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 .
This Office Action is in reply to Applicants’ correspondence of 02/02/2026.
Applicants’ remarks and amendments have been fully and carefully considered but are not found to be sufficient to put the application in condition for allowance. Any new grounds of rejection presented in this Office Action are necessitated by Applicants’ amendments. Any rejections or objections not reiterated herein have been withdrawn in light of the amendments to the claims or as discussed in this Office Action.
This Action is made FINAL.
Please Note: The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Election/Restrictions
In the reply filed on 07/09/2025 Applicants elected with traverse the invention of Group 1 (claims directed to methods of detecting)
The requirement was deemed proper and made FINAL as set forth on pages 2 of the Office Action of 10/07/2025.
Claims 18, 19 and 23 remain withdrawn from further consideration pursuant to 37 CFR 1.142(b), as set forth on pages 2 of the Office Action of 10/07/2025.
Information Disclosure Statement
The listing of references in the specification (e.g.: see pages 14-15 of the specification as filed) is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Withdrawn Claim Rejections - 35 USC § 112 - Indefiniteness
The rejections of claims under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as set forth on pages 3-4 of the Office Action of 10/07/2025, are withdrawn in light of the amendments to the claims.
Claim Rejections - 35 USC § 112 – Indefiniteness
Newly Applied as Necessitated by Claim Amendments
Claims 20 and 24 are 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.
Claims 20 is unclear over recitation of the limitation “a substrate for the exonuclease(s)” (encompassing a plurality of exonucleases). Claim 20 depends from claim 2; claim 2 recites “an exonuclease” (i.e.: a single exonuclease). As such there is not proper antecedent bases for a plurality of exonucleases as encompassed by the rejected claim.
Claims 20 and 24 are each unclear over recitation limitations related to aspects of the reporter molecule that are set forth in relation to an exonuclease. Claim 20 recites “the reporter oligonucleotide is not a substrate for the exonuclease”, and claim 24 recites “the reporter oligonucleotide is not a substrate for the RNAse T”. The claims depend from claim 2, where claim appears to conditionally require an exonuclease (i.e.: claim 2 recites “if a cleaved reporter oligonucleotide is produced in step (a), monoribonucleotides are produced … by an exonuclease”. Where the claims encompassed methods in which there is no requirement for an exonuclease (e.g.: if the reporter is not cleaved, then monoribonucleotides are not generated, and thus no exonuclease is required), it is unlcear how the limitations of claim 20 and 24, as set forth above, are intended to be interpreted.
Withdrawn Claim Rejections - 35 USC § 103
The rejections of claims under 35 U.S.C. 103, as set forth on pages 5-11 of the Office Action of 10/07/2025, are withdrawn in light of the amendments to the claims. New rejections of the amended claims are set forth below as necessitated by the claim amendments. Applicants’ arguments (p.7-10 of the Remarks of 02/02/2026) in traversal of the rejection of claims set forth in the previous Office Action are addressed below as they may be germane to the newly set forth rejections.
New Claim Rejections - 35 USC § 102
Necessitated by Claim Amendments
The amended claims are directed to methods in which several of the steps of the claims are conditional (i.e.: recitation of the term “if”) upon particular previous elements, where those previous elements are not themselves required to be present. For example, in claim 1, the limitations specify that “the Cas protein and the gRNA cause the collateral activity of the Cas protein to cleave the reporter oligonucleotide, thereby producing a cleaved reporter oligonucleotide”, but this action is completed/performed “if the target polynucleotide comprising the target nucleic acid sequence is present in the sample”. Additionally, claim 1 recites “hydrolyzing the cleaved reporter oligonucleotide to generate monoribonucleotides therefrom”, but this step is performed “if a cleaved reporter oligonucleotide is produced in step (a)”. Finally with regard to the conditional elements, claim 1 recites “characterizing the sample as containing the target polynucleotide with the target nucleic acid sequence”, but this characterization is performed “if monoribonucleotides are detected in step (c)”.
In this regard the claims are properly rejected where the encompass alternative embodiments in which there is no reporter cleavage, no monoribonucleotides are generated, it is determined that no monoribonucleotides are generated, and no characterization of the sample as containing the target is made.
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)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claim(s) 1, 2, 4, 5, 8,10, 15, 20 and 22 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Kellner et al (2019).
Regarding claims 1, Kellner et al teaches methods that include combining a Cas protein that has collateral nuclease activity, and is coupled to a programmable crRNAs that is specific for a target, and a reporter oligonucleotide that is cleaved by the collateral nuclease activity in the presence of the target (e.g.: Fig 1). Relevant to the encompassed embodiments of the claims as detailed above, Kellner et al exemplifies embodiments in which there is no target nucleic acid present in the sample (e.g.: p.3008, Figure a - “-ssRNA target”; Figures b, c, - samples with “0” ZIKV ssRNA concentration (aM); Figure d – sample with “0” Synthetic DNA 1 target concentration (aM)) using LwaCas13a (relevant to claim 22) in the analysis of ssRNA (relevant to claim 10). Additionally, Kellner et al teaches that analyses can include a negative control “in which the target molecule is known to be absent” (e.g.: p.3005). These samples, without a target nucleic acids, detect the absence of reporter oligonucleotide cleavage (i.e.: no fluorescence, indicating the reporter oligonucleotide with a fluorophore and a quencher is intact (relevant to claims 2 and 4)); where the reporter is intact (i.e.: not cleaved) such a reading is evidence that no monoribonucleotides are generated.
Relevant to claim 5, Kellner et al teaches reporters that are ribonucleotide polymers (e.g.: p.2997).
Relevant to claim 8, Kellner et al teaches a reporter that is composed or ribonucleotides and deoxyribonucleotides, and includes phosphorothioate bonds (relevant to claim 20).
Relevant to claim 15, Kellner et al teaches forming a Cas-crRNA complex prior to addition of the reporter oligonucleotide (e.g: see the order of addition of reagents in Box 2 on p. 2990; see the order of addition of reagents in the Box on p. 3007).
New Claim Rejections - 35 USC § 103
Claim(s) 1, 2, 4, 5, 7, 8,10, 15, 20, 21 and 22 is/are rejected under 35 U.S.C. 103 as being unpatentable over Kellner et al (2019).
Kellner et al teaches methods including combining a Cas protein that has collateral nuclease activity, and is coupled to a programmable crRNAs that is specific for a target, and a reporter oligonucleotide that is cleaved by the collateral nuclease activity in the presence of the target (e.g.: Fig 1), and the analysis of samples without a target nucleic acid, as detailed with regard to claims (1, 2, 4, 5, 8,10, 15, 20 and 22).
Further relevant to the instant rejection of claims 7 and 21, Kellner et al exemplifies reporter molecules that include a plurality of consecutive adenosines, including a reporter molecule that includes only adenosine bases (e.g.: p.2997 - PsmCas13b (/56-FAM/rArArArArA/3IABkFQ/) ).
Kellner et al does not particularly teach a reporter with 10 consecutive adenosines (claim 7) or a reporter that is at least 50 nucleotides in length with at least 60% adenosines (claim 21).
However, it would have been prima facie obvious to someone with ordinary skill in the relevant art before the effective filing date of the rejected claims to have used any reporter molecule, including the molecules encompassed by the rejected claims, in the methods of Kellner et al. Where the prior art demonstrated the use of a variety of different reporter molecules, different report molecules are obvious variations of the methods of the prior art. Where the prior art demonstrates the use of different reporters, and a person of ordinary skill has good reason to pursue the known options within his or her technical grasp for optimization of relevant reagents. If this leads to the anticipated success, it is likely the product not of innovation but of ordinary skill and common sense to provide routine optimization.
Response to Remarks
Applicants’ arguments in traversal of the rejections of the previous Office Action (p. 9 of the Remarks of 02/02/2026) provide an assertion of the improvement in results provided by the inventive methods. Relevant to the rejection set forth above, the Remarks assert that an advantage of rA-rich reporters (as encompassed by the rejected claims) is that a large number of AMP molecules may be generated by the exonuclease, and free AMP is readily converted to ATP which may be used in many luciferase-based assays”. This argument is not persuasive to withdraw the rejection because the asserted improvements require elements that are not part of the methods of the rejected claims. As set forth earlier in this Office Action, the rejected claims do not require that monoribonucleotides are generated using an exonuclease, or are detected in a luciferase based assay. The claims encompasses the lack of detection of the target nucleic acid, and so the argument is not commensurate in scope with required limitations of the rejected claims.
Claim(s) 1, 13, 14 and 17 is/are rejected under 35 U.S.C. 103 as being unpatentable over Kellner et al (2019) in view of Chen et al (2018) and Carter et al (US PG Pub 20040229242).
Relevant to the innate rejection of claim, Kellner et al teaches methods that include combining a Cas protein that has collateral nuclease activity, and is coupled to a programmable crRNAs that are specific for a target, and a reporter oligonucleotide that is cleaved by the collateral nuclease activity in the presence of the target (e.g.: Fig 1). Kellner teaches detection of the presence of the target nucleic acid by detection of fluorescence from a label of a reporter molecule that is separated form a quencher on the reporter molecule upon cleavage of the reporter in the presence of the target.
Further relevant to the instant rejection of claims 17, Kellner et al exemplifies the analysis of Zika virus (a human pathogen) variants (e.g.: Fig. 3), and suggests the analysis of bacterial genotypes (e.g.: p.2992 – Applications).
Kellner et al does not teach the detection of monoribonucleotides generated from the cleaved reporter molecule in the detection of the presence of the target nucleic acid.
However, the generation of mononucleotides by the collateral cleavage activity of CRISPR associated (Cas) proteins was known in the prior art, and the detection of generated monoribonucleotides was known in the prior art.
Relevant to the limitations of the claims, Chen et al teaches the analysis of biochemical aspects of collateral cleavage activity of Cas proteins. Relevant to the limitations of the claims, Chen et al provides evidence that the collateral cleavage of non-specific single stranded nucleic acids (e.g.: reporter molecules as recited in the instantly claimed methods) by Cas protein results in the complete degradation of the single stranded nucleic acids into monomers (e.g.: Fig 2A, Fig 3, Supplementary Material Figs S4, S5, S6, S9, S15).
Further relevant to the limitations of the rejected claims, Carter et al teaches that the detection of a nucleic acid can be achieved by the detection of monomers generated from an oligonucleotide, and teaches detection of monomers, such as AMP, using a system to create ATP from liberated AMP, where the ATP thus produced is then used to drive an easily-assayable ATP detection reaction (e.g.: para 0025). Further relevant to claims 13 and 14, the reference teaches that a luciferase/luceiferin system can be used as reporter system that generates light when the ATP is present (e.g.: para 0058; para 0096).
It would have been prima facie obvious to someone with ordinary skill in the relevant art before the effective filing date of the rejected claims to have recognized that in the detection of a target nucleic acid via collateral cleavage of a reporter molecule, as taught by Kellner et al, the reporter molecule may be digested to mononucleotides, as taught by Chen et al. It would have been prima facie obvious to one of ordinary skill in the relevant art before the effective filing date of the rejected claims to have used the AMP detection of Carter et al to detect mononucleotides generated by the collateral cleavage of a reporter molecule in the methods of target detection taught by Kellner et al in view of the teachings of Chen et al. The skilled artisan would have been motivated to detect monoribonucleotides generated by collateral cleavage based on the expressed teachings of Carter et al that such elements area detectable element from a source nucleic acid molecule. The skilled artisan would recognize that such a detection may offer an alternative means of detection that does not require the specific labeling of a reporter molecule, thus simplifying the construction of reagents. The skilled artisan would have a reasonable expectation of success based on the expressed teachings of Chen et al that collateral cleavage (i.e.: target-activated, nonspecific single stranded nuclease activity) is a functionality of a variety of Cas enzymes.
Response to Remarks
Applicants’ arguments in traversal of the rejections of the previous Office Action (p. 8 of the Remarks of 02/02/2026) provide an assertion that Carter et al does not teach aspects of the claimed invention. Relevant to the rejection set forth above, Applicants have argued that Carter et al does not teach the use of a single strand specific endonuclease to detect monoribonucleotides. This argument is not persuasive. Carter et al specifically teaches (para 0025):
A single-strand specific RNA exonuclease is added to digest any liberated RNA oligonucleotides down to monomers. A system to generate ATP from any liberated AMP is added. The ATP thus produced is then used to drive an easily-assayable ATP detection reaction.
As such the Examiner maintains that Carter et al is relevant to the diagnostic detection of monoribonucleotides. Furthermore, it is noted that the claims are not rejected as they may require an additional exonuclease, because the claims are rejected in view of the teachings of Chen et al which teaches that collateral cleavage by a Cas protein results in mononucleotides from the molecules subjected to the collateral cleavage. In this regard the argument that the claimed methods include “a further step” that “comprises using a separate reagent (a single strand specific endonuclease) to reduce the reporter oligonucleotide to monomers” is not commensurate in scope with the requirements of the claims (e.g.: claim 1 does not require any “separate reagent” to generate monomers.
Applicants’ further argue that incorporating the ATP detection of Carter et al is not a simple substitution because Carter et al teaches only capturing a target and using RNase H, whereas the claimed methods includes multiple steps. In this regard it is initially noted, as detailed above, that the rejected claims do not require any steps of using a separated reagent to reduce a reporter to monomers. The Examiner maintains that where Chen et al suggests that the methods of Kellner et al will produce monomer via collateral cleavage, the detection of any such monomers is established to be routine and conventional as demonstrated by the expressed teachings of Carter et al.
Claim(s) 9 is/are rejected under 35 U.S.C. 103 as being unpatentable over Kellner et al (2019) in view of Chen et al (2018) and Carter et al (US PG Pub 20040229242) as applied to claims 1, 13, 14 and 17, above, and further in view of Mondal et al (2017) (cited on the IDS of 10/05/2022).
Kellner et al in view of Chen et al and Carter et al renders obvious method of target nucleic acid sequence detection including collateral cleavage of an RNA reporter, and detection of monoribonucleotides generated from the cleaved reporter via Cas protein dependent collateral cleavage of the reporter for the detection of the presence of the target nucleic acid sequence. Further relevant to the rejection of claim 9, Kellner et al teaches methods comprising the amplification of the target nucleic acid including the addition of rNTPs (e.g.: Fig. 2; p.2990 – Box 2), and Carter et al teaches generating ATP from generated AMP for detection purposes (e.g.: para 0025).
Kellner et al in view of Chen et al and Carter et al does not teach removal of residual nucleotide triphosphates, however such a step was known in the prior art related to monoribonucleotide detection as is taught by Mondal et al.
Mondal et al teaches that prior to detection of diagnostic AMP, ATP is depleted from an assay system (e.g.: Fig 1; p.332 - Assay Principle and Format).
It would have been prima facie obvious to someone with ordinary skill in the relevant art before the effective filing date of the rejected claims to have used the techniques of Mondal et al, including removal of residual nucleotide triphosphates, in the AMP-based detection of collateral cleavage of a reporter rendered obvious by Kellner et al in view of Chen et al and Carter et al. The skilled artisan would have been motivated to incorporate the steps of Mondal et al based on the expressed teachings of Mondal et al that such steps are part of detection methods suitable for use in a bioluminescent assay, which offers several advantages over other assay systems (p.339).
Conclusion
No claim is allowed.
The relevant art made of record and not relied upon is considered pertinent to applicant's disclosure. Liu et al (2012) teaches that collateral cleavage by a Cas protein generates detectable mononucleotides (e.g.: Scheme 1).
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to STEPHEN THOMAS KAPUSHOC whose telephone number is (571)272-3312. The examiner can normally be reached M-F, 8am-5pm.
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Stephen Kapushoc
Primary Examiner
Art Unit 1683
/STEPHEN T KAPUSHOC/Primary Examiner, Art Unit 1683
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