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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.
Status of claims
The applicant’s response filed 8/11/2025 has been entered.
Claims 1-2 have been amended.
New claim 25 has been added.
Notes: On 5/6/2024, the applicant elected species:
Election 1: RNA guided nuclease,
Election 1a: Cas12a and Cas9.
Election 2: Single guide RNA (sgRNA).
In summary, claims 1-25 are pending and examined in the office action. Non-elected species are withdrawn.
Claim Rejections - 35 USC § 112
New Matter
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
Amended claims 1-2 and dependent claims 3-25 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claims 1 and 2 are amended to recite an element “immediately flanking”. The element is not in the original claims, nor disclosed and described in the specification. In fact, the word “immediately” or “immediate” does not appear anywhere in the specification.
In the art, “immediately flanking” refers to directly flanking. The element of directly flanking is not in the original claims, nor disclosed and described in the specification.
Such a claim is rejected on the ground that it recites elements without support in the original disclosure under 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph, Waldemar Link, GmbH & Co. v. Osteonics Corp., 32 F.3d 556, 559, 31 USPQ2d 1855, 1857 (Fed. Cir. 1994); In re Rasmussen, 650 F.2d 1212, 211 USPQ 323 (CCPA 1981). See MPEP § 2163.06 - § 2163.07(b) for a discussion of the relationship of new matter to 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph.
The applicant is required to either point to the basis of the limitation in the disclosure, or to delete the limitation in response to this Office action.
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.
Amended claims 1-2 and dependent claims 3-25 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 pre-AIA the applicant regards as the invention.
Note: since the preamble of the claim recites “a method for making a precise edit”, said “precise edit” is deemed a result of the method steps, and only exits after the method steps are performed.
Regarding claims 1 and 2
Each of the claims is amended to recite: “said precise edit comprising the sequence of the donor DNA” (step c).
However, each of the claims also recites: “wherein said donor DNA comprises said precise edit” (the 2nd wherein clause).
According to MPEP, the term "comprise" is an open-ended, inclusive term that means an invention includes the element listed, but is not limited to it (can include additional item(s)). Thus, “said precise edit comprising the sequence of the donor DNA” in step c means that said precise can include other item(s) in addition to the donor DNA sequence.
In this case, the “said donor DNA comprises said precise edit” in the 2nd wherein clause of each claim is contradicting to the “said precise edit comprising (the sequence of) the donor DNA” in step c. Thus, the claims are deemed indefinite.
Appropriate corrections and clarifications are required.
Dependent claims do not cure the deficiency, thus are included.
For compact prosecution, by BRI and definiteness (and not contradicting), in this office action only, the claims are interpreted that the 2nd wherein clause (the contradicting wherein clause) of each claim is not deemed valid and is not considered for merit, even though such wherein clause appears to recite structural limitations.
Claim 2 is additionally rejected for the following reason:
The claim recites “donor DNA in the preamble”, and “at least one donor DNA” in steps a and b. The claim also recites “the donor DNA molecule” (note by the examiner: not “a donor DNA molecule”) in step c, line 2 (and in the last line of the claim).
However, in view of the amended claim 1, “where said donor DNA molecule comprises the donor DNA”, “donor DNA molecule” and “donor DNA” clearly do not mean the same thing, and have different scopes and/or different structures --- “donor DNA molecule” comprises “donor DNA” and can also comprise other component(s); but “donor DNA” is a part of “donor DNA molecule”.
Thus, the “at least one donor DNA” in step a or step b does not serve as a proper antecedent base for the “the donor DNA molecule” in step c; there is insufficient antecedent basis for the “the donor DNA molecule”. Hence, the claim is deemed indefinite. See MPEP 7.34.05.
If the applicant intents that the “donor DNA molecule” means the “donor DNA”, it is suggested to delete the term “molecule” and recite “donor DNA” in all steps and wherein clause(s) in claim 2.
Appropriate correction and clarification are required.
For compact prosecution, from the context of the claim, by BRI and definiteness (proper antecedence base), in this office action only, the claim is interpreted that the “the donor DNA molecule” is the same as “the donor DNA”, in step c.
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 set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied 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 non-obviousness.
Claims 1-2, 4-7, 11-12, 15-18, 22-23 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al (Targeted mutagenesis using the Agrobacterium tumefaciens-mediated CRISPR-Cas9 system in common wheat. BMC Plant Biology, 1-12, 2018), in view of Yamamoto et al (Making ends meet: Targeted integration of DNA fragments by genome editing. Chromosoma. 127(4): 405–420, 2018), Filippova et al (Review. Guide RNA modification as a way to improve CRISPR/Cas9-based genome-editing systems. BioChimie. P49-60, published online 9/4/2019), and Upadhyay et al (RNA-Guided Genome Editing for Target Gene Mutations in Wheat. Genes/Genomics/Genetics, 3:2233-2238, 2013).
As analyzed above, claims 1-2 are deemed indefinite. For compact prosecution, the claims are interpreted as that the 2nd wherein clauses (the contradicting wherein clauses) are not valid, thus:
Amended claim 1 is drawn to a method comprising the steps of
a. introducing into a wheat cell
i. at least one donor DNA molecule, and
ii. at least one RNA guided nuclease or RNA guided nickase, and
iii. an RNA selected from the group consisting of:
1. at least one singleguideRNA (sgRNA); and
2. at least trans-activating CRISPR RNA (tracrRNA) and CRISPR RNA (crRNA), and
b. incubating the wheat cell to allow for introduction of said at least one donor DNA into said target region of the genome by homologous recombination, and
c. selecting a wheat cell comprising said precise edit in said target region, said precise edit comprising the sequence of the donor DNA in said target region,
wherein said donor DNA molecule comprises the donor DNA flanked by at least 30 bases that are each at least 80% identical to the sequence in the target region immediately flanking the insertion site of the donor DNA,
for making a precise edit in a target region of the genome of wheat through introduction of at least one donor DNA into said target region (preamble).
Note: “donor DNA molecule” can comprise “donor DNA” (solely comprise; or comprise additional element(s)).
Amended claim 2 is drawn to a method comprising the steps of
a. introducing into a wheat cell
i. at least one donor DNA, and
ii. at least one RNA guided nuclease or RNA guided nickase, and
iii. an RNA selected from the group consisting of:
1. at least one singleguideRNA (sgRNA); and
2. at least one tracrRNA and crRNA, and
b. incubating the wheat cell to allow for introduction of said at least one donor DNA into said target region of the genome by homologous recombination,
c. selecting a wheat cell comprising said precise edit in said target region. said precise edit comprising the sequence of the donor DNA in said target region, and
d. regenerating a wheat plant from said selected wheat cell,
wherein said donor DNA is flanked by at least 30 bases that are each at least 80% identical to the sequence in the target region immediately flanking the insertion site of the donor DNA,
for producing a wheat plant comprising a precise edit in a target region of its genome though introduction of donor DNA in said target region (preamble).
Note: since in claim 1, “donor DNA molecule” can comprise “donor DNA” with or without additional element(s)), and claim 1 does not recite any specific additional element(s). Thus, claim 2 recites essentially the same steps as claim 1 does, plus the step “d. regenerating a wheat plant from said selected wheat cell”.
According to the specification (p13, 3rd para), the terms "donor DNA molecule", "repair DNA molecule” or "template DNA molecule" all used interchangeably herein mean a DNA molecule having a sequence that is to be introduced into the genome of a cell. The specification does not define “donor DNA”. From the context of the claim 1, a “donor DNA” is comprised in the “donor DNA molecule” (the first wherein clause), and is a part or a whole part of the “donor DNA molecule”.
Zhang et al teach a method of introduction of donor DNA molecules into a target region of the genome of wheat, and producing a wheat plant comprising a donor DNA (inserted/introduced DNA) in the target region by using a CRISPR/Cas9 system. “CRISPR/Cas9 system has been widely used to precisely edit plant genomes”. Thus, the edit is considered precise (p1, Abstract).
Specifically, the method comprises introducing into a wheat cell/protoplast target genes for insertion or deletion/donor DNA molecule;
a gene encoding and expressing Cas9 (reading on the limitations of claims 1-2, 4-5, 15-16); and
sgRNAs (synthetic guide RNA, p7, fig 3)
(p1, abstract; p2, right col, Methods).
The wheat cells are incubated with the target genes/donor DNAs to allow the introduction of the target genes (p3, left col, 1st para).
The cells having the mutations (targeted insertions or deletions) are selected (by PCR, p3, left col, 2nd and 3rd paras, right col, 1st para). “CRISPR/Cas9 system has been widely used to precisely edit plant genomes” (p1, Abstract). Thus, the edit is considered precise.
A plasmid comprises an expression cassette comprising the genes/donor DNAs, the Cas9 encoding DNA sequence and the sgRNA encoding sequence is used for the introduction (p2, right col, 2nd para).
The target genes/donor DNAs are pre-selected for efficient targeting (p2, left col, 3rd para); and the sgRNAs are also pre-selected and designed to recognize the specific region of the coding sequence of the target genes for efficient targeting (p3, right col, last para, p4, left col, 1st para).
The site-specific introduction was successful, and the target genes are in the wheat genome, the efficiency was as high as 54.17% (Results in p3, right col, last para; p4-5, whole pages; p10, right col, last para).
Zhang et al further suggest that the method can be used for inserting transgenes (reading on donor DNA molecule, p7, left col, 1st para).
Zhang et al teach and demonstrated that the lines comprising the targeted mutants and transgenes were successfully regenerated (p5, left col, 1st para), teaching the step d. of claim 2. Thus, the producing of a wheat plant comprising the genes/donors in the genome was also successful.
Thus, Zhang et al teach and/or suggest the claim limitations, except
(1) are silent that the introduction of donor DNA is by homologous recombination (new limitation);
(2) are silent that wherein said donor DNA is flanked by at least 30 bases that are each at least 80% identical to the sequence in the target region immediately (new matter) flanking the insertion site of the donor DNA (newly modified limitation); and
(3) do not explicitly teach that the combination of at least one single guideRNA (sgRNA), and trans-activating CRISPR RNA (tracrRNA), and CRISPR RNA (crRNA).
Regarding (1) and (2), Zhang et al teach and demonstrated that “off-target mutations were not detected in regions that were highly homologous to the sgRNA sequences” (p1, Abstract). Thus, the process of Zhang et al at least comprise some part of homologous recombination. And Zhang et al teach a strong motivation to perform homologous recombination to avoid or reduce off-target mutations, and teach an advantage of homologous recombination.
Nevertheless, Yamamoto et al teach using Crispr-Cas9, sgRNA and donor DNA to editing genomes including plant genomes (p2233, Abstract; p2, 1st para; p4, 3rd para). Yamamoto et al teach methods of homologous recombination (p9, 1st para to p12, 3rd para).
Yamamoto et al particularly teach a method of homologous recombination with short homology arms, in which the arm length is 30-100 bases homology (reading on at least 30 bases) with the target DNA, and demonstrated that homologous recombination (HDR) can work with only 50 bases of homology. Yamamoto et al also teach that the advantage is to facilitate the introduction of the donor DNA, and increase the homology-directed repair (HDR) (p10, 2nd para to p11, 2nd para).
Note: any inserted sequence is a donor DNA sequence. Since both the donor DNA sequence and the target region of the wheat genome comprise the at least 30 base sequence that is homologous (or at least 80% identical), the target region and the donor DNA are immediately/directly flanking to each other.
Regarding (3), Filippova et al teach that the main components of the CRISPR/Cas9 system are CRISPR-Cas9 and guide RNA (gRNA) composed of two noncoding RNAs: a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) (p50, left col, 2nd para). Hence, since Zhang et al using CRISPR-Cas9, the gRNA of Zhang et al might comprise both a crRNA and a tracrRNA.
Upadhyay et al teach a method of using RNA-guided genome editing to make mutations in wheat (p2233, Title, Abstract). Specifically, Upadhyay et al teach that CRISPR RNA (crRNA) and trans activating CRIPR RNA (tracr RNA) had been used in other species (p2233, right col, 1st para), and teach using a complex comprising the combination of crRNA and tracr RNA as a chimeric guide RNA (cgRNA), along with Cas9, to make site specific mutations in target regions of wheat genome, and achieved success (Results in p2235, right col), including making site specific insertions in T. aestivum (common wheat) (p2236, left col, last para, right col, 1st para).
Yamamoto et al also suggest that crRNA/tracRNA can be combined into a single guide RNA (sgRNA) that targets Cas9 to its site of cleavage (p3, last para).
Regarding dependent claims, Zhang et al teach a plasmid comprising an expression cassette comprising genes/donor DNAs, Cas9 encoding DNA sequence, and the sgRNA encoding sequence, is used for the introduction (p2, right col, 2nd para), reading on the limitation of claims 6-7 and 17-18.
Zhang et al teach that the target genes/donor DNAs are pre-selected for efficient targeting (p2, left col, 3rd para); and the sgRNAs are also pre-selected and designed to recognize the specific region of the coding sequence of the target genes for efficient targeting (p3, right col, last para, p4, left col, 1st para), reading on the limitations of claims 11 and 22.
Zhang et al teach that Agrobacterium carrying the expression cassette is used for introduction/transformation (p3, left col, 1st para), reading on the limitations of claims 12 and 23.
An invention would have been obvious to one ordinary skill in the art if any teaching, suggestion or motivation in prior art leading the one to combine the teaching(s) or suggestion(s) of the cited references to arrive the claimed invention.
In this case, it would have been obvious for one ordinary skill in the art to modify the invention of Zhang et al, such that the method is/or include editing by homologous recombination as suggested by Zhang et al and taught by Yamamoto et al, and the donor DNA comprises an arm of at least 30 bases identical to a sequence in the target region also as taught by Yamamoto et al. One ordinary skill in the art would have been motivated to do so for the advantage of reducing off-target mutations as taught and demonstrated by Zhang et al, and facilitating introduction of donor DNA and increasing HDR efficiency, as taught by Yamamoto et al.
It also would have been obvious for one ordinary skill in the art to modify the invention of Zhang et al, such that the introduction of donor DNA of Zhang et al further comprises the combination of tracrRNA and crRNA as suggested by Yamamoto et al, and taught and demonstrated by Filippova et al and Upadhyay et al. One ordinary skill in the art would have been motivated to do so because Upadhyay et al had demonstrated success in making site specific insertion of donor DNA in wheat editing by using the combination.
The expectation of success would have been high because Zhang et al achieved success. The modifications have had demonstrated advantages, thus had been expected to improve the method of Zhang et al.
Therefore, the invention would have been obvious to one ordinary skill in the art.
Claims 3 and 14 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al in view of Filippova et al, Upadhyay et al and Yamamoto et al, as applied to claims 1-2 above, and further in view of Allen et al (CA 3033373, published 2/22/2018, filed 8/17/2018).
Claims 1-2 have been analyzed above.
Claims 3 and 14: after step b. the wheat cell is incubated on a medium comprising a selection agent.
Zhang et al in view of Filippova et al, Upadhyay et al and Yamamoto et al teach using PCR to select mutants/transformants, but do not teach using selection agent for the selection.
Allen et al teach a method comprising using Cas9 and single guide RNA to edit plants ([0063]). The plants include wheat ([0067]). Allen et al teach using a selection agent in a medium after editing/transformation to select mutants/transformants, and the advantage that the phenotype mutants/transformants are changed, for example, the mutants/transformants obtain resistance traits ([0153], [0214]). Allen et al also teach using PCR to select mutants/transformants ([213]).
In this case, it would have been prima facie obvious to one ordinary skill in the art to modify the invention rendered obvious by Zhang et al in view of Filippova et al, Upadhyay et al and Yamamoto et al, such that such that a selection agent is used as an alternative of PCR to select mutants as taught by Allen et al. One ordinary skill in the art would have been motivated to do so because if the mutants/transformants have resistance traits, the selection agents are able to kill the non-transformants, thus only the transformants survive, as taught by Allen et al. The expectation of success would have been high because selecting transformant by selection agents had been a mature method as taught by Allen et al, for example.
Therefore, the claims would have been obvious to one ordinary skill in the art.
Claims 9 and 20 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al in view of Filippova et al, Upadhyay et al and Yamamoto et al as applied to claims 1-2, 6 and 17 above, and further in view of Gao et al (WO 2018202199, published 11/8/2018, filed 5/7/2018).
Claims 1-2, 6 and 17 have been analyzed above.
Claims 9 and 20 depend on claims 6 and 17, wherein a polynucleotide sequence encoding the at least one nuclease is sequence optimized for expression in wheat.
Zhang et al in view of Filippova et al, Upadhyay et al and Yamamoto et al do not teach codon optimization.
Gao et al teach a method comprising using Cas9 to edit plants including wheat (Example 7 in p69, last para). Gao et al teach codon optimization and teach the optimized sequence of the DNA of interest for expression in cereal plants (p17, SEQ ID NO: 2; p18, SEQ ID NO: 14).
Gao et al teach the advantage that "Codon optimization" implies that the codon usage of a DNA or RNA is adapted to that of a cell or organism of interest to improve the transcription rate of said recombinant nucleic acid in the cell or organism of interest. The skilled person is well aware of the fact that a target nucleic acid can be modified at one position due to the codon degeneracy, whereas this modification will still lead to the same amino acid sequence at that position after translation, which is achieved by codon optimization to take into consideration the species-specific codon usage of a target cell or organism” (p29, 1st para).
It would have been prima facie obvious to one ordinary skill in the art to modify the invention rendered obvious by Zhang et al in view of Filippova et al, Upadhyay et al and Yamamoto et al, such that such that the coding sequence of the DNA of interest is optimized for the expression in the specific organism, as taught by Gao et al. One ordinary skill in the art would have been motivated to do so because the codon optimization improves the transcription rate of said recombinant nucleic acid in the cell or organism of interest, as taught by Gao et al. The expectation of success would have been high because codon optimization had been a mature method as taught by Gao et al, for example.
Therefore, the claims would have been obvious to one ordinary skill in the art.
Claims 10 and 21 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al in view of Yamamoto et al, Filippova et al, and Upadhyay et al, as applied to claims 1-2 above, and further in view of Liang et al (Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nature Communication. p1-5, 2016).
Claims 1-2 have been analyzed above.
Claims 10 and 21: wherein the at least one RNA guided nuclease and the at least one sgRNA are introduced into said cell as ribonucleoprotein (RNP) assembled outside said cell (Note: no donor DNA is in the RNP).
Zhang et al additionally suggest by citing references that Cas9 and guide RNA can be formed to a ribonucleoprotein complex (p10, left col, last para).
Thus, Zhang et al in view of Yamamoto et al, Filippova et al, and Upadhyay et al suggest but do not explicitly teach such a ribonucleoprotein complex.
Liang et al teach making a Cas9/sgRNA ribonucleoprotein (RNP) complex before the RNP is introduced to wheat cells by bombardment (p4, right col, last para; p5, left col, 1st para).
Liang et al further teach that “the most important advantage of CRISPR/Cas9 RNP mediated genome editing is the elimination of transgene integration and small DNA insertions in the mutants generated (p4, left col, 3rd para).
Liang et al achieved success of the DNA-free editing (p1, Abstract; p2-4, Results).
It would have been prima facie obvious to one ordinary skill in the art to modify the invention rendered obvious by Zhang et al in view of Yamamoto et al, Filippova et al, and Upadhyay et al, such that such that the nuclease and the sgRNA is formed to a RNP outside of the cells, and the RNP is delivered to the wheat cells, as suggested by Zhang et al and taught by Liang et al. One ordinary skill in the art would have been motivated to do so to take the advantage of the elimination of transgene integration and small DNA insertions in the mutants generated as taught by Liang et al. The expectation of success would have been high because such RNP had been made and delivered to wheat cells, and achieved successful DNA-free editing, as demonstrated by Liang et al.
Therefore, the claims would have been obvious to one ordinary skill in the art.
Claims 13 and 24 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang et in view of Yamamoto et al, Filippova et al, and Upadhyay et al as applied to claims 1-2 above, and further in view of Cigan et al (WO2017155714, published 9/14/2017, filed 2/27/2017).
Claims 1-2 have been analyzed above.
Claims 13 and 24: wherein the at least one RNA guided nuclease comprises a nuclear localization signal.
Zhang et al in view of Yamamoto et al, Filippova et al, and Upadhyay et al do not teach that the RNA guided nuclease (Cas9) comprises a nuclear localization signal.
Cigan et al teach a method comprising using Cas9 nucleases and single guide RNA (sgRNA) to edit plants including wheat (p2, 2nd para; p74, 6th para). Cigan et al suggest that the Cas9 nuclease can comprise a nuclear localization signal (p21, 2nd para), and teach making a Cas nuclease comprising a nuclear localization signal for a cereal plant (maize, Example 2 in p93).
Cigan et al teach that the nuclear localization signal (NLS) may drive accumulation of a Cas9 protein in a detectable amount in the nucleus where the editing occurs (p21, 2nd para), which is an advantage for the Cas editing.
It would have been obvious to one ordinary skill in the art to modify the invention rendered obvious by Zhang et al in view of Yamamoto et al, Filippova et al, and Upadhyay et al, such that such that the nuclease/Cas comprises a nuclear localization signal, as taught by Cigan et al. One ordinary skill in the art would have been motivated to do so that the nuclear localization signal drives the nuclease to the nucleus to facilitate the editing, as taught by Cigan et al. The expectation of success would have been reasonably high because such the mechanism and the method had been taught, and the sequences of nuclear localization signals have been made in a cereal plant.
Therefore, the claims would have been obvious to one ordinary skill in the art.
Claims 8 and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al in view of Yamamoto et al, Filippova et al, and Upadhyay et al, as applied to claims 1-2, 6 and 17 above, and further in view of HX Zhang et al (Genome Editing with mRNA Encoding ZFN, TALEN, and Cas9. Molecular Therapy Vol. 27, p735-746, 4/2019).
Claims 1-2, 6 and 17 have been analyzed above. Claims 6 and 17 recite that the at least one nuclease or the at least one single guide RNA (sgRNA, elected species) is introduced into said cell encoded by a nucleic acid molecule.
Claims 8 and 19 depend on claims 6 and 17, wherein the nucleic acid is an RNA molecule.
Zhang et al teach that the nuclease and sgRNA are encoded by a nucleic acid molecule, but Zhang et al in view of Yamamoto et al, Filippova et al, and Upadhyay et al do not teach “by an RNA molecule”.
HX Zhang et al teach that Cas9 is encoded by DNA in cell including plant cells (p735-p736, Principles of Genomic Editing).
HX Zhang et al also teach that Cas9 is encoded by mRNA in vitro (p735, Abstract; p737, right col, last para; p738, left col, 1st para). HX Zhang et al further teach and demonstrated that the in vitro mRNA encoded nucleases had produced high editing efficiency (p740, Table 3).
It would have been obvious to one ordinary skill in the art to modify the invention rendered obvious by Zhang et al in view of Yamamoto et al, Filippova et al, and Upadhyay et al, such that the nuclease/Cas are encoded by RNA molecules in vitro as an alternative of by DNA molecules in plant cells, as taught by HX Zhang et al. One ordinary skill in the art would have been motivated to do so because HX Zhang et al demonstrated that the mRNA encoded nucleases had produced high editing efficiency in vitro. The expectation of success would have been high because the in vitro mRNA encoded nucleases had been demonstrated and had produced high efficiency of editing by HX Zhang et al.
In addition, according to the specification (p10, 5th para), an RNA molecule may be merely an alternative to a DNA molecule for encoding a nuclease or a sgRNA. Thus, the applicant merely claims what the prior art had taught.
Therefore, the claims would have been obvious to one ordinary skill in the art.
New claim 25 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al in view of Yamamoto et al, Filippova et al, and Upadhyay et al, as applied to claims 2, and further in view of Cui et al (An optimised CRISPR/Cas9 protocol to create targeted mutations in homoeologous genes and an efficient genotyping protocol to identify edited events in wheat. BMC Plant Methods. p1-12, 2019.10). The PDF of the publish date is also attached.
Claim 2 has been analyzed above.
New claim 25 limits claim 2, wherein said wheat comprises three subgenomes, wherein said target region comprises a target gene has at least three homeologous copies in the wheat genome, and wherein one or more alleles of said target gene in one or more of said subgenomes comprise said precise edit.
Zhang et al in view of Yamamoto et al, Filippova et al, and Upadhyay et al teach claim 2 but do not teach the matter of claim 25.
Cui et al teach a method of using CRISPR-Cas9 and sgRNA to edit homoeologous genes of wheat (p1, Abstract).
Cui et al teach that wheat genome has 3 subgenomes (A, B and D) (p2, left col, 2nd para), and teach using the CRISPR-Cas9 and sgRNA to edit homoeologous genes in the 3 subgenomes, and achieved success (p2, left col, 3rd to 4th para; p8-12, Methods; p2, right col, 1st para to p5, right col, 1st para).
Specifically, Cui et al teach a detail method of identifying edit events of three homoeologous genes in the three subgenomes (p6, fig 2). Cui et al demonstrated different editing efficiency, some genes were edited with high efficiencies (p3, right col, 2nd para to p4, left col, 1st para).
Thus, it would have been obvious to one ordinary skill in the art to modify the invention rendered obvious by Zhang et al in view of Yamamoto et al, Filippova et al, and Upadhyay et al, such that the CRISPR-Cas and sgRNA system is used to target and edit all homeologous copies in the three wheat genomes as taught by Cui et al. One ordinary skill in the art would have been motivated to do so because Cui et al demonstrated success of different efficiencies in wheat. The expectation of success would have been high because Zhang et al achieved success, and Cui et al demonstrated success of different efficiencies in wheat, both in wheat. Thus, such modification would have been expected to be successful.
Therefore, the claim would have been obvious to one ordinary skill in the art.
Remarks
The following references were previously filed or relevant to instant application, thus, are filed but not cited by the examiner:
Zhang et al (CA 3121327, filed 4/12/2019). Zhang et al teaches using Cas nuclease and sgRNA to edit wheat cells, and suggest using Cas12a.
Brower-Toland et al (US 20180105819, published 4/19/2018, filed 10/18/2017).
Kudithipudi et al (WO 2020023864, filed 7/26/2019).
Response to Arguments
The applicant significantly amended independent claims 1-2, and added new claim 25. The applicant then argues that the cited references do not teach or suggest the amended claims.
In view of the amended claims, the examiner made 112 rejections for new matter and indefiniteness, as analyzed above. The examiner also significantly modifies or re-write the 103 rejections, and made a new rejection to new claim 25 citing a new reference, as analyzed above.
Thus, the arguments to the previous rejection are no longer applicable.
Conclusion
No claim is allowed.
Contact information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to WAYNE ZHONG whose telephone number is (571)270-0311. The examiner can normally be reached 8:30am to 5:00pm EST.
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/Wayne Zhong/
Primary Examiner, Art Unit 1662