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 .
Status of claims / Response to Amendment
This office action is in response to an amendment filed on August 12, 2026.
Claims 1-15 were previously pending. Applicant amended claims 1-5, 7 and 10-14; cancelled claim 9.
Claims 1-8 and 10-15 are currently pending and under consideration.
Applicant's cancellation of claim 9 obviated the previously presented objection to specification.
Applicant's claim amendments overcame the following rejections:
Rejection of Claim 9 under 35 U.S.C. 112(b), as being incomplete for omitting essential elements;
Rejection of Claims 1-15 under 35 U.S.C. 112(b), as being indefinite;
Rejection of Claim 9 under 35 U.S.C. 112(a);
Rejection of Claims 10-11 under 35 U.S.C. 101;
Rejection of claim 9 under 35 U.S.C. 103.
All other previously presented rejections are maintained for reasons given in the "Response to Arguments" below.
Applicant's amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
This office action contains new grounds for rejection necessitated by amendment.
Priority
For the instant claims 1-8 and 10-15 in this U.S. Application, the applicant claims priority of Foreign Application EP 21198612.0, which has a filling date on September 23, 2021.
Response to Arguments
Applicant's arguments filed on August 12, 2026 have been fully considered.
35 U.S.C. § 103 rejection
In the Non-Final Office Action mailed on May 28, 2026:
Claims 1-8 and 10-15 are rejected under 35 U.S.C. 103 as being unpatentable over Brown, in view of Wang, Shon, Lohmann, and Zhang, as evidenced by Bailey.
These rejections are maintained in this Office Action for reasons below.
First, Applicant argues that "Wang does not establish that its Cas9 nickase could be substituted for Brown's BrdU/UV nicking step while retaining Brown's Exonuclease III digestion with predictable results." (Remarks, pages 6-7).
This argument has been considered but is not persuasive. Applicant's argument is based on the incorrect assumption that a prima facie case of obviousness requires a single reference to provide the entire reason to modify the prior art and arrive at the claimed invention. However, it is well established that the sources for rationales or reasons to modify or combine prior art teachings need not be limited to the cited references themselves. See MPEP 2143, subsection I, and MPEP 2144.
Examiners can take into account “the background knowledge possessed by a person having ordinary skill in the art.” KSR, 550 U.S. at 418. “A person of ordinary skill in the art is also a person of ordinary creativity, not an automaton.” KSR, 550 U.S. at 421, 82 USPQ2d at 1397. “[I]n many cases a person of ordinary skill will be able to fit the teachings of multiple patents together like pieces of a puzzle.” Id. at 420, 82 USPQ2d at 1397. Office personnel may also take into account “the inferences and creative steps that a person of ordinary skill in the art would employ.” Id. at 418, 82 USPQ2d at 1396.
Here, the 103 rejection is still deemed proper because a skilled artisan, in view of the prior art and background knowledge in the field, would have found it obvious to substitute Brown’s DNA-nicking approach using BrdU labeling and UV light, with Wang’s Cas9 nickase approach, while retaining Brown’s exonuclease digestion step.
Brown teaches a known combination of two functional elements:
a nicking element that creates nicks in one DNA strand, and
an exonuclease digestion element that digests the nicked strand to expose single-stranded DNA for probe hybridization.
Brown teaches BrdU labeling and UV light as the nicking element. Wang teaches Cas9 nickase as another known nicking element for creating strand-specific nicks in double-stranded DNA.
Thus, a skilled artisan would have readily recognized that Brown’s BrdU labeling/UV nicking element and Wang's Cas9 nickase serve the same function of creating nicks in dsDNA that can provide initiation sites for exonuclease digestion. This represents a simple substitution of one known DNA nicking element for another to obtain the predictable result of creating nicks on a DNA strand that can serve as an initiation site for exonuclease digestion.
A skilled artisan would have reasonably expected that nicks created by Cas9 would be suitable substrates for exonuclease digestion. This is supported by Bailey, which teaches that Exo III catalytically degrades one strand of the double helix starting at nicks (p. 175, lines 8-10). Wang also explicitly discusses the 3’ flap produced by Cas9 nickase can be digested by exonuclease (page 1567, left-hand col, lines 34-37).
Therefore, a skilled artisan would have reasonably expected the substituted Cas9 nicking element to function with the exonuclease digestion step of Brown.
Applicant has not identified, and the record does not show, that the nick created by Cas9 nickase is structurally different from the nick created by Brown’s BrdU labeling/UV nicking in a way that would make the Cas9-generated nick unsuitable for exonuclease digestion. Rather, both approaches create nicks in dsDNA as initiation sites for exonuclease digestion.
Applicant further argues that dependent claims 2 and 16 "additionally require the absence of a helicase enzyme." Applicant asserts that Wang uses a helicase enzyme to achieve sufficient unwinding of target DNA for probe hybridization, which is contrary to the requirement of the claims. (Remarks, page 9)
This argument is not persuasive because it mischaracterizes the rejection. The proposed modification in the rejection does not involve helicase enzyme at all. Rather, the rejection relies on replacing Brown’s DNA-nicking approach using BrdU labeling with UV light, with Wang’s Cas9 nickase approach, while retaining Brown’s exonuclease digestion step.
Because the DNA is made single-stranded by exonuclease digestion in the modified method, a skilled artisan would have understood that helicase is not needed to unwind the DNA for probe hybridization. In other words, exonuclease digestion and helicase unwinding serve the same general purpose of making the target DNA sequence available for probe binding. One or the other may be used; both are not required.
In this case, as supported by facts, a skilled artisan familiar with Brown’s method and in view of Wang’s would have been motivated to adopt the Cas9 nickase element, but not the Rep-X helicase element, for two reasons.
First, using helicase instead of exonuclease does not bring any additional benefit. As discussed in the rejection, the benefits suggested by Wang, such as improvement in cleaving specificity and not requiring any treatment in live cells before cell fixation and applicability to patient tissue samples (page 1574, left-hand col, lines 1-12), are mainly directed to the difference between the nicking elements, i.e., BrdU labeling/UV nicking vs. Cas9 nickase. Thus, there is no apparent substantial benefit from replacing Brown’s exonuclease digestion with Wang’s helicase unwinding step.
Second, exonuclease is the more cost-effective option. The Rep-X helicase enzyme in Wang is not commercially available and requires additional time and cost to express and purify (see Wang p. 1575, method details on Rep-X expression and purification). Without a substantial benefit from using this helicase, a skilled artisan would have retained Brown’s exonuclease digestion step, which uses a commercially available exonuclease that can be easily sourced (Brown, page 12, right-hand col, para 3, lines 18-19, Exonuclease III (NEB), list price for 5,000 units of Exonuclease III on NEB is $77.00, see www. neb.com/en-us/products/m0206-exonuclease-iii-e-coli).
Accordingly, in the absence of secondary evidence of non-obviousness that helicase was essential to Wang, or a skilled artisan would not expect the combination of Cas plus exo to work, the exclusion of helicase in dependent claims is obvious in view of the proposed combination of prior art elements, because the rejection relies on Cas9-based nicking and exonuclease digestion, not helicase-mediated unwinding.
For the reasons above, Applicant's arguments are unpersuasive. Accordingly, since all the claims remain rejected under the same statutory basis and in view of the same prior art references with the same rationales and teachings, the previously set forth rejections under 35 USC § 103 are properly maintained and updated to reflect the recent claims amendment.
Maintained 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.
Claims 1-8 and 10-15 are rejected under 35 U.S.C. 103 as being unpatentable over Brown (Brown et al. A tissue-specific self-interacting chromatin domain forms independently of enhancer-promoter interactions. Nat Commun 9, 3849 (2018). doi.org/10.1038/s41467-018-06248-4) , in view of Wang (Wang et al., Genome oligopaint via local denaturation fluorescence in situ hybridization. Mol Cell. 2021 Apr 1;81(7):1566-1577.e8. doi: 10.1016/j.molcel.2021.02.011. Epub 2021 Mar 2. PMID: 33657402; PMCID: PMC8026568; cited as NPL document #2 in IDS filed 03/21/2024),
Shon (Shon et al., The nucleotide sequence of the replication origin beta of the plasmid R6K. J Biol Chem. 1982 Nov 25;257(22):13823-7. PMID: 6292210),
Lohmann (Lohmann et al., Detection of short repeated genomic sequences on metaphase chromosomes using padlock probes and target primed rolling circle DNA synthesis. BMC Molecular Biol 8, 103 (2007). doi.org/10.1186/1471-2199-8-103),
Zhang (Zhang et al., Strand break-induced replication fork collapse leads to C-circles, C-overhangs and telomeric recombination. PLoS Genet. 2019 Feb 4;15(2):e1007925. doi: 10.1371/journal.pgen.1007925. PMID: 30716077; PMCID: PMC6382176), as evidenced by
Bailey (Bailey et al., (2010). Chromosome Orientation Fluorescence In Situ Hybridization or Strand-Specific FISH. In: Bridger, J., Volpi, E. (eds) Fluorescence in situ Hybridization (FISH). Methods in Molecular Biology, vol 659. Humana Press, Totowa, NJ. ; doi.org/10.1007/978-1-60761-789-1_12).
A) Brown teaches a method for fluorescent in situ hybridization (FISH), referred to as RASER (resolution after single-strand exonuclease resection)-FISH. RASER-FISH maintains nuclear fine-scale structure by replacing heat denaturation with exonuclease digestion, and is suitable for high and super-resolution imaging analysis (page 12, right-hand col, para 3).
Regarding claim 1, Brown teaches a method for fluorescent in situ hybridization (FISH), comprising the steps of
targeted denaturing of a double stranded sample DNA using an exonuclease as denaturing agent (page 12, right-hand col, para 3, lines 6-8, exonuclease digestion targeting a single strand);
hybridizing one or more FISH probes with the denatured sample DNA of (a) (page 12, right-hand col, para 3, lines 12-14; 21-22); and
detecting the binding of hybridized FISH probes (page 13, left-hand col, lines 5-6).
Brown teaches that the principle of RASER-FISH is adapted from chromosome orientation FISH (CO-FISH) (page 12, right-hand col, para 3). This involves two steps:
(1) creating nicks on a single DNA strand of dsDNA using strand-specific labeling of BrdU in combination with UV light;
(2) digesting the nicked strand using exonuclease III, an enzyme that degrades one strand of dsDNA using the nicks created in step (1) as initiation sites (see Bailey, page 174-176; Fig. 1).
Although Brown does not explicitly teach using RNA-guided DNA endonuclease, this feature would have been obvious in view of the knowledge in the art, as a skilled artisan would understand that endonucleases, such as a Cas9-guide RNA complex, can also be used as alternative approach to generate single-stranded nicks in a dsDNA.
The use of sequence-specific endonucleases together with exonuclease digestion to generate single-stranded DNA for probe hybridization is well-known in the art. See Shon (page 13825, left-hand col, para 2); see also Lohmann (Figure 1); see also Zhang (Fig. 3A).
Shon discloses employing restriction endonuclease enzymes (EcoRI or BamHI) that create sequence-specific nicks on a double-stranded DNA; followed by Phage T7 exonuclease digestion to yield single-stranded DNA for primer binding (page 13825, left-hand col, para 2).
Lohmann teaches fluorescence in situ hybridization and detection of target DNA strand, using sequence-specific restriction enzyme followed by lambda exonuclease digestion to obtain single-stranded DNA for probe hybridization (Figure 1).
Zhang teaches providing single stranded DNA for probe hybridization by digesting DNA with restriction enzymes, followed by digestion with Exo III (Exonuclease III), a 3’ to 5’ exonuclease that remove nucleotides from blunt end or break/gap in double stranded DNA to generate single stranded DNA (Fig 3A-B).
Accordingly, the general strategy of first applying an endonuclease element to create breaks in dsDNA, followed by directional exonuclease digestion to digest one strand while leaving the other strand intact for oligonucleotide hybridization, was well-known in the art. A variety of known break-creating elements and exonuclease elements for strand-specific digestion were available and used to carry out this process. Therefore, a skilled artisan in view of the prior art, exercising ordinary creativity would have understood that a known break-creating endonuclease could be combined with a known exonuclease to achieve strand-specific digestion.
Wang specifically teaches using a Cas9 nickase to expose single-stranded DNA regions for probe hybridization in FISH (Graphical Abstract). Wang further teaches that the RASER FISH method (disclosed by Brown) digests DNA nonspecifically and requires a BrdU treatment step in live cells, and in contrast its method using Cas9 nickase provides improvement in cleaving specificity and does not require any treatment in live cells before cell fixation and therefore can also be applied to patient tissue samples (page 1574, left-hand col, lines 1-12).
Accordingly, a skilled artisan in view of the teachings in the art, would have found it prima facie obvious before the effective filing date of the claimed invention to modify Brown’s FISH method to substitute the DNA-nicking approach using BrdU labeling with UV light, with Wang’s Cas9 nickase approach, to create single strand nicks for single-stranded exonuclease digestion.
This represents a simple substitution of one known DNA nicking approach for another to obtain predictable results, because both approaches serve the same function of creating strand-specific nicks in double-stranded DNA that provide initiation sites for exonuclease digestion. see MPEP 2141.
The skilled artisan would have been motivated to make this modification, in view of the additional benefits for using Cas9 nickase suggested by Wang, such as improvement in cleaving specificity and compatibility with patient samples.
The person of ordinary skill would have had a reasonable expectation of success in making such a modification because a skilled artisan would have understood that nicks created by Cas9 would be suitable substrates for exonuclease digestion. This is supported by Bailey, which teaches that Exo III catalytically degrades one strand of the double helix starting at nicks (p. 175, lines 8-10). Wang also explicitly discusses the 3’ flap produced by Cas9 nickase can be digested by exonuclease (page 1567, left-hand col, lines 34-37).
Therefore, a skilled artisan would have reasonably expected that Cas9 could be used to facilitate strand-specific digestion by ExoIII because Cas9 creates nicks, which are digestion initiation sites for ExoIII.
B) Regarding claim 2, it recites “wherein the method does not involve the use of a helicase enzyme to unwind the double stranded sample DNA during step (a).”
This limitation is obvious in view of the combined teachings in Brown and Wang. As discussed above, the proposed modification of Brown uses exonuclease to digest the nicked DNA strand and does not need helicase to unwind the DNA.
Regarding claim 3, Wang teaches a Cas9 enzyme, specifically a Cas9 nickase comprising an H840A mutation (Graphical Abstract; methods, e1, Alt-R S.p. Cas9 H840A Nickase V3 ).
Regarding claim 4, Shon teaches T7 bacteriophage exonuclease to yield single-stranded DNA (Shon. page 13825, left-hand col, para 2), and Lohmann using lambda exonuclease digestion to obtain single-stranded DNA for probe hybridization (Figure 1).
A skilled artisan would have found it obvious to use either T7 exonuclease or lambda exonuclease in place of Exo III in the modified method these enzymes perform the same function of generating single-stranded DNA. This represents a simple substitution of one known exonuclease for another to obtain the predictable result of producing single-stranded DNA.
Regarding claim 5, it recites carrying out endonuclease digestion and exonuclease digestion at the same time. This limitation would have been obvious because the advantages of performing reaction steps at the same time are readily apparent, including reduced processing time and reduced likelihood of human error. Performing simultaneous digestion of DNA substrate using multiple enzymes is a common time saving procedure (see New England Biolabs: Single-temperature Double Digest. protocols.io ; https://dx.doi.org/10.17504/protocols.io.cjhuj5; 2014). Such modification would have constituted routine optimization by a person of ordinary skill in the art.
Accordingly, a skilled artisan would have found it obvious to perform the recited digestion steps simultaneously, and the claimed order of steps would therefore have been obvious over the cited references in the absence of secondary considerations.
One having ordinary skill in the art would have had a reasonable expectation of success because performing optimization of a known process, is well within the skill level of the ordinary artisan.
Regarding claim 6, it recites performing denaturation and probe hybridization steps simultaneously. This limitation would have been obvious because the advantages of performing reaction steps at the same time are readily apparent, including reduced processing time and reduced likelihood of human error. Such modification would have constituted routine optimization by a person of ordinary skill in the art.
Further, as taught by Brown and Wang, the enzyme digestion reactions involving Cas9 nickase (see Wang, page e7, para 1), Exonuclease III (see Brown page 12, right-hand col, para 3, lines 18-19), and probe hybridization (see Brown, page 12, right-hand col, para 3, lines 21-22) are all suitable for incubation under the same temperature condition at 37°C. Accordingly, a skilled artisan, in view of these teachings, would have found it obvious to combine these steps into a simultaneous reaction process in order to simply the workflow and reduce assay time.
Regarding claim 7, Wang teaches contacting of the sample DNA with the RNA-guided DNA endonuclease and a guide RNA (sgRNA), wherein the sgRNA has a sequence targeting one or more sequences known to be present in the sample DNA (Wang, Figs.1- 2).
Regarding claim 8, Brown teaches no heat- assisted denaturing is used (Brown, page 12, right-hand col, para 3).
Regarding claims 10-11, the claims recite a method comprising contacting a double stranded sample DNA with an RNA-guided DNA endonuclease and an exonuclease as denaturing agents. These claims would have been obvious in view of Brown and Wang for the same reasons discussed above with respect to claims 1 and 3, which apply Cas9 enzyme and exonuclease treatment to dsDNA in FISH methods.
Regarding claims 12-15, the claims recite FISH kits comprising the same elements and features used in the FISH methods of claims 1, 3, 4, and 15. Accordingly, these claims would have also been obvious in view of Brown, Wang, Shon and Lohmann for the same reasons discussed above with respect to the corresponding method claims.
Conclusion
No claims are allowed.
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.
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/TIAN NMN YU/Examiner , Art Unit 1681 /AARON A PRIEST/Primary Examiner, Art Unit 1681
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