DETAILED ACTION
CONTINUED EXAMINATION UNDER 37 CFR 1.114 AFTER FINAL REJECTION
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant’s submission of RCE and amendment filed on October 6, 2025 have been entered. The claims pending in this application are claims 1-20 wherein claims 10-12, 16, and 17 have been withdrawn due to the restriction requirement mailed on September 5, 2024. Claims 1-9, 13-15, and 18-20 will be examined.
Claim Objections
Claim 1 is objected to because of the following informality: “generating the target nucleic acid fragment comprising the sequence of interest which is protected from exonuclease digestion by the simultaneous binding of both the first and second gRNA-CAS complexes at its respective ends; and at least one non-target nucleic acid fragment that is not simultaneously bound by both the first and second gRNA-CAS complexes; wherein the first and the second gRNA-CAS complexes remain bound to the ends of the target fragment after cleavage and during the subsequent exonuclease treatment in step c)” in step b) should be “generating cleaved nucleic acid molecules comprising the target nucleic acid fragment which is protected from exonuclease digestion by the simultaneous binding of both the first gRNA-CAS complex and the second gRNA-CAS complex 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”.
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, 4-8, 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) cleaving the nucleic acid molecule using a first guide RNA-CRISPR-associated protein (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), thereby generating cleaved nucleic acid molecules comprising the target nucleic acid fragment which is protected from exonuclease digestion by the simultaneous binding of the first gRNA-CAS complex and the second gRNA-CAS complex 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; 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).
Regarding claims 7 and 8, 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) and Shuber et al., teach to cleave a population of nucleic acids using a complex comprising a first Cas endonuclease, a first guide RNA, a crRNA and a tracrRNA and a complex comprising a second Cas endonuclease, a second guide RNA, a crRNA and a tracrRNA and the first Cas endonuclease and the second Cas endonuclease can be Cas9 (see columns 1-10, Figures 1-5 and claims 1-19), Shuber et al., must disclose that at least one of the first gRNA-CAS complex and the second gRNA-CAS complex is capable of inducing a DNA double-stranded break (DSB) as recited in claim 7 and both the first gRNA-CAS complex and the second gRNA-CAS complex are capable of inducing a DSB as recited in claim 8.
Therefore, Shuber et al., teach all limitations recited in claims 1, 2, 4-8, and 13-15.
Claim 9 is rejected under 35 U.S.C. 102(a)(2) as being anticipated by Shuber et al., as applied to claims 1, 2, 4-8, and 13-15 above and as evidence by Jinek et al., (Science, 337, 816-821, 2012).
Since Jinek et al., teach that 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 and each nuclease domain of the Cas9 protein is specialized for cutting one strand of the double helix (see page 817) and claim 9 does not require that a third gRNA-CAS complex is different from a first gRNA-CAS complex or a second gRNA-CAS complex, Shuber et al., as evidence by Jinek et al., disclose that in step b) at least one of the first gRNA-CAS complex and second gRNA-CAS complex is a nickase that nicks one strand of the nucleic acid molecule, and wherein the nucleic acid molecule is further contacted with at least a third gRNA-CAS complex that nicks the complement strand of the one strand of the nucleic acid molecule at a region that is complementary to a region of the one strand of the nucleic acid molecule nicked by the first gRNA-CAS complex or the second gRNA-CAS complex as recited in claim 9.
Response to Arguments
In page 6, last paragraph bridging to page 7, third paragraph of applicant’s remarks, applicant argues that “[I]nitially, Applicant notes that claim 1 has been amended to recite that the ‘first and the second gRNA-CAS complexes remain bound to the ends of the target fragment after cleavage and during the subsequent exonuclease treatment in step c)’ Thus, claim 1, as amended now recites that the first and the second gRNA-CAS complexes that cleave the nucleic acid molecule remain as the same complexes that protect the target nucleic acid fragment against exonuclease cleavage. Shuber does not disclose this limitation. In other words, the fact that active gRNA-CAS complexes are capable of protecting the target nucleic acid fragment after cleavage against exonuclease degradation is not disclosed by Shuber. In fact, Shuber only cites that ‘Protection of the ends of the target nucleic acid may include the binding of the Cas complexes to one or both ends. The Cas complexes that bind to the ends of the target nucleic acid may be catalytically inactive.’ (Column 3, lines 35-38). It should be noted here that the subsequent phrase in Shuber that ‘Protection of the ends of the target nucleic acid may include cleavage of the target nucleic acids at one or both ends’ (Column 3, lines 38-40), does not disclose that the cleavage is performed by the same gRNA-CAS complexes. At least for these reasons, Applicant submits that the present claims are novel over Shuber. This deficiency of Shuber is not remedied by Jinek. Jinek does not disclose the use of the same type of active gRNA-CAS complexes to both generate a target nucleic acid fragment from a nucleic acid molecule and protect it against exonuclease cleavage. In view of the above remarks, Applicant submits that the claims are novel”.
The above arguments have been fully considered but they are not persuasive toward the withdrawal of the rejection. Since Shuber et al., teach that “[P]rotection of the ends of the target nucleic acid may include the binding of the Cas complexes to one or both ends” and “[F]IG. 2 illustrates the method 201. A population 203 of nucleic acids 205a, 205b, including a target nucleic acid 207, is provided. The target nucleic acid 207 is protected 211 by allowing Cas complexes 213a, 213b to bind to sequences at the ends of the target nucleic acid 207. The target nucleic acid 207 may be a portion of larger nucleic acid molecule, and the ends of the target nucleic acid 207 may not be the ends of a nucleic acid molecule, i.e., the ends may not be free 5’ phosphate groups or free 3’ OH groups. Binding of the Cas complexes to the ends of the target nucleic provides protection against exonuclease digestion. Nucleic acids 205a, 205b in the population 203 are then degraded 221, but the target nucleic acid 207 is protected from degradation. Preferably, degradation occurs via exonuclease digestion. The target nucleic acid 207 may then be detected by any suitable means” (see column 3, lines 35-37 and column 6, last paragraph, and Figure 2), Shuber et al., must disclose that the target nucleic acid fragment is protected against exonuclease cleavage by binding to both the first gRNA-CAS complex and the second gRNA-CAS complex to both 5’ and 3’ ends of the target nucleic acid fragment (ie., a nucleic acid fragment that binds both the first gRNA-CAS complex and the second gRNA-CAS complex can provide a protection against an exonuclease digestion) and the first gRNA-CAS complex and the second gRNA-CAS complex that cleave the nucleic acid molecule remain bound to the 5’ and 3’ ends of the target fragment after said cleaving the nucleic acid molecule using a first gRNA-CAS complex and a second gRNA-CAS complex, and applicant’s argument “claim 1, as amended now recites that the first and the second gRNA-CAS complexes that cleave the nucleic acid molecule remain as the same complexes that protect the target nucleic acid fragment against exonuclease cleavage. Shuber does not disclose this limitation. In other words, the fact that active gRNA-CAS complexes are capable of protecting the target nucleic acid fragment after cleavage against exonuclease degradation is not disclosed by Shuber” is incorrect. Furthermore, applicant has no evidence to show that the phrase “Protection of the ends of the target nucleic acid may include cleavage of the target nucleic acids at one or both ends” in Shuber et al., (see column 3, lines 38-40) discloses that the cleavage is performed by different gRNA-CAS complexes. In addition, the correct interpretation for the phrase “[T]he Cas complexes that bind to the ends of the target nucleic acid may be catalytically inactive” in Shuber et al., (see column 3, lines 37 and 38) is that “[T]he Cas complexes that bind to the ends of the target nucleic acid are either catalytically inactive or catalytically active”.
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, 4-8, 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 7, fourth paragraph bridging to page 8, first paragraph of applicant’s remarks, applicant argues that “[A]s discussed above, neither Shuber nor Jinek teach or suggest the use of the same type of active gRNA-CAS complexes to both generate a target nucleic acid fragment from a nucleic acid molecule and protect it against exonuclease cleavage, as required in independent claim 1. Further, Shuber does not disclose or teach that a catalytically active gRNA- CAS complex remains bound to the target nucleic acid for a sufficient long time to protect it against exonuclease cleavage. Therefore, a person of ordinary skill in the art would not have a reasonable expectation of success to perform step b) by incubating the first gRNA-CAS complex and the second gRNA-CAS complex and the nucleic acid molecule together for the required length of time as recited in claim 3”.
The above arguments have been fully considered but they are not persuasive toward the withdrawal of the rejection because Shuber et al., teach all imitations recited in claim 1 (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 1-8, 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.
Conclusion
No claim is allowed.
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/FRANK W LU/
Primary Examiner, Art Unit 1683
February 17, 2026