DETAILED CORRESPONDENCE
Application Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
2. Applicant’s amendment to the claims filed on 02/17/2026 in response to the Non-Final Rejection mailed on 11/14/2025 is acknowledged. This listing of claims replaces all prior listings of claims in the application.
3. Claims 1-26 are pending.
4. Claims 21-26 stand withdrawn pursuant to 37 CFR 1.142(b).
5. Applicant’s remarks filed on 02/17/2026 in response to the Non-Final Rejection mailed on 11/14/2025 have been fully considered and are deemed persuasive to overcome at least one of the rejections and/or objections as previously applied.
The text of those sections of Title 35 U.S. Code not included in the instant action can be found in the prior Office Action.
Drawings
5. The objection to the Drawings under 37 CFR 1.821 is withdrawn in view of the replacement drawings filed on 02/17/2026 to incorporate appropriate sequence identifiers.
Claim Rejections - 35 USC § 112(b)
6. The rejection of claim 14 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, for lack of antecedent basis is withdrawn in view of applicants’ amendment to the claims.
Claim Rejections - 35 USC § 103
7. The rejection of claims 1-2 and 4-20 under 35 U.S.C. 103 as being unpatentable over Bikard et al. (WO 2014/124226 A1; cited on IDS filed on 06/07/2022), Boyle et al. (WO 2015/148680 A1; cited on IDS filed on 06/07/2022), Zhang et al. (Genome Biology, 2017; cited on IDS filed on 06/07/2022) and Pyne et al. (Applied and Environmental Microbiology, 2015; cited on IDS filed on 06/07/2022) is maintained for the reasons of record and the reasons set forth below.
8. With respect to claim 1, Bikard et al. teach methods for selectively reducing the amount of antibiotic resistant and/or virulent bacteria in a mixed bacteria population comprising modifying a bacterial plasmid gene for antibiotic resistance with a Pro-Ag system comprising a first plasmid encoding a Cas9 protein and a second plasmid encoding a gRNA cassette comprising a promoter for constitutive expression of a gRNA having a sequence that hybridizes toa target genomic sequence on a target sequence in the bacteria wherein the target sequence in the bacteria confers antibiotic resistance [see Abstract; paragraphs 0004-0007].
With respect to claim 2, Bikard et al. teach the method wherein the copies of the gRNA cassette increase via a positive feedback loop allowing for self-amplification of the Pro-Ag system [see paragraph 0052].
With respect to claim 4, Bikard et al. teach wherein the bacterial gene for antibiotic resistance is a beta-lactamase gene [see paragraph 0039].
With respect to claim 6, Bikard et al. teach the method wherein the bacteria is E. coli [see paragraph 0033].
With respect to claim 9, Bikard et al. teach the method wherein a promoter drives constitutive expression of the gRNA [see paragraphs 0004-0007 and 0043].
With respect to claim 10, Bikard et al. teach the method wherein the bacteria is in a subject [see paragraphs 0007 and 0040].
With respect to claim 11, Bikard et al. teach the method wherein the bacteria is on the skin or a mucosal surface (interpreted as a solid surface or liquid) [see paragraphs 0047-0048].
With respect to claims 12-13, Bikard et al. teach the method wherein the second plasmid further comprises at least one cargo sequence such as GFP [see paragraph 0052].
With respect to claim 14, Bikard et al. teach the method wherein the at least one cargo sequence is not flanked by the first and second homology sequences [see paragraph 0055].
With respect to claim 15, Bikard et al. teach the method wherein the second plasmid comprises a dual Pro-AG system comprising a further gRNA cassette comprising a further promoter for constitutive expression of a further gRNA having a further sequence that hybridizes to a further target genomic sequence on the target plasmid in the bacteria [see Abstract; paragraphs 0004-0007; 0053].
With respect to claim 16, Bikard et al. teach the method wherein the second plasmid comprises a nested Pro-AG system comprising a further gRNA cassette comprising a further promoter for constitutive expression of a further gRNA having a further sequence that hybridizes to a further target genomic sequence on the target plasmid in the bacteria [see Abstract; paragraphs 0004-0007; 0053].
With respect to claim 17, Bikard et al. teach methods for selectively reducing the amount of antibiotic resistant and/or virulent bacteria in a mixed bacteria population comprising modifying a bacterial plasmid gene for antibiotic resistance with a Pro-Ag system comprising a first plasmid encoding a Cas9 protein and a second plasmid encoding a gRNA cassette comprising a promoter for constitutive expression of a gRNA having a sequence that hybridizes toa target genomic sequence on a target sequence in the bacteria wherein the target sequence in the bacteria confers antibiotic resistance [see Abstract; paragraphs 0004-0007].
With respect to claim 18, Bikard et al. teach methods for selectively reducing the amount of antibiotic resistant and/or virulent bacteria in a mixed bacteria population comprising modifying a bacterial plasmid gene for antibiotic resistance with a Pro-Ag system comprising a first plasmid encoding a Cas9 protein and a second plasmid encoding a gRNA cassette comprising a promoter for constitutive expression of a gRNA having a sequence that hybridizes toa target genomic sequence on a target sequence in the bacteria wherein the target sequence in the bacteria confers antibiotic resistance [see Abstract; paragraphs 0004-0007].
With respect to claim 19, Bikard et al. teach the method wherein the gene confers antibiotic resistance [see Abstract; paragraphs 0004-0007].
With respect to claim 20, Bikard et al. teach the method wherein the gene encodes a virulence factor [see paragraph 0038].
However, Bikard et al. does not teach the method of claim 1, wherein said Cas9 protein comprises an inducible Cas9 protein, and a first homology arm and a second homology each flanking opposite ends of the gRNA cassette in the second plasmid, wherein the first homology arm and the second homology arm have sequences that hybridize to respective sequences on opposite sides of a cut site for the Cas9 protein on the target genomic sequence; and (iii) an inducible lRed DNA repair cassette, wherein inducing expression of the Cas9 protein effects association of the Cas9 with the gRNA to form an endonuclease complex and cleavage of the target genomic sequence at the cut site, and inducing expression of the lRed DNA repair cassette effects integration of a copy of the gRNA cassette into the cut site by homology directed repair, thereby modifying the bacterial plasmid gene to inhibit antibiotic resistance; the method of claim 5, wherein the antibiotic is ampicillin or gentamicin; the method of claim 5, wherein the antibiotic is ampicillin or gentamicin; the method of claim 7, wherein the Cas9 protein is induced with anhydrotetracycline; the method of claim 8, wherein the lRed DNA repair cassette is induced with arabinose; the method of claim 9, wherein a Tet promoter drives constitutive expression of the gRNA; the method of claim 15, a further first homology arm and a further second homology arm each flanking opposite ends of the further gRNA cassette in the second plasmid, wherein the further first homology arm and the further second homology arm have sequences that hybridize to respective sequences on opposite sides of a cut site for the Cas9 protein on the further target genomic sequence; the method of claim 16, a further first homology arm outside the gRNA cassette on a side of the plasmid adjacent the second homology arm, and a further second homology arm outside the gRNA cassette on an opposite side of the plasmid adjacent the further gRNA, wherein the further first homology arm and the further second homology arm have sequences that hybridize to respective sequences on opposite sides of a cut site for the Cas9 protein on the further target genomic sequence; the method of claim 17, a further first homology arm and the further second homology arm have sequences that hybridize to respective sequences on opposite sides of a cut site for the Cas9 protein on the further target genomic sequence; and the method of claim 18, wherein said Cas9 protein comprises an inducible Cas9 protein, and a first homology arm and a second homology each flanking opposite ends of the gRNA cassette in the second plasmid, wherein the first homology arm and the second homology arm have sequences that hybridize to respective sequences on opposite sides of a cut site for the Cas9 protein on the target genomic sequence; and (iii) an inducible lRed DNA repair cassette, wherein inducing expression of the Cas9 protein effects association of the Cas9 with the gRNA to form an endonuclease complex and cleavage of the target genomic sequence at the cut site, and inducing expression of the lRed DNA repair cassette effects integration of a copy of the gRNA cassette into the cut site by homology directed repair, thereby inhibiting a bacterial plasmid gene.
Boyle et al. teach engineered genetic systems and methods to confer the ability to confer the ability to target and degrade undesirable nucleic acids in an organism using an inducible Cas9 protein wherein it is induced by tetracycline and its derivatives (anhydrotetracycline) and constitutive expression is driven by a Tet promoter [see paragraphs 0003, 0017, and 0060]. Boyle et al. further teach various antibiotic resistant transformants including ampicillin [see paragraph 0039]. It would have been obvious for one of ordinary skill in the art to substitute the beta lactams of Bikard et al. that uses penicillin with the ampicillin of Boyle et al. because ampicillin is an obvious variant of penicillin commonly used in biotechnology.
Zhang et al. teach methods for improving the efficiency of precise CRISPR/Cas9 mediated gene editing and homology directed repair knocking comprising using a first homology arm and a second homology arm each flanking opposite ends of a gRNA cassette in a second plasmid, wherein the first and second homology arms have sequences that hybridize to respective sequence on opposite sides of a cut site for the Cas9 protein [see p. 4, column 1, paragraph 2; p. 10, column 1, paragraph 1; p. 12, column 2, paragraph 2]. Zhang et al. teach that the improved targeting strategies are broadly applicable in generating precise knockin or reporter animals and human cell lines for basic research and disease modeling [see p. 13, column 2].
Pyne et al. teach that coupling traditional lRed recombineering with CRISPR/Cas9 system leads to simplified construction of markerless chromosomal gene replacement in E. coli [see p. 5113, column 1, paragraph 2]. Pyne et al. teaches an inducible lRed cassette using arabinose that provides a simpler, less labor intensive, and allows efficient production of gene replacement mutants [see Abstract; p. 5105, column 1].
Before the effective filing date of the claimed invention, it would have been obvious for one of ordinary skill in the art to combine the teachings of Bikard et al., Boyle et al., Zhang et al. and Pyne et al. to arrive at the claimed invention because Bikard et al. teach a methods for inhibiting antibiotic resistance in bacteria using Cas9/gRNA systems. Boyle et al. teach similar methods using inducible Cas9 proteins. Zhang et al. teach improving the efficiency of CRISPR-Cas9 gene editing through the introduction of flanking homology arms, and Pyne et al. teach that coupling traditional lRed recombineering with CRISPR/Cas9 system leads to simplified construction of markerless chromosomal gene replacement in E. coli. One of ordinary skill in the art would have had a reasonable expectation of success, a reasonable level of predictability, and would have been motivated to combine the teachings of Bikard et al., Boyle et al., Zhang et al. and Pyne et al. because Boyle et al. acknowledges that Cas9 can be controlled by an inducible promoter and one would desire to do so in order to control the timing of Cas9 expression for genetic modification. Zhang et al. acknowledges that first and second homology arms flanking the target site results in a two to five fold increase in HDR mediated modification, and Pyne et al. acknowledges that coupling traditional lRed recombineering with CRISPR/Cas9 system leads to simplified construction of markerless chromosomal gene replacement in E. coli. Therefore, the above invention would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention.
9. The rejection of claim 3 under 35 U.S.C. 103 as being unpatentable over Bikard et al. (WO 2014/124226 A1; cited on IDS filed on 06/07/2022), Boyle et al. (WO 2015/148680 A1; cited on IDS filed on 06/07/2022), Zhang et al. (Genome Biology, 2017; cited on IDS filed on 06/07/2022) and Pyne et al. (Applied and Environmental Microbiology, 2015; cited on IDS filed on 06/07/2022) as applied to claims 1-2 and 4-20 above, and further in view of Gill et al. (US Patent Application Publication 2017/0369870 A1; cited on PTO-892 mailed on 11/14/2025) is maintained for the reasons of record and the reasons set forth below.
10. The relevant teachings of Bikard et al., Boyle et al., Zhang et al. and Pyne et al. are set forth above.
However, the combination of Bikard et al., Boyle et al., Zhang et al. and Pyne et al. do not teach the method of claim 3, wherein the first plasmid encoding an inducible Cas9 protein is present in a low copy number than the second plasmid and the bacterial plasmid.
Gill et al. is in the field of genetic engineering using targeting nucleases such as Cas9, wherein the editing efficiencies of the Cas9 is increased by expressing the Cas9 in a low copy number plasmid [see paragraphs 0172-0173].
Before the effective filing date of the claimed invention, it would have been obvious for one of ordinary skill in the art to combine the teachings of Bikard et al., Boyle et al., Zhang et al., Pyne et al., and Gill et al. according to the teachings of Gill et al. to use a low copy plasmid for expressing the inducible Cas9 in the methods of Bikard et al., Boyle et al., Zhang et al. and Pyne et al. because Bikard et al., Boyle et al., Zhang et al. and Pyne et al. teach methods for inhibiting antibiotic resistance in bacteria using Cas9. Gill et al. teach the editing efficiencies of the Cas9 is 10ncreaseed by expressing the Cas9 in a low copy number plasmid. One of ordinary skill in the art would have had a reasonable expectation of success, a reasonable level of predictability, and would have been motivated to combine the teachings of Bikard et al., Boyle et al., Zhang et al., Pyne et al., and Gill et al. because Gill et al. acknowledges the editing efficiencies of the Cas9 is increased by expressing the Cas9 in a low copy number plasmid. Therefore, the above invention would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention.
Response to Remarks Regarding Prior Art Rejections
11. Beginning on p. 11 of applicants’ remarks, applicants in summary contend that the skilled artisan would have no motivation to combine Bikard with Zhang because Bikard does not concern homology directed repair because Bikard does not contemplate the insertion of genetic material at all and one would not arrive at the invention without impermissible hindsight bias. Furthermore, applicants contend that the systems of Bikard and Zhang differ so substantially because Bikard uses prokaryotic genomes and Zhang uses eukaryotic genomes and their systems differ in their methods of delivery. Applicants further contend that Bikard did not think to employ homology arms due to the failure of others to employ this technique to improve inactivation of antibiotic resistance genes.
These arguments are found to be not persuasive because it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971). Furthermore, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). In the instant case, as acknowledged by Zhang, up until the time of Zhang, the precise genome editing via HDR was relatively inefficient. However, Zhang et al. teach methods for improving the efficiency of precise CRISPR/Cas9 mediated gene editing and homology directed repair knocking comprising using a first homology arm and a second homology arm each flanking opposite ends of a gRNA cassette in a second plasmid, wherein the first and second homology arms have sequences that hybridize to respective sequence on opposite sides of a cut site for the Cas9 protein. This discovery led to more efficient targeting via HDR and Zhang acknowledges the broad applicability of the method to other cell types. As such, the level of skill of one of ordinary skill in the art of genetic engineering is high, and one of ordinary skill in the art would have a reasonable expectation of success and predictability to apply the methods to other cell types (i.e. prokaryote and eukaryote) via delivery systems suited for those types of cell types.
As stated above, Boyle et al. acknowledges that Cas9 can be controlled by an inducible promoter and one would desire to do so in order to control the timing of Cas9 expression for genetic modification. Zhang et al. acknowledges that first and second homology arms flanking the target site results in a two to five fold increase in HDR mediated modification, and Pyne et al. acknowledges that coupling traditional lRed recombineering with CRISPR/Cas9 system leads to simplified construction of markerless chromosomal gene replacement in E. coli. Accordingly, the invention would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention.
Conclusion
12. Status of the claims:
Claims 1-26 are pending.
Claims 21-26 stand withdrawn pursuant to 37 CFR 1.142(b).
Claims 1-20 are rejected.
No claims are in condition for an allowance.
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to PAUL J HOLLAND whose telephone number is (571)270-3537. The examiner can normally be reached Monday to Friday from 8AM to 5PM.
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/PAUL J HOLLAND/Primary Examiner, Art Unit 1656
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