DETAILED ACTION
Response to Amendment
Applicant’s response to the office action filed on May 20, 2026 has been entered. The claims pending in this application are claims 1-6 and 10-20 wherein claims 10-12, 16, and 17 have been withdrawn due to the restriction requirement mailed on September 5, 2024. The objection not reiterated from the previous office action is hereby withdrawn in view of applicant’s amendment filed on May 20, 2026. Claims 1-6, 13-15, and 18-20 will be examined.
Claim Objections
The disclosure is objected to because of the following informality: “gRNA-CAS” in “a first gRNA-CAS complex” is an abbreviation. It can be used after the whole phrase representing the abbreviation appears once.
Appropriate correction is required.
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.
Claims 1, 2, 4-6, and 13-15 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Shuber et al., (US Patent No. 10,081,829 B1, priority date: June 13, 2017).
Regarding claims 1, 2, 4-6, and 13-15, Shuber et al., teach a method for enrichment of a target nucleic acid fragment from a sample comprising a nucleic acid molecule, wherein the target nucleic acid fragment comprises a sequence of interest, and wherein the method comprises the steps of: a) providing the sample comprising the nucleic acid molecule, wherein the nucleic acid molecule comprises the sequence of interest; b) generating cleaved nucleic acid molecules comprising the target nucleic acid fragment which is protected from exonuclease digestion by the simultaneous binding of both a first gRNA-CAS complex and a second gRNA-CAS complex (ie., a complex comprising a first Cas endonuclease and a first guide RNA and a complex comprising a second Cas endonuclease and a second guide RNA) at both 5’ and 3’ ends of the target nucleic acid fragment, and at least one non-target nucleic acid fragment that is not simultaneously bound by both the first RNA-CAS complex and the second gRNA-CAS complex, wherein the first gRNA-CAS and the second gRNA-CAS complexes remain bound to both 5’ and 3’ ends of the target fragment of the cleaved nucleic acid molecules after said cleaving the nucleic acid molecule using a first gRNA-CAS complex and a second gRNA-CAS complex, and both the first gRNA-CAS complex and the second gRNA-CAS complex are capable of inducing a double stranded break (DSB); c) contacting the cleaved nucleic acid molecules obtained in step b) with an exonuclease and allowing the exonuclease to digest the at least one non-target nucleic acid fragment; and d) optionally purifying the target nucleic acid fragment comprising the sequence of interest from the digest obtained in step c) as recited in claim 1 wherein the method does not comprise a further step of protecting the target nucleic acid fragment, or the ends of the target nucleic acid fragment (ie., by binding both the first gRNA-CAS complex and the second gRNA-CAS complex to ends of the target nucleic acid fragment), prior to exonuclease digestion in step c) as recited in claim 2, at least one of the first gRNA-CAS complex and the second gRNA-CAS complex comprises a Cas9 protein as recited in claim 4, the at least one of the first gRNA-CAS complex and the second gRNA-CAS complex comprises a single guide RNA (sgRNA) as recited in claim 5, at least one of the first gRNA-CAS complex and the second gRNA-CAS complex comprises a CRISPR RNA (crRNA) and a trans-activating
(tracrRNA) as separate molecules as recited in claim 6, the method is performed in parallel for multiple nucleic acid samples as recited in claim 13, the nucleic acid molecule is genomic DNA (ie., a target nucleic acid from the genome of a pathogen) as recited in claim 14, and the nucleic acid molecule is a nucleic acid molecule obtainable from a plant, animal, human or microorganism as recited in claim 15 (see columns 1-10, Figures 1-5 and claims 1-19).
Therefore, Shuber et al., teach all limitations recited in claims 1, 2, 4-6, and 13-15.
Response to Arguments
In page 7, second paragraph bridging to page 8, second paragraph of applicant’s remarks, applicant argues that “[I]nitially, Applicant notes that claim 1 has been amended to incorporate the limitations previously recited in claim 8. Specifically, claim 1 has been amended to specify that the first and second Cas nucleases are capable of inducing a double stranded break. Shuber is a hypothetical disclosure of using ‘binding proteins’, such as a Cas endonuclease, to protect the ends of the nucleic acid of interest, see column 1, lines 57 - 63 of Shuber. Shuber does not disclose any working examples. Shuber however makes clear that a catalytically inactive Cas endonuclease protein is envisioned, see e.g. column 3, line 25 and column 4, line 29 of Shuber:
‘The Cas endonuclease is catalytically inactive’ Shuber thus discloses that the protection requires binding of a catalytically inactive Cas protein. According to the Office Action, Shuber discloses that the Cas complexes that bind to the end of the target nucleic acid are ‘either catalytically inactive or catalytically active’. Applicant respectfully disagrees. The disclosure in Shuber that ‘The Cas complexes that bind to the ends of the target nucleic acid may be catalytically inactive’ is no unambiguous disclosure that both first and second gRNA-CAS complex are capable of inducing a double- stranded break (DSB) as recited in amended claim 1.
In the absence of any working examples, Shuber merely expresses the thought that the Cas9 complex may be catalytically inactive. There is no teaching or suggestion that Cas could, instead, be active. Even if a person of skill in the art would consider that this disclosure could apply to a ‘catalytically active’ Cas complex (quod non), it remains unclear which one of the first and the second complexes may not be catalytically inactive. In addition, in (the hypothetical) case that one of the complexes is not catalytically inactive, it further remains unclear whether the RuvC domain, the HNH domain or both domains of the Cas protein would then be a catalytically active domain. Applicant notes that the activity of both domains is required for generating a double-stranded break. Hence, Shuber at least does not disclose that: - The first gRNA-CAS complex is capable of inducing a double-stranded break; and - The second gRNA-CAS complex is capable of inducing a double-stranded break. The Office Action on page 5 suggests that Shuber ‘must’ disclose that the first gRNA-CAS complex and the second gRNA-CAS complex is capable of inducing a double- stranded break and refers to column 1-10, Figure 1-5 and claims 1-19 of Shuber. It is however not disclosed in Shuber that the binding proteins as referred to in column 1, lines 57 - 63 of Shuber, can cleave both strands. In contrast, Shuber discloses the use of catalytically inactive proteins, as outlined above. For at least these reasons, Applicant submits that the claims are novel of Shuber”.
The above arguments have been fully considered but they are not persuasive toward the withdrawal of the rejection. Although applicant argues that “[S]huber however makes clear that a catalytically inactive Cas endonuclease protein is envisioned, see e.g. column 3, line 25 and column 4, line 29 of Shuber: ‘The Cas endonuclease is catalytically inactive’ Shuber thus discloses that the protection requires binding of a catalytically inactive Cas protein”, since the specification teaches that “[T]ype II CRISPR-CAS systems include a signature Cas9 protein, a single protein (about 160KDa), capable of generating crRNA and specifically cleaving duplex DNA. The Cas9 protein typically contains two nuclease domains, a RuvC-like nuclease domain near the amino terminus and the HNH (or McrA-like) nuclease domain near the middle of the protein. Each nuclease domain of the Cas9 protein is specialized for cutting one strand of the double helix (Jinek et al, 2012, Science 337 (6096): 816-821). The Cas9 protein is an example of a CAS protein of the type II CRISPR/-CAS system and forms an endonuclease, when combined with the crRNA and a second RNA termed the trans-activating crRNA (tracrRNA), which targets the invading pathogen DNA for degradation by the introduction of DNA double strand breaks (DSBs) at the position in the pathogen genome defined by the crRNA” (see paragraph [0106] of US 2022/0033879 A1, which is US Publication of this instant case) while Shuber et al., teach that “[F]IG. 5 illustrates the method 101. A population 103 of nucleic acids 105a, 105b, including a nucleic acid of interest 107, is provided. The nucleic acids 105a, 105b include numerous target sequences 109a, 109b, and 109c for a set of Cas complexes 113a, 113b, 113c,
but the nucleic acid of interest does not contain a target sequence. The population 103 is exposed 111 to the set of Cas complexes 113a, 113b, and 113c, which are targeted to the various target sequences 109a, 109b, and 109c. The nucleic acids 105a, 105b are then digested 121 by the Cas complexes 113a, 113b, and 113c. Most nucleic acids 105a, 105b are digested into small
fragments, but the nucleic acid of interest 107, which was not targeted by a Cas complex, remains intact. The nucleic acid of interest 107 may then be detected by any suitable means. The Cas complexes include a Cas endonuclease and a guide RNA. For example, the Cas endonuclease may be Cas9, Cpf1, C2c1, C2c3, C2c2, CasX, or CasY, including sequence variants of Cas9, Cpf1, C2c1, C2c3, C2c2, CasX, or CasY. Preferably, the Cas endonuclease is Cas9. The Cas endonuclease may be from any bacterial species” (see column 10, second and third paragraph), the Cas endonucleases in the first gRNA-CAS complex and the second gRNA-CAS complex taught by Shuber et al., can be either catalytically inactive or catalytically inactive and Shuber et al., disclose that both the first gRNA-CAS complex and the second gRNA-CAS complex are capable of inducing a double stranded break (DSB) as recited in claim 1.
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.
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.
Claims 3, 18, and 20 are rejected under 35 U.S.C. 103 as being unpatentable over Shuber et al., as applied to claims 1, 2, 4-6, and 13-15 above, and further in view of Jinek et al., (Supplementary Material for Science, 337, 816-821, 2012, pages 1-37).
The teachings of Shuber et al., have been summarized previously, supra.
Shuber et al., do not disclose that at least one of i) step b) is performed by incubating the first gRNA-CAS complex and the second gRNA-CAS complex and the nucleic acid molecule together for about 1 min to about 18 hours at about 10-90 °C; and ii) step c) is performed by incubating the cleaved nucleic acid molecule with the exonuclease for about 1 minute to about 12 hours at about 10-90°C as recited in claim 3, step b) is performed by incubating the first gRNA-CAS complex and the second gRNA-CAS complex and the nucleic acid molecule together for about 60 minutes as recited in claim 18, and step b) and/or step c) is performed at about 37°C as recited in claim 20.
Jinek et al., (Supplementary Material) teach that a Cas 9 assay is performed at 37°C for 60 minutes (see pages 2 and 3).
Therefore, it would have been prima facie obvious to one having ordinary skill in the art at the time the invention was made to have performed the methods recited in claims 3, 18, and 20 by incubating the first gRNA-CAS complex and the second gRNA-CAS complex and the nucleic acid molecule together at 37°C for 60 minutes in view of the prior arts of Shuber et al., and Jinek et al., (Supplementary Material). One having ordinary skill in the art would have been motivated to do so because Shuber et al., teach that CAS in a gRNA-CAS complex can be Cas 9 (see column 3, third paragraph) and Jinek et al., (Supplementary Material) have successfully performed a Cas 9 assay at 37°C for 60 minutes (see pages 2 and 3). One having ordinary skill in the art at the time the invention was made would have a reasonable expectation of success to perform the methods recited in claims 3, 18, and 20 by incubating the first gRNA-CAS complex and the second gRNA-CAS complex and the nucleic acid molecule together at 37°C for 60 minutes wherein CAS in each of the first gRNA-CAS complex and the second gRNA-CAS complex is Cas 9 in view of the prior arts of Shuber et al., and Jinek et al., (Supplementary Material) in order to make the cleaved nucleic acid molecules of step c) of claim 1.
Response to Arguments
In page 8, fifth and sixth paragraphs of applicant’s remarks, applicant argues that “[T]he arguments related to Shuber are outlined above. In view of Shuber, a person of skill in the art would not reasonably expect that a gRNA-CAS complex that can introduce a double-stranded break would protect the end of the cleaved fragment, instead of dissociating after cleavage. These deficiencies are not remedied by Jinek. Accordingly Applicant submits that the claims are not obvious over Shuber in view of Jinek”.
The above arguments have been fully considered but they are not persuasive toward the withdrawal of the rejection because Shuber et al., teach that both the first gRNA-CAS complex and the second gRNA-CAS complex are capable of inducing a double stranded break (DSB) as recited in claim 1 since Shuber et al., disclose either catalytically inactive Cas endonuclease or catalytically active Cas endonuclease such as Cas9 (see above Response to Arguments related to the rejection under 35 USC 102 (a) (2)).
Claim 19 is rejected under 35 U.S.C. 103 as being unpatentable over Shuber et al., in view of Jinek et al., (Supplementary Material) as applied to claims 1-6, 13-15, 18, and 20 above, and further in view of Klein et al., (US 2016/0348164 A1, published on December 1, 2016).
The teachings of Shuber et al., and Jinek et al., (Supplementary Material) have been summarized previously, supra.
Shuber et al., and Jinek et al., (Supplementary Material) do not disclose that step c) is performed by incubating the cleaved nucleic acid molecule with the exonuclease for about 30 minutes as recited in claim 19.
Klein et al., teach that Exonuclease I digestion reaction is performed at 37°C for 30 minutes (see paragraph [0224]).
Therefore, it would have been prima facie obvious to one having ordinary skill in the art at the time the invention was made to have performed the method recited in claim 19 by incubating the cleaved nucleic acid with the exonuclease for about 30 minutes in view of the prior arts of Shuber et al., Jinek et al., (Supplementary Material), and Klein et al.. One having ordinary skill in the art would have been motivated to do so because Shuber et al., teach to incubate the cleaved nucleic acid molecule with exonuclease I (see column 7, last paragraph) while Klein et al., teach that Exonuclease I digestion reaction is performed at 37°C for 30 minutes (see paragraph [0224]). One having ordinary skill in the art at the time the invention was made would have a reasonable expectation of success to perform the method recited in claim 19 by incubating the cleaved nucleic acid with the exonuclease for about 30 minutes in view of the prior arts of Shuber et al., Jinek et al., (Supplementary Material), and Klein et al., in order to digest the at least one non-target nucleic acid fragment of the cleaved nucleic acid in step c) of claim 1.
Response to Arguments
In page 8, last paragraph bridging to page 9, first paragraph of applicant’s remarks, applicant argues that “[I]n view of the arguments presented above, Applicant submits that the claims are not obvious over Shuber in view of Jinek. The deficiencies of Shuber and Jinek are not remedied by Klein. Accordingly, Applicant submits that the claims are not obvious over Shuber in view of Jinek and Kelin”.
The above arguments have been fully considered but they are not persuasive toward the withdrawal of the rejection because Shuber et al., teach that both the first gRNA-CAS complex and the second gRNA-CAS complex are capable of inducing a double stranded break (DSB) as recited in claim 1 since Shuber et al., disclose either catalytically inactive Cas endonuclease or catalytically active Cas endonuclease such as Cas9 (see above Response to Arguments related to the rejection under 35 USC 102 (a) (2)).
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Frank Lu, Ph. D., whose telephone number is (571)272-0746. The examiner can normally be reached Monday to Friday, 9 AM to 5 PM.
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/FRANK W LU/
Primary Examiner, Art Unit 1683
July 2, 2026
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