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 .
Claims Status
Claims 21-77 is/are cancelled. Claims 1-20 is/are currently pending. Claims 1-20 is/are under examination.
Claim Rejections - 35 USC § 112
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.
Claim 19 is 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.
Claim 19 recites a list of agents present in a media which selects against expression of URA3 (claim 18, on which claim 19 depends, requires that “the protein that complements the auxotrophy for the nutrient” is URA3 from K. lactis). Claim 19 includes, but does not require, the agent 5-FOA. 5-FOA is the only agent known to select against URA3 expression (see Gnugge, 2017, Table I). It is unclear, based on the art, how media comprising alpha-aminoadipate, canavanine, fluoroacetamide, 5-fluorocytosine, D-histidine, antifolate media, or 5-fluoroanthranilic acid and not 5-FOA could select against URA3 expression, as required by claim 19. As such, the metes and bounds of claim 19 are indefinite.
112(a):
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.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
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 of carrying out his invention.
Written Description:
Claims 1-2 and 5-20 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 applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
To satisfy the written description requirement, a patent specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention. See, e.g., Moba, B.V, v. Diamond Automation, Inc., 325 F.3d 1306, 1319, 66 USPQ2d 1429, 1438 (Fed. Cir. 2003); Vas-Cath, Inc. v. Mahurkar, 935 F.2d at 1563, 19 USPQ2d at 1116. Possession may be shown in a variety of ways including description of an actual reduction to practice, or by showing that the invention was "ready for patenting" such as by the disclosure of drawings or structural chemical formulas that show that the invention was complete, or by describing distinguishing identifying characteristics sufficient to show that the applicant was in possession of the claimed invention. See, e.g., Pfaff v. Wells Eiees., Inc., 525 U.S. 55, 68, 119 S.Ct. 304, 312, 48 USPQ2d 1641,1647 (1998); Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406; Amgen, Inc. v. Chugai Pharm., 927 F. 2d 1200, 1206, 18 USPQ2d 1016, 1021 (Fed. Cir. 1991) (one must define a compound by "whatever characteristics sufficiently distinguish it”).
Claim 1 encompasses a “population of gene-edited cells” wherein the cells are of any organism (not limited to fungal or bacterial organisms). Claims 2 and 5-6 claim a bacterial cell in the method of claim 1, and claims 7-20 depend on claim 1 and read on the breadth of claim 1, encompassing a “population of gene-edited cells” wherein the cells are bacterial cells. However, the disclosure only sufficiently describes “a population of gene-edited cells” wherein the cells are fungal cells.
The specification and drawings disclose embodiments of the claimed methods using fungal cells (see Figs. 1-4C; paragraphs [272]-[292]). While the specification does disclose that “looping out” deletion techniques can be used in bacteria (paragraph [141]), and does disclose dominant selectable markers for use in bacteria (paragraph [159], “plasmids contain as a selectable marker the bacterial drug resistance marker AMPr or BLA gene”), the specification does not disclose bacterial genes required for prototrophy for a nutrient which can be selected for and against. A search of the art does not reveal bacterial genes used in the art to modulate prototrophy for a nutrient and which could be selected for and against (see Elander, 1979, and Dong, 2014: Dong page 3 teaches that deletion of both pyrR and pyrF in G. kaustophilus can result in auxotrophy for uracil and resistance to 5-fluoroorotate, but neither Elander nor Dong teach a single gene which is responsible for prototrophy of a nutrient and can be both selected for and against). The disclosure does not provide any description of use of the claimed methods in cells which are neither bacterial nor fungal, and as such, it cannot be determined whether the applicants were in possession of any embodiment of the claimed invention used in non-fungal cells. The art does describe genes required for nutrient prototrophy whose expression can be both selected for and against in yeast (see Gnugge, 2017). Based on the state of the art and the lack of description in the disclosure of elements of the invention which were not known in the art (specifically regarding applications in bacteria), an artisan would not be able to determine that the applicants were in possession of claimed embodiments of the claimed methods wherein the claimed methods produce genetically modified bacteria or other non-fungal cells. An artisan would only be able to determine that the applicants were in possession of the claimed methods used in fungal cells.
Scope of Enablement:
Claims 1-2 and 5-20 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for the claimed methods used to create genetically-modified fungal cells, does not reasonably provide enablement for the claimed methods used to create genetically-modified bacterial cells. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
The factors to be considered in determining whether a disclosure would require undue experimentation include:
A) The breadth of the claims;
(B) The nature of the invention;
(C) The state of the prior art;
(D) The level of one of ordinary skill;
(E) The level of predictability in the art;
(F) The amount of direction provided by the inventor;
(G) The existence of working examples; and
(H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure.
In re Wands, 8 USPQ2d, 1400 (CAFC 1988) and MPEP 2164.01.
The breadth of the claims:
With respect to claim breadth, the standard under 35 U.S.C. §112, first paragraph, entails the determination of what the claims recite and what the claims mean as a whole. As such, the broadest reasonable interpretation of the claimed method is that every claimed methodological step, structural components required to enact these methodological steps, and structural components resulting from the methodological steps can be found, used, and/or produced in bacterial cells. This includes, specifically, a single gene responsible for prototrophy of a nutrient, whose expression can be selected for and against. A skilled artisan would not know how to use the method with a reasonable expectation of success based solely on what is disclosed in the specification.
The amount of direction provided by the inventor and the level of predictability in the art:
The specification and drawings disclose embodiments of the claimed methods using fungal cells which encompass all of the claimed elements (see Figs. 1-4C; paragraphs [272]-[292]). Regarding the claimed methods as applied to bacterial cells, the specification teaches that “looping out” deletion techniques can be used in bacteria (paragraph [141]), and does disclose dominant selectable markers for use in bacteria (paragraph [159], “plasmids contain as a selectable marker the bacterial drug resistance marker AMPr or BLA gene”). However, the specification does not disclose bacterial genes required for prototrophy for a nutrient which can be selected for and against. The art at the time of filing does not provide enabling guidance for a single bacterial gene required for prototrophy for a nutrient which can be selected for and against; the closest approximation taught by the art is a dual-knockout of pyrR and pyrF in G. kaustophilus which results in auxotrophy for uracil and resistance to 5-fluoroorotate (see Dong, page 3). Claim 1 requires that the target gene locus for integration is “required for prototrophy for the nutrient”, that the prototrophy of that nutrient (and thus expression of that gene required for prototrophy) be selected for (see claim 1 part (c)), and that expression of the gene required for prototrophy be selected against (see claim 1 part (e)). Therefore, a bacterial gene fulfilling those requirements is required in order to enable the claimed methods using bacterial cells. However, the art does not teach such genes, and the present disclosure does not provide examples of such genes (see Dong, 2014, and Elander, 1979). The specification as filed does not provide guidance that overcomes this unpredictability within the art.
The existence of working examples:
What is enabled by the working examples is narrow in comparison to the breadth of the claims: The specification discloses working examples of the claimed methods, encompassing all required method steps and structural requirements, using fungal cells (see paragraphs [272]-[292])..
The quantity of experimentation needed to make or use the invention:
The standard of an enabling disclosure is not the ability to make and test if the invention works but one of the ability to make and use with a reasonable expectation of success. A patent is granted for a completed invention, not the general suggestion of an idea (MPEP 2164.03 and Chiron Corp. v. Genentech Inc., 363 F.3d 1247, 1254, 70 USPQ2d 1321, 1325-26 (Fed. Cir. 2004). The instant specification is not enabling because one cannot follow the guidance presented therein, or within the art at the time of filing, and practice the claimed method without first making a substantial inventive contribution. Given that the nature of the invention requires the identification and modification of a single gene responsible for prototrophy of a nutrient and whose expression can be selected for and against, a person having ordinary skill in the art would have to perform extensive analysis of the genomes of a number of bacterial species representative of the known populations of bacteria and further experimentation to identify one or more genes which each individually fulfill these functional requirements, in order to demonstrate the invention could be used with a reasonable expectation of success in bacteria. The amount of experimentation required for enabling guidance, commensurate in scope with what is claimed, goes beyond what is considered ‘routine' within the art, and constitutes undue further experimentation in order to use the method with a reasonable expectation of successfully treating any CNS disorder or neurodegenerative disease. Therefore, Claims 1-2 and 5-20 are rejected under 35 U.S.C. 112, first paragraph, for failing to meet the enablement requirement.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
Claim(s) 1-4, 7-13, 16-20 is/are rejected under 35 U.S.C. 103 as being unpatentable over Nguyen (2017), in view of Mans (2018), Mans (2015), Gnugge (2017), Walther (2003), Rose (1996), Voth (2003).
Regarding claim 1, Nguyen teaches a method for producing a population of gene-edited cells free of some or all gene-editing system molecules, comprising:
introducing an integrating nucleic acid construct into a population of cells that comprise a target gene of interest and that are prototrophic for a nutrient (Fig. 2: the cells are prototrophic for the nutrient leucine because of expression of the gene LEU2), wherein the integrating nucleic acid construct integrates into a gene that is required for prototrophy for the nutrient (Fig. 2: the nucleic acid is integrated into the LEU2 locus), and wherein the integrating nucleic acid construct comprises a first sequence encoding a gene-editing protein (Cas9, see Fig. 2), a second sequence encoding a dominant selectable marker (NAT, selected for using nourseothricin; Fig. 2 and page 2), and a pair of repeat nucleotide sequences flanking the first and second sequences (Fig. 2: the description of Fig. 2A states that the direct repeats are the “EU” portions of “LEU…” and “…EU2” in the construct);
co-introducing a non-integrating plasmid comprising a third sequence encoding a gene-editing nucleic acid (gRNA) that introduces an edit into the gene of interest (page 3, Supp. Fig. S1: soCPEC expression system);
selecting for expression of the dominant selectable marker to produce a population of cells that are auxotrophic for the nutrient (page 9, Materials and Methods);
removing the integrating nucleic acid construct from the population of cells by growing the cells on media that selects for prototrophy for the nutrient (leucine) to produce a population of cells that comprise the edited gene of interest and that are free of the integrating nucleic acid construct (page 9, Materials and Methods).
Regarding claim 2, Nguyen teaches that the method is performed in fungal cells (see Title).
Regarding claims 7-11, Nguyen teaches that the gene-editing protein is Cas9 (Fig. 2).
Regarding claim 12, Nguyen teaches that the gene-editing nucleic acid is a guide RNA (page 3, Supp. Fig. S1).
Regarding claim 13, Nguyen teaches that the gRNA is an sgRNA (see page 9 “single guide”).
Regarding claim 16, Nguyen teaches that the dominant selectable marker is nourseothricin N-acetyl transferase (NAT) (Fig. 2; page 2).
While Nguyen does teach that the method comprises introducing a non-integrating plasmid comprising a gRNA in the described “soCPEC” system, Nguyen does not teach that the introduction of this separate gRNA-containing plasmid occurs after selection with the dominant selectable marker of part (b) or that the separate gRNA-containing non-integrating plasmid comprises a fourth nucleotide sequence encoding a protein that complements the auxotrophy for the nutrient.
Mans (2018) teaches a method for removing gRNA-encoding plasmids from S. cerevisiae cells engineered to express Cas9. Mans (2015) is used herein to describe further the genetically-engineered S. cerevisiae cells, as Mans (2018) uses the S. cerevisiae cells described in Mans (2015).
Regarding claim 1, Mans (2018) teaches a yeast cell (S. cerevisiae) which is engineered such that a Cas9-coding sequence is integrated into the genome (Table 1). Mans (2018) teaches a method of introducing a non-integrating plasmid comprising a gRNA-encoding sequence and a sequence encoding a protein that complements auxotrophy for a nutrient in the cell (URA3, complementing auxotrophy for uridine: Table 2, in particular plasmids pROS10 and pMEL10, encoding URA3 and KlURA3, respectively). Mans (2018) also teaches that the Cas9 sequence and natNT2 (nourseothricin N-acetyl transferase) are integrated into the CAN1 gene (page 2). As taught by Mans (2015), these cells are selected for by providing media containing nourseothricin and canavanine (page 2, a break in CAN1 confers resistance to canavanine; pages 3-4, selecting for transformed cells with nourseothricin). It would have been obvious to an artisan at the time of filing that the S. cerevisiae strains should be continuously grown on nourseothricin and canavanine-containing media in order to prevent spontaneous elimination or migration of the spCas9-natNT2 cassette. Mans (2018) also teaches that the gRNA-encoding plasmids should be eliminated by growing the plasmid-bearing yeast strain in media which selects against expression of the protein that complements auxotrophy (pages 11-12: on page 12, see “10: Low efficiency plasmid removal”). Mans (2018) further teaches that the spCas9-natNT2 integrated expression cassette could be removed by targeting gRNA to sequences flanking the integrated expression cassette (Table 3).
Regarding claims 3-4, Mans teaches that the cells are Saccharomyces cerevisiae (Title).
Regarding claim 17, Mans (2018) teaches that the gene required for prototrophy is CAN1 (page 2). Mans (2018) also teaches that the cells are auxotrophic for uracil (IMX581, IMX664, IMX672, and IMX673, shown in Table 1, have the genotype ura3—52, which results in an auxotrophy for uracil, as URA3 is required for uracil production).
Regarding claim 18, Mans (2018) teaches that the protein that complements the auxotrophy for a nutrient (uracil) is KlURA3 (see Table 2: plasmid pMEL10).
Regarding claim 19, Mans (2018) teaches that the gRNA-encoding plasmids should be eliminated by growing the plasmid-bearing yeast strain in media which selects against expression of the protein that complements auxotrophy (pages 11-12: on page 12, see “10: Low efficiency plasmid removal”). While Mans (2018) does not recite the counterselection agent for URA3 (page 12 teaches that plasmids comprising URA3 can be removed by counterselection), Gnugge teaches that 5-FOA was known in the art to counterselect URA3 (Table 1 of Gnugge).
Regarding claim 20, Mans (2018) teaches cells which are auxotrophic for uracil (ura3—52), arginine (can1Δ), and/or tryptophan (trp1—289) (Table 1).
While the cells of Mans are S. cerevisiae, and not C. albicans as in Nguyen, it would have been obvious that the methods of Nguyen could be used in S. cerevisiae. Both Mans and Nguyen use homologous recombination to integrate a Cas9-NAT expression cassette into a target gene locus. Because these similar methods are used effectively in C. albicans and S. cerevisiae, it would have been obvious that the Cas9-NAT expression cassette of Nguyen could be used in the yeast species of Mans, and vice versa. Regarding methods of introducing foreign DNA into yeast cells, Walther teaches that methods used for “transformation of…C. albicans rely on established methods for the yeast Saccharomyces cerevisiae” (Abstract). It therefore would have been further obvious that the methods of modifying a C. albicans yeast cell of Nguyen could be used in the S. cerevisiae cells of Mans, with well-established methodological modifications made as needed (see Abstract of Walther: specific alterations of methodologies were known in the art which would allow for methods using C. albicans to be used with S. cerevisiae, and vice versa).
Moreover, it was known in the art that “loop out” systems (used in Nguyen to scarlessly remove the integrated sequence through recombination of direct repeat sequences) could be used effectively in S. cerevisiae (see Rose, 1996, page 670). It was also known in the art that multiple genes required for nutrient prototrophy could be selected for and against in yeast systems of gene regulation (see Gnugge, 2017, Table 1, which teaches that URA3, TRP1, LYS2, and MET15 were known auxotrophic marker genes with known agents for selection and counterselection). It was also known in the art that, in S. cerevisiae, a URA3 gene could be disrupted through insertion of another sequence (and selected for this disruption using 5-FOA), and subsequently restored through “looping out” of the inserted sequence (and selected for this “looping out” with uracil-deficient growth media (see Voth, 2003, which teaches multiple disruptions of URA3 (ura3::…), and teaches that disruption of URA3 allows for determination of target locus-specific integration and recombination-mediated excision, see Fig.1, Table 1, page 989; see also Gnugge, URA3 was known in the art to be a selectable and counterselectable marker). It thus would have been obvious to an artisan that the C. albicans “loop out” system of Nguyen could be modified such that the cell was S. cerevisiae and the target integration locus was any selectable or counterselectable prototrophic or auxotrophic marker in S. cerevisiae (or C. albicans, if used), such as URA3.
It would additionally be obvious to modify the methods of Nguyen to use non-integrating gRNA-encoding plasmids as in Mans (2018). Nguyen teaches a “soCPEC” “LEUpOUT” wherein the Cas9-NAT cassette (additionally comprising a first gRNA) is introduced in a first plasmid, and co-introduced with a second plasmid encoding a second, non-integrating gRNA, as described above. However, while Nguyen teaches that the non-integrating gRNA plasmid comprises a bacterial selection gene (carbenicillin resistance, see supplemental methods), Nguyen does not teach that the non-integrating soCPEC gRNA plasmid comprises a yeast selection gene. As yeast selection genes were well-known in the art, it would have been obvious to an artisan to add a yeast selection gene to the soCPEC gRNA plasmid in order to select for cells transfected with the plasmid. This would be beneficial to the method of Nguyen, as the method taught by Nguyen can only determine whether the Cas9-NAT integration cassette is present in the cell and whether the Cas9-NAT integration cassette has been removed from the cell. A mechanism to select for cells which have been transfected with the non-integrating gRNA plasmid would inherently select for and enrich a population of cells containing the genomic modification produced by the Cas9-gRNA complex. Mans (2015) and (2018) combined teach a method of producing and selecting for a ura3 (lacking URA3 function) yeast cell with a Cas9-NAT cassette integrated into an endogenous gene locus (as in Nguyen, with the exception of the ura3 genotype) and a second, subsequent method of transfecting these genetically-modified yeast cells with non-integrating plasmids encoding one or more gRNAs, wherein the non-integrating plasmids additionally encode a selectable prototrophy and/or auxotrophy marker (including KlURA3). Mans (2018) teaches that the selectable marker, KlURA3, is used to select for cells containing the gRNA plasmid (by removing uracil from the growth medium) and to select for cells which removed the gRNA plasmid (by adding 5-FOA to the growth medium). Given that the use of a non-integrating plasmid comprising the URA3 gene in a ura3 yeast cell enables for the selection of both presence and absence of the non-integrating plasmid, it would have been obvious to a person of ordinary skill in the art at the time of filing that the non-integrating gRNA plasmid of the “soCPEC” method of Nguyen should be modified to further comprise the URA3 gene.
As it would have been obvious to an artisan that the method of Nguyen should be modified to add a yeast selectable and counterselectable marker to the “soCPEC” gRNA plasmid of Nguyen, at least two possibilities would have been obvious solutions: the target integration gene locus and the selectable/counterselectable marker gene of the non-integrating plasmid could be different or the same. Using two different genes would allow for simultaneous transfection of the integrating nucleic acid and the non-integrating nucleic acid, with the selection agents used in tandem (for example, integration into the TRP1 locus and inserting the URA3 gene into the non-integrating nucleic acid). However, multiple reagents would need to be acquired and a variety of growth media with different combinations of selective agents would need to be made, incurring higher costs. If the target integration locus and the selection gene on the non-integrating plasmid were the same gene (e.g., integration into the URA3 locus and the non-integrating gRNA plasmid comprises the URA3 gene), only the selecting and counterselecting agents (e.g., uracil and 5-FOA for URA3) for that one gene would need to be obtained, and a much more limited number of types of growth media would need to be made, cutting costs. Such a system, however, would not allow for simultaneous co-transfection, as simultaneous co-transfection of the two plasmids would not enable selection for proper genomic integration of the integrating nucleic acid and for uptake of the non-integrating gRNA plasmid. However, a step-wise transfection, wherein the population of cells are first transfected with the integrating nucleic acid, enriched for cells with the integrated nucleic acid, and then transfected with the non-integrating gRNA plasmid, as in Mans (2018), allows the additional benefit of producing a population of cells which stably expresses Cas9 and is broadly applicable and modular; such cells could be used in a variety of methods of CRISPR-Cas genetic editing, as any gRNA sequence could be introduced, to target any sequence (as seen in Mans, 2018, which teaches that the Cas9-expressing yeast cells drawn from a single parent population could be transfected with a variety of different gRNA-encoding plasmids).
Given the extensive characterization of URA3 selection and counterselection systems in S. cerevisiae, it would have been obvious to a person of ordinary skill in the art that the spCas9-natNT2 expression cassette of Mans (2018) and (2015) could have been integrated into the URA3 gene locus instead of the CAN1 locus; such cells would exhibit the same ura3 phenotype as the cells described in Table 1 of Mans (2018). This would be beneficial, as URA3 expression can be selected for and against, while CAN1 expression can only be selected against with canavanine, allowing for the selection of cells in which spCas9-natNT2 has been properly integrated into the target locus (as in Mans 2015, using nourseothricin and canavanine) and for the selection of cells in which the spCas9-natNT2 expression cassette had been properly removed (as described in Table 3 of Mans 2018, gRNAs are used to target and excise the integrated Cas9-natNT2 sequence with no mechanism taught by Mans for definitively determining that the integrated sequence had been removed).
Moreover, it would have been obvious to modify Nguyen such that the target gene locus for insertion of the Cas9-NAT expression cassette was URA3 and not LEU2, as LEU2—like CAN1 of Mans—is not both selectable and counterselectable, while URA3 is. Integration of the expression cassette of Nguyen into the URA3 locus would thus allow for selection of cells with integration into the desired gene locus (in addition to selection for the presence of the cassette through nourseothricin selection media) and for selection of cells with proper excision of the integrated sequence—as a result of the inherent properties of LEU2, the methods of Nguyen as described can only be used to select for presence of the integrated sequence through nourseothricin selection and for excision of the integrated sequence through leucine-deficient media (Fig. 2). It would have been obvious to an artisan that such a modification of Nguyen was possible because the direct repeats necessary for the “loop out” system are not both endogenously present in the target gene locus. From the depiction in Fig. 2 of Nguyen, the upstream (US_LEU2) and downstream (DS_LEU2) sequences are used as homology arms for homologous recombination, replacing the native LEU2 locus with the expression cassette. The direct repeats “LEU…” and “…EU2” of the expression cassette recombine at the “EU” direct repeats, excising the intervening sequence and an “EU” repeat and restoring “LEU2”. Given the nomenclature used by Nguyen, it is clear that the functional gene is “LEU2” and not “LEUEU2”—if the direct repeats “EU” were both present in the native gene, “LEUEU2” would be the native gene. It is thus obvious that a direct repeat sequence, equivalent to “EU” of Nguyen, can be synthesized for any sequence. For example, to create a “LEUpOUT” system as in modified Nguyen wherein the target integration locus is URA3, based on the nomenclature used by Nguyen, an artisan would find obvious that the integration cassette would be designed such that the Cas9-NAT cassette was flanked by the direct repeats “URA…” and “…RA3”, wherein “URA…” and “…RA3” are homologous to the 5’ and the 3’ halves of the URA3 gene, respectively, just as “LEU…” and “…EU2” are homologous to the 5’ and the 3’ halves of the LEU2 gene in Nguyen, and wherein “URA…” and “…RA3” have homology at the “RA” sequence, just as the “LEU…” and “…EU2” sequences of Nguyen have homology at the “EU” sequence.
In conclusion, it would have been obvious to modify the non-integrating gRNA “soCPEC” plasmids of Nguyen to comprise the KlURA3 or URA3 selection gene of Mans (2018); it would have been obvious to modify the method of Nguyen such that the Cas9-NAT expression cassette was integrated in the yeast cell genome before transfection of the yeast cells with the modified non-integrating gRNA “soCPEC” plasmid; it would have been obvious that the methods of Nguyen could be used in S. cerevisiae; and it would have been obvious to modify Nguyen and Mans such that the target integration gene locus was URA3.
Claim(s) 14-15 is/are rejected under 35 U.S.C. 103 as being unpatentable over Nguyen (2017), Mans (2018), Mans (2015), Gnugge (2017), Walther (2003), Rose (1996), Voth (2003) as applied to claim 1 above, and further in view of Verruto (WO2016109840A9).
Nguyen, Mans, Gnugge, Walther, Rose, and Voth are described above and combined render obvious the limitations of claim 1. However, Nguyen and Mans do not teach that the CRISPR endonuclease integrated into the yeast cell genome is a Type I CRISPR Cas nuclease.
Verruto teaches that Type I CRISPR Cas nucleases can be used in Saccharomyces.
Regarding claims 14-15, Verruto teaches a method of generating a genome editing Saccharomyces cell line (claims 59 and 62), comprising introducing a nucleic acid encoding a Type I CRISPR Cas nuclease, including Cas3 and Cas10 (claim 40), into a Saccharomyces host cell and identifying and selecting Saccharomyces cells expressing the CRISPR/Cas gene editing system (claims 35-36).
As presented in Verruto, Cas3 and Cas10 are obvious alternatives and variants of Cas9 in methods requiring an RNA-guided endonuclease. It therefore would have been obvious to an artisan that the methods of Nguyen and Mans could be modified by replacing the Cas9-coding sequence with a Cas3- or Cas10-coding sequence in the integrating expression cassette.
Conclusion
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/AFRICA M MCLEOD/ Examiner, Art Unit 1635
/RAM R SHUKLA/ Supervisory Patent Examiner, Art Unit 1635