harvesting seeds from the cross.
Jacobs et al. in view of Gaudelli et al. teaches genome edited transgenic plants modified to contain at least one newly introduced sPAM site, as discussed above.
However, they did not describe a method of crossing a first genome-edited crop plant to a second crop plant and harvesting seed from the cross.
Afzal et al. describes hybrid as the progeny of cross between two inbred lines selected on the basis of genetic divergence and their specific- and general-combining ability (page 671, para 2). Any plant including the genome edited transgenic plant as described by Jacobs et al. in view of Gaudelli et al., can be crossed (backcrossed) with specific elite line(s), especially if the original transgenic plant population shows poor agronomic or adaptability performance, as described by Afzal et al. (page 673, para 2, line 12-13). Afzal et al. also describes various seed production techniques including production of hybrid seeds, along with various management practices of seed production of agronomic crops (page 656, first para, line 2; page 671, para 2).
Before the effective filing date, it would have been obvious to any person with ordinary skill in the art to modify the methods taught by Afzal et al. to develop hybrid soybean lines, by crossing the genome-edited plant comprising newly introduced PAM site(s) (as described above) to an elite line for commercial hybrid seed production. Harvesting seeds is implied in these breeding methods especially when crossing is involved between two sexually propagated plants and/or the seeds are the main product, as in commercially important soybean plant.
Before the effective filing date, an ordinarily skilled artisan would have been motivated to develop hybrid lines by crossing the genome-edited plant to an elite line for commercial hybrid seed production in commercially important soybean plant.
Response to Applicant’s arguments
The argument set forth in Applicant’s reply on 12/19/2025 to the rejection of claims under 35 U.S.C. 103 has been fully considered but is not found persuasive.
The Applicant argues that, “the combination of Jacobs and Gaudelli fails to teach a modified transgenic locus wherein the modification comprises a sPAM site in the DNA junction polynucleotide of the modified transgenic locus, and wherein the sPAM site is absent from the original transgenic locus” (response, page 10, para 1, last 3 lines) as “Jacob actually introduces an original, unmodified transgenic locus while utilizing PAM sites pre-existing in the genome” (page 10, last para, line 5-6) and “Gaudelli fails to remedy this deficiency as Gaudelli describes methods of programmable base editing of AT to GC in genomic DNA without DNA cleavage” (page 11, last para, line 1-2).
The Examiner disagrees. All PAM sites are around the DNA junction where the Cas endonuclease cleaves one or both the DNA standard(s), depending on the type of Cas nuclease used. The Cas9 nuclease, as used by Jacobs et al.(page 4, Fig. 1) and the Applicant (Spec, page 73, para 00219), are not positioned exactly at the junction but about 4-5 nucleotide apart from the site where the nuclease makes the double stranded break, as described by Jacobs et al. (Fig. 1). The sequence(s)/site(s) newly created by base editing, as described by Gaudelli et al., was/were not used for the base editing by dCAS9 nuclease (or any other endonuclease without nuclease activity). Those new site(s) was/were created to be used as (newly introduced/created) PAM sites which was/were not present before (base editing) in the genome of the original transgenic plant. These newly created PAM site(s) would have been used by a different Cas enzyme for genome editing and thus, satisfying all the limitations of claim 2. Jacobs et al. teaches the reason for introducing PAM sites, and Gaudelli is just teaching a method that can be utilized to make those new sites which can be used as PAM sites.
Moreover, new site(s), which is to be used as new PAM site(s), can also be introduced in specific location(s) (e.g. at the start and at the end of a transgene) using conventional CRISPR-Cas based genome editing to remove the transgene by crossing the genome edited plant with a previously created stable transgenic plant expressing specific Cas9 endonuclease, as described by Hua et al. (page 1004, right column, para 2, line 14-16), which recognizes the newly introduced PAM site(s), as described above. This method would have enabled an ordinarily skilled artisan to edit (including removing a transgene) a specific gene (including a transgene) in a plant genome where it was simply not possible due to lack of suitable PAM site(s) around the target sequence and/or to improve genome editing efficiency by introducing a more efficient PAM site for a specific Cas endonuclease. DNA cleavage is not a required limitation in any of the claims.
Conclusion
No claim is allowed.
Communication
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Jay Chatterjee
Patent Examiner
Art Unit 1662
/Jay Chatterjee/Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Units 1661 & 1662
1Tang et al. (Class 2 CRISPR/Cas: an expanding biotechnology toolbox for and beyond genome editing, 2018, Cell Biosci., 8:59) provides the evidence that Cas9 is a class 2 type II RNA dependent DNA endonuclease (RdDe) (page 2, Fig. 1).