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 1-10, 12, 14, 16-21, 23-25 and 43 are pending and are examined in this office action.
Claim Objections
Claim 4 is objected to because of the following informalities:
In claim 4 line 4, claim recite “polynucleotide of in step” which is grammatically incorrect, applicants are advised to change it to “polynucleotide of step” by deleting the term “in”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112 - Indefiniteness
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.
Claims 1-10, 12, 14, 16-21 and 23-25 are 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. All dependent claims are included in these rejections unless they include a limitation that overcomes the deficiencies of the parent claim.
Regarding claims 1, 25 and 43, claim recite “directed to” renders claim indefinite since it is not clear whether it means it recognize target sequence or is able to bind to the target sequence or it is sent to the target sequence so that the genome editing occurs. Therefore, the meets and bounds of the term “directed to” cannot be determined in the method of producing a genome edited plant cell.
Claims 1, 21 and 25, claim recite “second insertion does not comprise the donor DNA template polynucleotide or fragment thereof” renders claim indefinite since it is not clear in the claim how does the insertions in first genomic site differs from insertions from second genomic sites. For example, the fragment of a DNA template would comprise any fragment as small as single nucleotide. Thus, the insertion in first genomic site would be the same insertions in second genomic sites since a genomic sequence would only comprise G, C, T or A. Furthermore, it is not clear since a genomic sequence would only comprise G, C, T or A, how to know whether the insertions in second nucleotide does not comprise the any fragment of the any donor DNA template polynucleotide.
Regarding claim 9, claim ends in “optionally wherein a first Cas nuclease which recognizes the first gRNA and a second Cas nuclease recognizes the second gRNA.” It looks like the part of sentence is providing optional path of introduction in the method; however, it is not clear whether the claim is reciting about the Cas nuclease and its specific recognition of Cas nuclease or it is reciting about the steps where the Cas nuclease is introduced.
In claim 10 applicant recites “wherein the two distinct Cas nucleases comprise first and a second Cas nuclease” wherein the first is introduced as an RNP, since for second gRNA the sentence ends at “is introduced” it is not clear how this component is introduced. Is this introduced as expression cassette or RNP or any other method? Therefore, the metes and bound of the claim cannot be determined.
Inc claim 20 line 1, claim recites “method of any one of claim 1” renders claim indefinite since there is only one method in claim 1. Therefore, it is advised to delete “any one of”.
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)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Anticipated by Shi et al.
Claims 25 and 43 are rejected under 35 U.S.C. 102 (a)(1) and/or (a)(2) as being anticipated by Shi et al. (WIPO International Pub. No.: WO 2016/007948 Al, Pub. Date: 14 January 2016).
Claim 43 is directed to a kit comprising a first guide (gRNA) directed to a first genomic site and a donor DNA template and second gRNA directed to second genomic DNA site in a plant cell. Claim 25 is directed to the gRNAs and the DNA template are in a plant cell.
Regarding claim 43, Shi et al. discloses a guide RNA construct, gRNA3, was made for targeting the genomic target site CTS3 (SEQ ID NO:453) of maize plant cell, located 710-bp upstream of the ZmARGOSB. Another guide RNA, gRNA2, designed to target the genomic target site CTS2 (SEQ ID NO:452) located in the 5'-UTR of ZmARGOSO8 (Figure 9). Shi et al. discloses the polynucleotide modification template contained a 400-bp genomic DNA fragment derived from the upstream region of CTS3, Zm-GOS2 PRO:GOS2 INTRON and a 360-bp genomic DNA fragment derived from the downstream region of CTS2 (Figure 9) (page 109, lines 1-6).
Shi et al. discloses the gRNA3 and gRNA2, the Cas9 cassette, the polynucleotide modification template and the PMI selection marker were used to transform immature embryo cells wherein multiple promoter swap events were identified (page 109, lines 6-11).
Since applicant has not defined the term “kit”, the combination of the gRNA3 and gRNA2 along with the donor template that binds to CT3 and CTS2 genomic sites in maize plant cell anticipates the claim.
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.
Therefore Shi et al. anticipates the claims 25 and 43.
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 Shi et al.
Claims 1-4, 7-9, 12, 14, 16-19, 21 and 23- 24 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 Shi et al.
Claim are directed to a method of producing a genome edited plant cell comprising introducing a first guide (gRNA) directed to a first genomic site and a donor DNA template and second gRNA directed to second genomic DNA site 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.
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 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 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 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 1a nd 3 below).
Yoshimi et al. teaches introduction of the two sgRNA in mouse embryo (page283, last paragraph) and screening of survived embryo for the mutations using PCR and sequence analysis (page 284).
Yoshimi et al. does not teach the method was in plant cell.
Shi et al. teaches introducing guide RNAs targeting 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 taught by Shi et al. for more precise genome editing as taught by Yoshimi et al. by using two guide RNA and DNA template to cause insertion of template in one site and insertion, deletion and substitution on other 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 district 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, 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).
Therefore, it would have been obvious to select the insertions of the fragment of donor template insertions by NHEJ which would not require the DNA template to comprise homology arms complementary to DNA.
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 19, Yoshimi et al. teaches their sgRNA at first target site integrates KI cassette by HDR dependent method (page 281, left last paragraph, see figure above).
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).
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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).
Obvious over Yoshimi et al. and further in view of 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 a first genomic site and a donor DNA template and second gRNA directed to second genomic DNA site 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 targeting mice and rat cells using CRISPR-Cas9 wherein the method comprise 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 which resulted in 20–33% (Knock-ins) KI efficiency among G0 pups (page 277, Abstract).
Shi et al. teaches introducing guide RNAs targeting 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-ARGOSB with Zm-GOS2 PRO:GOS2 INTRON which were identified by PCR screening (page 109, lines 1-14).
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).
Obvious over Yoshimi et al. and further in view of Shi et al. and Cemak et al.
Claims 1 and 20 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 Cemak et al. (US Patent Application Publication No.: US 2020/0407754 A1, Publication date: Dec. 31 , 2020).
Claim are directed to a method of producing a genome edited plant cell comprising introducing a first guide (gRNA) directed to a first genomic site and a donor DNA template and second gRNA directed to second genomic DNA site 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 further drawn to the method includes the plant cell comprising homology dependent repair enhancing polypeptides.
Regarding claim 1, 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).
Shi et al. teaches introducing guide RNAs targeting 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-ARGOSB with Zm-GOS2 PRO:GOS2 INTRON which were identified by PCR screening (page 109, lines 1-14).
Regarding claim 20, Yoshimi et al. and Shi et al. does not teach their method includes the plant cell comprising homology dependent repair enhancing polypeptides.
Cemak et al. teaches method of increasing HDR mediated genome modification of a target editing site by using SSAP, exonuclease, single stranded DNA binding protein (SSB) (claim 174). Cemak et al. teaches significant increase in precise genome editing attributable to HDR, and a decrease in insertion and deletion indel) editing attributable to non - homologous end joining NHEJ) , as shown in Table 4A.
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 in its method of genome editing taught by Shi et al. for more precise genome editing as taught by Yoshimi et al. to yield predicable result of developing a method of producing a gene edited plant. Furthermore, someone skilled in the art would use the agents for HDR enhancing polypeptides such as SSAP, exonuclease, single stranded DNA binding protein (SSB) in the method of increasing HDR mediated genome modification which were found to increase the HDR as taught by Cemak et al.
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, 19-20 and 23-25 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 2, 4-5, 7, 9, 17-18 and 20 of copending Application No. 18703773 (Herein referenced as ‘773) in view of Yoshimi et al.
Regarding claims 1, 3, 4, 8 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 does not expressly teach introducing second gRNA targeting a different genomic site to create a insertion, deletion or substitution.
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).
Therefore, someone skilled in the art would introduce second gRNA targeting a different genomic site to create a insertion, deletion or substitution that would increase the KI efficiency in genome editing. The method would produce the plant cell comprising the gRNAs and the donor template.
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 9 teaches introducing in callus comprising plan cell.
Regarding claims 19-20, the copending Application ‘773 claim 17 teaches HDR enhancing polypeptide for example SSAP or SSB etc.
Regarding claim 23, the copending Application ‘773 claim 20 teaches regenerating plant from edited callus.
Regarding claim 24, the copending Application ‘773 claim 18 teaches the plant cell is maize plant cell.
Regarding claim 43, the development of kit comprising first and second gRNA targeting two genomic sites with insertion of donor template in one site and any insertion, deletion and substitution in other site would have been obvious over the requirement of such components in copending application ‘773 and further in view of Yoshimi et al.
Claims 1, 5-7, 9-10, 16-18 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) in view of Yoshimi et al. 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 does not expressly teach introducing second gRNA targeting a different genomic site to create a insertion, deletion or substitution.
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).
Regarding claims 5 and 10, copending application ‘773 and Yoshimi 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 a polynucleotide encoding the protein as taught by copending Application ‘773 and Yoshimi 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 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 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, 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).
Therefore, it would have been obvious to select the insertions of the fragment of donor template insertions by NHEJ which would not require the DNA template to comprise homology arms complementary to DNA.
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.
Summary
No claim is allowed.
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/SANTOSH SHARMA/ Examiner, Art Unit 1663
/DAVID H KRUSE/ Primary Examiner, Art Unit 1663