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
Amendments to claims 1, 10, 16, 21 and 25 submitted on 05/15/2026 is acknowledged. Claims 1-10, 12, 14, 16-17, 21, and 23-25 are pending and are examined in this office action.
Rejections that are withdrawn
Objection to claim 4 is withdrawn in light pf applicant’s amendment of the claim to recite “polynucleotide of step” in line 4.
35 USC § 112 – Indefiniteness rejection is withdrawn in light of applicant’s amendment of claims 1, 25 and 43 to delete the indefinite term “directed to”, applicant’s amendment of claims 1, 21 and 25 by deleting “fragment thereof” and specifically reciting first target is in promoter and second target is in “the coding region, the non-coding region, or the negative-regulatory element of the second gene”, applicant’s amendment of claim 9 to recite “the at least two distinct Cas nucleases comprise” to clarify the step of the method, applicant’s amendment of claim 10 to clarify the sentence by deleting “is introduced” at end of sentence, and applicant’s cancellation of claim 20.
35 USC § 102 rejection over claims 25 and 43 has been withdrawn in light of applicant’s amendment of claim 25 to recite the plant cell in the editing system comprise a first genomic DNA site in a promoter of a first gene, a donor template lacks homology arms, a DNA molecule encoding a second gRNA targeting second gene wherein the prior art does not disclose the limitation of second gRNA targeting second gene and by applicant’s cancellation of claim 43.
Following 35 USC § 103 rejection has been modified in light of applicant’s amendment of claims 1 and 25 to recite the new limitation in the method to include a first genomic DNA site in a promoter of a first gene, a donor template lacks homology arms, and a second gRNA targets second gene. A new art He et al. has been included in this rejection to analyze the known method of developing and inserting DNA template lacking homology arm as recited in claims 1, 21 and 25.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Obvious over Yoshimi et al. and further in view of He et al. and Shi et al.
Claims 1-4, 7-9, 12, 14, 16-17, 21, 23- 24 and 43 are rejected under 35 U.S.C. 103 as being unpatentable over Yoshimi et al. (Published: 2021, Journal: Human Genetics 140:277–287, https://doi.org/10.1007/s00439-020-02198-4), and further in view of He et al. (Published: 2015, Journal: Plant Physiology, 169: 931–945), further in view of Shi et al.
Claims are directed to a method of producing a genome edited plant cell comprising introducing a first guide (gRNA) directed to a first genomic site of a first gene and a donor DNA template lacking homology arms and second gRNA directed to second genomic DNA site of second gene in a plant cell, and selecting the genome edited plant with an insertion of the template in first site and other deletion and insertions in second site.
Regarding claims 1 and 24, Yoshimi et al. teaches targeting mice and rat cells using CRISPR-Cas9 wherein the method comprises using a donor template with Cas9 and two single guide RNAs, one designed to cut the targeted genome sequences and the other to cut both the flanked genomic region and one homology arm of the dsDNA plasmid, which resulted in 20–33% (Knock-ins) KI efficiency among G0 pups (page 277, Abstract).
Yoshimi et al. teaches G0 KI mice carried NHEJ-dependent indel mutations at one targeting site of the genome (page 277, Abstract).
Yoshimi et al. teaches the combinatorial method of NHEJ and HDR mediated by the CRISPR-Cas9 system facilitates the efficient and precise KIs of plasmid DNA cassettes in mice and rats (page 277, Abstract) (see figures 1 and 3 below).
Yoshimi et al. teaches targeting parvalbumin (Pvalb) gene and tyrosine hydroxylase (Th) with a sgRNA-1 targeting terminal codon of the Pvalb or Th gene and SgRNA-2 targeting intro upstream of the terminal exon the PCR screening indicated the presence of indel mutation at the sgRNA-2 targeting intron region and integration (i.e. KI) of P2A-Cre cassette (page 281, left paragraphs 1-2).
Yoshimi et al. teaches insertion of whole donor vector sequence (i.e. not comprising the homology arm) in a rat genome by NHEJ mechanism alone in in the first step the sgRNA-2 and Cas9 induces double strand breaks followed by the DSB repair pathways wherein such method produced the insertion without requiring HDR (i.e. not requiring homology arms) (page 281, right first paragraph, Figure 3). Yoshimi et al. table 2 further showed the method of targeted insertions only using NHEJ and not requiring HDR for example ObLiGaRe, HITI, 2H2OP etc. Therefore, integration of a genomic region using NHEJ (i.e. not requiring homology arms) was known in the art. Yoshimi et al. teaches development #3 rat which carried the whole donor vector sequence insertion using NHEJ repair pathway alone (page 281, left paragraph 2, Figure 3).
Yoshimi et al. teaches introduction of the two sgRNA in mouse embryo (page 283, last paragraph) and screening of survived embryo for the mutations using PCR and sequence analysis (page 284).
Furthermore, He et al. teaches NHEJ mediated knock in (KI) of template without homology arm wherein the donor plasmids without homology arm was inserted in GAPDH locus (Figure 2A) (see part of the Figure 5D below) (page 3, right paragraph 3) producing efficient knock-in in the human cell lines genome (page 6, left paragraph 2). He et al. teaches the NHEJ mediated knock in is non directional and it accommodates large NDA inserts (page 6, right paragraph 2). He et al. teaches NHEJ mediated knock in (KI) had higher efficiency of insertion than HDR based approach (page 9, left paragraph 2). Therefore, NHEJ is efficient for integration of template without homology arm.
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Yoshimi et al. does not teach the method comprise targeting first gene at promoter region.
Shi et al. teaches modification promoter region of an endogenous ARGOS8 gene to attain desired expression patterns and eliminate the need for a transgene (page 107, lines 19-21). Shi et al. teaches introducing guide RNAs and donor template into the maize embryo cells where multiple promoter replacement events to replace the native promoter of Zm-ARGOS8 with Zm-GOS2 PRO:GOS2 INTRON which were identified by PCR screening (page 109, lines 1-14) and a donor template comprising Zm-GOS2 promoter leads to promoter swap (i.e. replacement) or GOS2 promoter insertion in maize (page 109, Table 2).
Shi et al. teaches in the repair mechanism to bring the broken ends together is the nonhomologous end-joining (NHEJ) pathway, the structural integrity of chromosomes is typically preserved by the repair, but deletions, insertions, or other rearrangements are possible (page38, lines 23-28). Shi et al. teaches insertions as a result of NHEJ (Figure 3A and 3B, page 9, lines 1-9).
Shi et al. teaches gRNA/maize optimized Cas9 endonuclease provide efficient system to target multiple chromosomal loci (i.e. genes) that are co-transferred into maize embryo that simultaneously mutagenize the multiple genes (page 96, lines 15-25).
Shi et al. further teaches introducing guide RNAs targeting genes MS26Cas-2 , LIGCas-3, and MS45Cas-2 target sites into maize embryo along with the Cas9 endonuclease expression cassette and which is examined by deep sequencing for the presence of imprecise NHEJ mutations (page 96, lines 15-30). Shi et al. teaches introducing guide RNAs and donor template into the maize embryo cells where multiple promoter replacement events to replace the native promoter of Zm-ARGOS8 with Zm-GOS2 PRO:GOS2 INTRON which were identified by PCR screening (page 109, lines 1-14).
Therefore, it would have been obvious to apply a known technique taught by Yoshimi et al. to a known and economically importance crop for example in maize which is ready for improvement for its method of genome editing including insertion of a template comprising a promoter of a gene Zm-ARGOS8 of maize and mutation in MS26Cas-2 , LIGCas-3, and MS45Cas-2 genes taught by Shi et al. and someone skilled in the art would insert the template by NHEJ pathway not requiring template with homology arms by more precise insertion as taught by Yosimi et al. and He et al. by using two guide RNA and DNA template to cause insertion of template in one gene site and insertion, deletion and substitution on other gene site to yield predicable result of developing a method of producing a gene edited plant.
Regarding claim 2, Shi et al. teaches regenerating identified callus events for insertions and confirming the event by PCR amplification and sequencing in T0 plants (page 108, lines 25-29, page 109, lines 15-21).
Regarding claims 3 and 4, Shi et al. teaches the plant cell comprise Cas9 encoding Cas nuclease (page 96, lines 15-30).
Regarding claim 7, Shi et al. teaches different types of endonucleases for example Type I, Type II, Type Ill, and Type IV endonucleases, which further include subtypes wherein Cas9 is type II system (page 23, lines 16-21, page 94, lines 14-19).
Therefore, designing a distinct nuclease to recognize the first and second gRNA is within the skill of the art.
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Regarding claims 8 and 9, Shi et al. teaches the Cas9 endonuclease expression cassette are co-transferred into maize embryo with different gRNAs (page 96, lines 1—25).
Regarding claim 12, Shi et al. teaches delivery of the Cas9 (as DNA vector) and guide RNA (as DNA vector) by co-delivering the DNA cassettes on a single or multiple Agrobacterium vectors and transforming plant tissues by Agrobacterium mediated transformation (page 97, lines 17-20).
Regarding claim 14, Shi et al. teaches their introduction was in maize callus (page 10, lines 17-31).
Regarding claims 16 and 17, Shi et al. teaches in the repair mechanism to bring the broken ends together is the nonhomologous end-joining (NHEJ) pathway, the structural integrity of chromosomes is typically preserved by the repair (page38, lines 23-28). Shi et al. teaches genomic insertions as a result of imperfect NHEJ (Figure 3A and 3B, page 9, lines 1-9).
Shi et al. teaches insertion of CaMV enhancer in front of endogenous promoter of FRM1 gene in order to enhance the expression of FTM1 gene leading to shortened maturity to the modulated plant (page 113, lines 5-10). Shi et al. claim 6 teaches a donor polynucleotide comprising the enhancer element in the method of genetic modification of the gene. Shi et al. teaches insertion of the 35S enhancer or MMV enhancer into an endogenous promoter region increase gene expression wherein guide polynucleotide/Cas endonuclease system is used to insert enhancer element (page 48, lines 13).
Therefore, it would have been obvious to develop the method for the insertions of the enhancer element, for example the 35S enhancer in a donor template by NHEJ which would not require the DNA template to comprise homology arms complementary to DNA in promoter of FRM1gene leading to its enhanced expression.
Regarding claim 21, Shi et al. teaches using deep sequencing for the presence of imprecise NHEJ mutations in gRNA targeting genes MS26, LIG and MS45 as described in Example 2 (page 96, lines 18-25, page 8, lines 28-32). The result of such mutation screening are shown in Figure 3A, 3B, for LIG gene, and Figure 6 showing mutation in genomic target site 55CasRNA-1 (page 13, lines 31-32).
Regarding claim 23, Shi et al. teaches regenerating identified callus events for insertions and confirming the vent by PCR amplification and sequence in T0 plants (page 108, lines 25-29, page 109, lines 15-21).
Regarding claim 43, The combination of the gRNA3 and gRNA2 of Shi et al. targeting the promoter region of Zm-ARGOS8 (page 109, lines 1-14), a donor template comprising Zm-GOS2 promoter wherein promoter swap (i.e. replacement) or GOS2 promoter insertion (page 109, Table 2) and Shi et al.’s disclosed gRNA targeted for genes MS26Cas-2 , LIGCas-3, and MS45Cas-2 target sites into maize embryo along with the Cas9 endonuclease expression cassette and which is examined by deep sequencing for the presence of imprecise NHEJ mutations (page 96, lines 15-30) would lead to the system comprising the guide RNA and template. Furthermore, someone skilled in the art would utilize the template without homology arms to integrate into the promoter region since Yoshimi et al. and He et al. teaches the NHEH method of insertion of template without homology arm is more efficient compared to the HDR method. That would lead to the plant ell comprising the genome editing system.
Regarding claim 25, Shi et al. discloses the gRNA3 and gRNA2, the Cas9 cassette, the polynucleotide modification template was used to transform immature maize plant embryo cells wherein multiple promoter swaps (or promoter replacement or insertions) events were identified in T0 plants (page 109, lines 6-11) (Figure 10D).
Since claim recites the second insertion comprise any insertions, any deletion and any substitution of any number of nucleotides, such changes can make any structure of the genome found in the maize plant disclosed by Shi et al. in any second genomic site.
Obvious over Yoshimi et al. and further in view of He et al., Shi et al. and Svitashev et al.
Claims 1, 5-6, 9-10 are rejected under 35 U.S.C. 103 as being unpatentable over Yoshimi et al. and further in view of Shi et al. and further in view of Svitashev et al. (Published: 2016, Journal: Nature com., 7:13274, DOI: 10.1038/ncomms13274).
Claim are directed to a method of producing a genome edited plant cell comprising introducing a first guide (gRNA) directed to the promoter of the first gene and a donor DNA template not comprising homology arm and second gRNA directed to second gene in a plant cell, and selecting the genome edited plant with a insertion of the template in first site and other deletion and insertions in second site. Claims are drawn to introducing the Cas nuclease and gRNA as RNP complex.
Regarding claim 1, Yoshimi et al. teaches insertion of whole donor vector sequence (i.e. not comprising the homology arm) in a rat genome by NHEJ mechanism alone in the first step the sgRNA-2 and Cas9 induces double strand breaks followed by the DSB repair pathways wherein such method produced the insertion without requiring HDR (i.e. not requiring homology arms) (page 281, right first paragraph, Figure 3). Yoshimi et al. table 2 further showed the method of targeted insertions only using NHEJ and not requiring HDR for example ObLiGaRe, HITI, 2H2OP etc. Therefore, integration of a genomic region using NHEJ (i.e. not requiring homology arms) was known in the art. Yoshimi et al. teaches development #3 rat which carried the whole donor vector sequence insertion using NHEJ repair pathway alone (page 281, left paragraph 2, Figure 3).
Yoshimi et al. teaches introduction of the two sgRNA in mouse embryo (page 283, last paragraph) and screening of survived embryo for the mutations using PCR and sequence analysis (page 284).
Furthermore, He et al. teaches NHEJ mediated knock in (KI) of template without homology arm wherein the donor plasmids without homology arm was inserted in GAPDH locus (Figure 2A) (see part of the Figure 5D below) (page 3, right paragraph 3) producing efficient knock-in in the human cell lines genome (page 6, left paragraph 2). He et al. teaches the NHEJ mediated knock in is non directional and it accommodates large NDA inserts (page 6, right paragraph 2). He et al. teaches NHEJ mediated knock in (KI) had higher efficiency of insertion than HDR based approach (page 9, left paragraph 2). Therefore, NHEJ is efficient for integration of template without homology arm.
Regarding claims 5 and 10, Shi et al. teaches the plant cell comprise Cas9 encoding Cas nuclease (page 96, lines 15-30). Shi et al. teaches the Cas9 endonuclease expression cassette are co-transferred into maize embryo with different gRNAs (page 96, lines 1—25).
Yoshimi et al. or Shi et al. does not teach the method include introducing Cas nuclease as a RNP complexed with first gRNA.
Svitashev et al. teaches creating RNP complex using a gRNA and Cas9 ribonucleoprotein complex to edit maize plant cell genome (page 5, left, second to last paragraph). Svitashev et al. teaches the method including RNP complex improves the genome editing frequency (page1, Abstract).
Therefore, someone skilled in the art would use the RNP complex in their method of genome editing in maize plant. Furthermore, someone skilled in the art would use modification of one site as RNP complex and other as an expression cassette as taught by Shi et al.
Regarding claim 6, Yoshimi et al. teaches the vector comprising Cas9 and two different guide RNAs wherein first and second targeting first and second site of the genome (page 283, right paragraph 2, page 281, right paragraph 1, page 280, left first paragraph, see Figure 3 above).
Regarding claim 9, Shi et al. teaches the Cas9 endonuclease expression cassette are co-transferred into maize embryo with different gRNAs (page 96, lines 1—25).
Response to Arguments
Applicants’ arguments filed 06/15/2026 have been fully considered but they are not persuasive.
Applicant argues claim 1 has been amended to include first and second guide RNA and the DNA template lacks homology arms (Applicant Arguments, page 14, paragraph2). Applicant argues the amendments clarify that the claimed method employs two distinct guide RNAs targeting distinct genomic DNA sites located in different genes, resulting in independent editing outcomes at each site, wherein insertion at the first genomic DNA site is donor-template-mediated using a donor DNA template that lacks homology arms and deletion at the second genomic DNA site occurs independently of the donor DNA template (Applicant Arguments, page 14, last paragraph).
Applicant assert Yoshimi discloses a Combi-CRISPR system that relies on coordinated cleavage of genomic DNA and a donor plasmid to improve knock-in efficiency. Applicant argues Yoshimi does not teach or suggest editing at genomic DNA sites located in two different genes using a donor DNA template lacking homology arms template (Applicant Arguments, page 15, second paragraph). Applicant assert while Shi, while directed to plant genome editing, targets multiple positions within the same gene locus and utilizes a donor DNA template with homology arms that spans both target sites to mediate a single homologous recombination event. Applicant argues Shi does not disclose or suggest editing at genomic DNA sites located in two different genes or achieving different editing outcomes at those genes. Applicant argues neither Yoshimi nor shi teaches first and second guide RNA targeted to two genes and donor template lacking homology arms (Applicant Arguments, page 15, last paragraph).
Applicant argues even combined, Yoshimi and Shi would not result in editing at two different genes with distinct and independent editing outcomes -namely, insertion at a first genomic DNA site in a promoter of a first gene and deletion at a second genomic DNA site in a coding region, non-coding region, or regulatory element of a second gene. Applicant argues nor do Yoshimi and Shi suggest limiting donor-template-mediated insertion to only the first genomic DNA site while the second genomic DNA site undergoes deletion independently of the donor DNA template. Applicant argues Yoshimi and Shi also do not suggest use of donor DNA template lacking homology arms (Applicant Arguments, page 16, first paragraph).
Applicant assert Svitashev teaches delivery of Cas nuclease in the form of ribonucleoprotein complex with guide RNA wherein a skilled in the art would not have motivated to utilize such RNP complexes in the method of Yoshimi and Shi et al. (Applicant Arguments, page 16, first paragraph).
Applicants’ arguments filed have been fully considered but they are not persuasive since regarding amendments targeting two specific site in a genome was known in the art which would comprise template insertion in the promoter of Zm-ARGOS8 gene and mutations in genes of LIG, MS26 and MS45. Furthermore, the teaching of Shi et al. and further in view of Yoshimi et al. and He et al. teaches such insertion of template would be carried out using NHEJ pathway using a template not comprising the homology arms lacking homology to the promoter, since He et al. showed the NHEJ is more efficient in insertions of a template compared to HDR, see analysis above.
Regarding argument on Yoshimi et al. teaches insertion of whole donor vector sequence (i.e. not comprising the homology arm) in a rat genome by NHEJ mechanism alone in in the first step the sgRNA-2 and Cas9 induces double strand breaks followed by the DSB repair pathways wherein such method produced the insertion without requiring HDR (i.e. not requiring homology arms) (page 281, right first paragraph, Figure 3). Yoshimi et al. table 2 further showed the method of targeted insertions only using NHEJ and not requiring HDR for example ObLiGaRe, HITI, 2H2OP etc. Therefore, integration of a genomic region using NHEJ (i.e. not requiring homology arms) was known in the art. Yoshimi et al. teaches development #3 rat which carried the whole donor vector sequence insertion using NHEJ repair pathway alone (page 281, left paragraph 2, Figure 3).
Furthermore, Shi et al. teaches genomic insertions as a result of imperfect NHEJ (Figure 3A and 3B, page 9, lines 1-9). Shi et al. showed targeting promoter of Zm-ARGOS8 gene and mutations in genes of LIG, MS26 and MS45. Shi et al. teaches gRNA/maize optimized Cas9 endonuclease provide efficient system to target multiple chromosomal loci (i.e. genes) that are co-transferred into maize embryo that simultaneously mutagenize the multiple genes (page 96, lines 15-25).
Therefore, it would have been obvious to apply a known technique taught by Yoshimi et al. to a known and economically importance crop for example in maize which is ready for improvement for its method of genome editing including insertion of a template comprising a promoter of a gene Zm-ARGOS8 of maize and mutation in MS26Cas-2 , LIGCas-3, and MS45Cas-2 genes taught by Shi et al. and someone skilled in the art would insert the template by NHEJ pathway not requiring template with homology arms by more precise insertion as taught by Yosimi et al. and He et al. by using two guide RNA and DNA template to cause insertion of template in one gene site and insertion, deletion and substitution on other gene site to yield predicable result of developing a recited method of producing a gene edited plant.
Following double patenting rejection has been modified in light of applicant’s amendment of claims 1 and 25 to recite the new limitation in the method to include a first genomic DNA site in a promoter of a first gene, a donor template lacks homology arms, and a second gRNA targets second gene.
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).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1-4, 8, 12, 14, 16-17 and 23-25 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 2, 4-5, 7 and 17-18 of copending Application No. 18703773 (Herein referenced as ‘773) in view of Shi et al.
Regarding claims 1, 3, 4, 8, 16-17 and 25, copending application ‘773 claims 1, 4-5 teaches a method of producing a genome edited plant cell comprising introducing one or more of polypeptide of a genome editing systems (i.e. guide RNA guided nuclease donor template, gRNA) and identifying a genome edited plant cell. Copending application ‘773 claims 1 recite wherein one or more gRNAs are introduced with double stranded oligonucleotides (i.e. template) lacking homology arms (step C), wherein the oligonucleotide is inserted by NHEJ (last step).
Copending application ‘773 does not expressly teach introducing second gRNA targeting a different genomic site to create an insertion, deletion or substitution and the method comprise targeting first gene at promoter region.
Shi et al. teaches modification promoter region of an endogenous ARGOS8 gene to attain desired expression patterns and eliminate the need for a transgene (page 107, lines 19-21). Shi et al. teaches introducing guide RNAs and donor template into the maize embryo cells where multiple promoter replacement events to replace the native promoter of Zm-ARGOS8 with Zm-GOS2 PRO:GOS2 INTRON which were identified by PCR screening (page 109, lines 1-14) and a donor template comprising Zm-GOS2 promoter leads to promoter swap (i.e. replacement) or GOS2 promoter insertion in maize (page 109, Table 2).
Shi et al. teaches in the repair mechanism to bring the broken ends together is the nonhomologous end-joining (NHEJ) pathway, the structural integrity of chromosomes is typically preserved by the repair, but deletions, insertions, or other rearrangements are possible (page38, lines 23-28). Shi et al. teaches insertions as a result of NHEJ (Figure 3A and 3B, page 9, lines 1-9).
Shi et al. teaches gRNA/maize optimized Cas9 endonuclease provide efficient system to target multiple chromosomal loci (i.e. genes) that are co-transferred into maize embryo that simultaneously mutagenize the multiple genes (page 96, lines 15-25).
Furthermore, Shi et al. teaches introducing guide RNAs targeting genes MS26Cas-2 , LIGCas-3, and MS45Cas-2 target sites into maize embryo along with the Cas9 endonuclease expression cassette and which is examined by deep sequencing for the presence of imprecise NHEJ mutations (page 96, lines 15-30). Shi et al. teaches introducing guide RNAs and donor template into the maize embryo cells where multiple promoter replacement events to replace the native promoter of Zm-ARGOS8 with Zm-GOS2 PRO:GOS2 INTRON which were identified by PCR screening (page 109, lines 1-14).
Therefore, it would have been obvious to apply a known technique taught by Copending application ‘773 to a plant cell which is ready for improvement for its method of genome editing including insertion of a template in a promoter of a gene Zm-ARGOS8 of maize and furthermore creating mutation in MS26Cas-2 , LIGCas-3, or MS45Cas-2 genes taught by Shi et al. and someone skilled in the art would insert the template by NHEJ pathway not requiring template with homology arms as taught by ‘773 leading to the predictable result of developing the method of producing genome edited plant cell.
Regarding claim 2, copending Application ‘773 claim 2 recite regenerating the genome edited plants.
Regarding claim 12, the copending Application ‘773 claim 7 teaches introduction is by using Agrobacterium mediated transformation.
Regarding claim 14, the copending Application ‘773 claim 1 teaches introducing in callus comprising plan cell.
Regarding claim 23, the copending Application ‘773 claim 2 teaches regenerating plant from edited callus.
Regarding claim 24, the copending Application ‘773 claim 18 teaches the plant cell is maize plant cell.
Regarding claims 16 and 17, the copending Application ‘773 claim 1 recite wherein the oligonucleotide is inserted by NHEJ (last step).
Furthermore, Shi et al. teaches in the repair mechanism to bring the broken ends together is the nonhomologous end-joining (NHEJ) pathway, the structural integrity of chromosomes is typically preserved by the repair, but deletions, insertions, or other rearrangements are possible (page38, lines 23-28). Shi et al. teaches insertions as a result of imperfect NHEJ (Figure 3A and 3B, page 9, lines 1-9).
Shi et al. teaches insertion of CaMV enhancer in front of endogenous promoter of FRM1 gene in order to enhance the expression of FTM1 gene leading to shortened maturity to the modulated plant (page 113, lines 5-10). Shi et al. claim 6 teaches a donor polynucleotide comprising the enhancer element in the method of genetic modification of the gene. Shi et al. teaches insertion of the 35S enhancer or MMV enhancer into an endogenous promoter region increase gene expression wherein guide polynucleotide/Cas endonuclease system is used to insert enhancer element (page 48, lines 13).
Therefore, it would have been obvious to develop the method for the insertions of the enhancer element, for example the 35S enhancer in a donor template by NHEJ which would not require the DNA template to comprise homology arms complementary to DNA in promoter of FRM1gene leading to its enhanced expression.
Claims 1, 5-7, 9-10 and 21 provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 and 4-5 of copending Application No. 18703773 (Herein referenced as ‘773), and further in view of Svitashev et al. and further in view of Shi et al.
Regarding claim 1, copending application ‘773 claims 1, 4-5 teaches a method of producing a genome edited plant cell comprising introducing one or more of polypeptide of a genome editing systems (i.e. guide RNA guided nuclease donor template, gRNA) and identifying a genome edited plant cell. copending application ‘773 claims 1 recite wherein one or more gRNAs are introduced with double stranded oligonucleotides (i.e. template) lacking homology arms (step C), wherein the oligonucleotide is inserted by NHEJ (last step).
Copending application ‘773 does not expressly teach introducing second gRNA targeting a different genomic site to create an insertion, deletion or substitution and the method comprise targeting first gene at promoter region.
Shi et al. teaches modification promoter region of an endogenous ARGOS8 gene to attain desired expression patterns and eliminate the need for a transgene (page 107, lines 19-21). Shi et al. teaches introducing guide RNAs and donor template into the maize embryo cells where multiple promoter replacement events to replace the native promoter of Zm-ARGOS8 with Zm-GOS2 PRO:GOS2 INTRON which were identified by PCR screening (page 109, lines 1-14) and a donor template comprising Zm-GOS2 promoter leads to promoter swap (i.e. replacement) or GOS2 promoter insertion in maize (page 109, Table 2).
Regarding claims 5 and 10, copending application ‘773 and Shi et al. does not teach the method to include introducing Cas nuclease as a RNP complexed with first gRNA.
Svitashev et al. teaches creating RNP complex using a gRNA and Cas9 ribonucleoprotein complex to edit maize plant cell genome (page 5, left, second to last paragraph). Svitashev et al. teaches the method including RNP complex improves the genome editing frequency (page1, Abstract).
Therefore, someone skilled in the art would use the RNP complex in their method of genome editing in maize plant. Furthermore, someone skilled in the art would use modification of one site as RNP complex and other as a polynucleotide encoding the protein as taught by copending Application ‘773 and Shi et al.
Regarding claim 6, Shi et al. Figure 1 C teaches a polynucleotide comprising a sequence encoding a Cas9 and sequence that express guide RNA.
Regarding claim 7, Shi et al. teaches different types of endonucleases for example Type I, Type II, Type Ill, and Type IV endonucleases, which further include subtypes wherein Cas9 is type II system (page 23, lines 16-21, page 94, lines 14-19).
Regarding claim 9, Shi et al. teaches the Cas9 endonuclease expression cassette are co-transferred into maize embryo with different gRNAs (page 96, lines 1—25).
Regarding claim 18, Shi et al. teaches their donor template has homology arms (Figures 4, 5 and 9) (page 9, lines 10-15).
Regarding claim 21, Yoshimi et al. teaches targeting mice and rat cells using CRISPR-Cas9 wherein the method comprises using a donor template with Cas9 and two single guide RNAs, one designed to cut the targeted genome sequences and the other to cut both the flanked genomic region and one homology arm of the dsDNA plasmid, which resulted in 20–33% KI efficiency among G0 pups (page 277, Abstract).
Yoshimi et al. teaches G0 KI mice carried NHEJ-dependent indel mutations at one targeting site of the genome wherein the HDR-dependent precise knock-ins (KIs) of the various donor cassettes spanning from 1 to 5 kbp such as EGFP, mCherry, Cre, and genes of interest, at the other exon site (page 277, Abstract).
Yoshimi et al. teaches genotyping analysis for PCR and sequence analysis (page 284) which found the mice carrying indel mutation at the sgRNA-2 targeting site and precisely repaired via HDR between the sgRNA-1 targeting genome (page280, right paragraphs 1-2).
This is a provisional nonstatutory double patenting rejection.
Response to Arguments
Applicants’ arguments filed 06/15/2026 have been fully considered but they are not persuasive.
Applicant argues claims of the present application are patentably distinct from the claims of the '773 application since the copending application does not teach or suggest the specific combination of features including editing at two different genes and achieving different editing outcomes at those genes, as required by the amended claims (Applicant Arguments, page 19, first paragraph).
Applicant argues Shi et al. and Svitashev et al. does not teach the recited arrangements (Applicant Arguments, page 19, paragraph 2).
Applicants’ arguments have been fully considered but they are not persuasive since copending application '773 claims 1 recite wherein one or more gRNAs are introduced with double stranded oligonucleotides (i.e. template) lacking homology arms (step C), wherein the oligonucleotide is inserted by NHEJ (last step). Creating another mutation in another gene and creating insertion in
Summary
No claim is allowed.
Applicants’ amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/SANTOSH SHARMA/ Examiner, Art Unit 1663
/DAVID H KRUSE/Primary Examiner, Art Unit 1663
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