DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status
Claims 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Claims 15, 17, 30-32, 34, and 36 are withdrawn from examination.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 2, 10-11, 16 and 19-25 are being examined.
All previous objections and rejections not set forth below are withdrawn in view of claim amendments or Applicant’s arguments.
Due to applicant’s amendments, the rejection is modified from the rejection set forth on pages 3-11 in the Office action dated 02/09/2026.
Claim Rejections - 35 USC § 103
Claims 2, 10-11 and 19-23 remain 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 signature PAM (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, i.e., within “18-500 base pairs in length comprised of both endogenous chromosomal DNA and heterologous transgenic DNA” (spec, p.9, para 0050), where the modified transgenic locus containing a GFP transgene has 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, bridging paragraph between left column and right column). The new sites developed due to efficient and permanent substitution of one or more (as recited in claim 1) C•G to T•A base pair(s) in different organisms including plants (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 of the invention, 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 target polynucleotide sequence in a specific way. A specific sPAM sequence may not be present in a specific target sequence or in a gene in a genome and, thus, new sPAM sequence(s) needs(s) to be created or introduced at the target polynucleotide sequence/gene. The newly introduced sites(s) would have been used as sPAM sites which were absent in the original transgenic plant, like the transgenic soybean plant comprising a heterologous polynucleotide sequence encoding GFP protein, as taught by Jacobs et al. It is irrelevant to the Cas endonuclease being used and CRISPR-Cas based gene editing technique how the newly introduced 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) using a different pre-existing PAM site(s) in a transgenic plant wherein the new PAM site(s) were not present in specific target sequence(s) in a plant genome. The newly created PAM site(s) can be used by the artisan to edit/modify/delete 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 Cas endonuclease (e.g., Cas9) that 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. The artisan can also be motivated to use the newly created PAM sites to remove any unwanted gene including antibiotic/herbicide resistance gene(s) in the original or first transgenic plant, as recited in claim 11.
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 remains rejected under 35 U.S.C. 103 as being unpatentable over Jacobs et al. in view of Gaudelli et al. and He 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. and He 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. and He et al. do 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) or SMG(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 remain rejected under 35 U.S.C. 103 as being unpatentable over Jacobs et al. in view of Gaudelli et al. and He 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. and He 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. and He et al. does 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 and 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. and He 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. and He 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 6/26/2026 to the rejection of claims under 35 U.S.C. 103 has been fully considered but is not found persuasive.
The Applicant argues, “Even assuming arguendo that base editing could incidentally create a PAM-like motif, Gaudelli provides no teaching or suggestion to do so deliberately at the DNA junction polynucleotide of a transgenic locus, as required by claim 2 as amended. Such reasoning improperly uses Applicant's own disclosure as a blueprint for combining the references. See In re McLaughlin, 443 F.2d 1392, 1395, 170 USPQ 209, 212
(CCPA 1971) (a reconstruction based on hindsight reasoning is improper where it includes
"knowledge gleaned only from applicant's disclosure") (response, p.10, para 1, line 6-12). The Applicant continues to argue, ““Jacobs does not teach any "reason for introducing PAM sites." Jacobs exploits pre-existing endogenous NGG PAM sites already present in the soybean genome to perform CRISPR/Cas9 editing”” (response, p.10, last para, line 3-6), “Hua et al. does not
teach introducing new PAM sites into a genome (response, p.11, first para, line 5-6), “The Examiner's stated motivation-to “expand the option of available PAM sequences operably linked to any specific genomic sequence" (Office Action, pg. 5) is entirely generic and does not provide any reason to specifically modify the junction polynucleotides of a pre-existing transgenic locus (response, bridging para between p.11-12) and “He et al. does not teach or suggest introducing a sPAM site in a DNA junction polynucleotide of a modified transgenic locus, as required by independent claim 2…” (response, p.12, last para, line 4-6).
The Examiner disagrees. The obviousness analysis and the motivation for an ordinarily skilled artisan to use any specific method and/or developing any new product need not be the same or explicitly taught by any of the prior art(s) used in rejections under 35 U.S.C. 103. See MPEP ¶2143.
Even though the cited prior arts do not explicitly teach all the limitations of the recited claims, those method steps are mere design choice that should be obvious to one of ordinary skill in the art, as described above.
A general state of the art regarding CRISPR-Cas based gene editing in soybean is described by Jacobs et al. while Gaudelli et al. describes targeted substitution of one or more specific nucleotide(s) in a target sequence which would include any pre-existing PAM site(s) to create new PAM site(s) in one or more target polynucleotide sequence(s) in a transgenic plant. Before the effective filing date of the invention, it was well known in the art the requirement of specific a protospacer adjacent motif (PAM) be present at any target site is a key limitation to use CRISPR-Cas technique using any specific Cas endonucleases including Cas9 (Hu et al., Evolved Cas9 variants with broad PAM compatibility and high DNA specificity, 2018, Nature, 556: 57–63; abstract). Thus, there is no hindsight involved, as alleged by the Applicant, for any ordinarily skilled artisan based on the general state of the art in view of the teachings of Jacobs et al. in view of Gaudelli et al. and He et al.
Moreover, newly edited sequence(s), which is/are to be used as new PAM site(s), can be introduced in specific location(s) (e.g. at the start and/or 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). 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.
Applicant is reminded that the test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981).
Conclusion
No claim is allowed.
THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Communication
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JAY CHATTERJEE whose telephone number is (703)756-1329. The examiner can normally be reached (Mon - Fri) 8.30 am to 5.30 pm..
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J.C.
/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).
Prosecution Insights