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 the Applications, Amendments and/or Claims
This action is written in response to applicant's correspondence(s) submitted on 12/30/2025. In the paper of 12/30/2025, Applicant amended claims 1-3, 6, 8, 14, 18-19, 21 and canceled claims 10 and 12-13 and 22 and added new claims 25-29. Accordingly, claims 1-9, 11, 14-21, 23-29 remain pending and under review.
Applicant’s statements submitted on 12/30/2025 that a request for correction of the filing receipt, and a corrected filing receipt was mailed on February 3, 2023 are acknowledged.
This paper contains new rejection(s) necessitated by claim amendments.
Response to Arguments
Moot and/or Withdrawn Objection(s) and/or Rejection(s)
The objection to the drawings because the text of Fig. 1 is illegible is withdrawn in view of the replacement drawing(s) submitted on 12/30/2025.
The rejections of claims 12 and 22 under 35 U.S.C. 101 because the claimed invention is directed to a judicial exception are moot based on cancellation of these claims.
The rejection of claim 22 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite is moot based on cancellation of this claim.
The rejections of claims 2-3, 6, 8-9, 14, 18-21 and 23-24 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for reasons stated in paragraphs 10-14 on pages of 10-11 of the Non-final Office action mailed on 09/30/2025, are withdrawn based on the amendments of claims 2, 3, 6 and 14.
The rejections of claims 18-21 and 23-24 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for reasons stated in paragraphs 15-16 on pages of 11-13 of the Non-final Office action mailed on 09/30/2025, are withdrawn based on the amendment made to claims 18, 19 and 21.
The rejection of claim 12 under pre-AIA 35 U.S.C. 102(a) as being anticipated by Hu et al. (pub July 25, 2019, Analytical Chemistry, 91(16), pp.10870-10878) is moot based on cancellation of this claim.
The rejection of claims 1-2, 4-9 and 14 under pre-AIA 35 U.S.C. 102(a) as being anticipated by Hu et al. (pub July 25, 2019, Analytical Chemistry, 91(16), pp.10870-10878) is withdrawn based on claim amendments. Applicant’s argument that Hu et al. do not teach nucleic acid reporter molecules that are blocked at the 3' termini is partially persuasive. The claims fail to recite any functional limitation provided by the instant 3’ termini block and this failure invokes a new rejection citing Carpenter et al. (WO2016/100955A2).
The rejection of claims 15-17 under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Hu et al. (pub July 25, 2019, Analytical Chemistry, 91(16), pp.10870-10878) in view of Polansky (US2004/0023207) is withdrawn for the same reasons as provided above for the withdrawal of the rejection under pre-AIA 35 U.S.C. 102(a) citing Hu et al. (2019).
The rejection of claim 12 under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Li et al. (April 24, 2018, Cell discovery, 4(20), pp. 1-4) in view of Luo et al. (2017, Talanta, 169, pp.57-63) and Polansky (US2004/0023207) is moot based on cancellation of this claim.
The rejection of claim 22 under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Li et al. (April 24, 2018, Cell discovery, 4(20), pp. 1-4) in view of Luo et al. (2017, Talanta, 169, pp.57-63) as applied for claim 1 above is moot based on cancellation of this claim.
Maintained Rejection(s)
The rejections of claims 1-9, 11, 14, 15-18 and 24 under 35 U.S.C. 101 are maintained as the instant nucleic acid reporter molecules blocked at the 3' termini encompassed circularized nucleic acid molecules.
The rejection of claims 1-9, 11 and 14-17 under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Li et al. (April 24, 2018, Cell discovery, 4(20), pp. 1-4) in view of Luo et al. (2017, Talanta, 169, pp.57-63) and Polansky (US2004/0023207) is maintained.
The rejection of claims 18-21 and 23-24 under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Li et al. (April 24, 2018, Cell discovery, 4(20), pp. 1-4) in view of Luo et al. (2017, Talanta, 169, pp.57-63) as applied for claim 1 above, is maintained.
Argument(s)
Applicant's arguments filed 12/30/2025 have been fully considered but they are not fully persuasive because of the following.
Applicant argues that detection system described in Hu et al. is fundamentally different from the claimed nucleic acid detection system as Hu et al. fails to disclose each and every limitation of amended claim 1 (see Remarks of 12/20/2025, pg 10, 2nd para).
Specifically, Hu et al. teach a double-stranded DNA substrate is engineered to include a poly (AT-TA) segment at the 3' end of one strand and a biotin group at the 5' end of the complementary strand. Following Cas9-mediated cleavage, uncleaved DNA substrates retaining the poly(AT-TA) segment serve as templates for CuNP formation (see Remarks of 12/30/2025, pg 10, 1st para and pg 11, third paragraph of section 4).
Applicant further argues that the poly(AT-TA) segment of the target nucleic acid of Hu et al. does not constitute a 3' block, as this segment does not structurally prevents enzymatic extension at the 3' terminus (see Remarks, pg 10, 4th paragraph) and that Hu et al. do not teach a polymerase capable of adding a polynucleotide tail to nucleic acid fragments.
Hu et al. detects the presence of uncleaved DNA substrates that retain a pre-incorporated poly(AT-TA) segment and signal generation depends on the absence of cleavage by the Cas effector protein (pg 11, third paragraph of section 4).
While Applicant remarks relating to the teachings of Hu et al. as stated above are correct, it is noted that the instant claims are simply directed to a system and method of providing the system,
the system comprising of
(i) nucleic acid reporter molecules blocked at the 3' termini,
(ii) a target activatable-CRISPR-Cas system comprising an effector protein and one or more guide sequence,
the effector protein capable of cleaving nucleic acid reporter molecules that are blocked at the 3' termini to generate nucleic acid fragments,
the guide sequence capable of targeting the effector protein to a target sequence of a target nucleic acid, and
(iii) a polymerase capable of transferring nucleotides to nucleic acid fragments so as to form polynucleotide tails attached to the nucleic acid fragments.
Any detection system of a cited prior art reference that discloses the three features of the instant system i.e. (i) a 3’-termi blocked nucleic acid, (ii) a CRISPR-Cas system comprising an effector protein and one or more guide sequence and (iii) a polymerase that could provide the functional limitations of the instant claims 1-9, 11, 14-117 and 25-29 read over the claims, even if the cited reference lacks disclosure of these functions.
Applicant’s arguments (see Remarks of 12/30/2025, pg 11, 1st paragraph) that Hu et al. do not teach: (iii) target-activated cleavage of such reporter molecules; and (iv) polymerase-mediated formation of polynucleotide tails on generated nucleic acid fragments, are not found to be persuasive for claims 1-9, 11, 14-17 and 25-29, as these arguments relate to processes, while the instant claims 1-9, 11, 14-17 and 25-29 are simply directed to a system comprising reporter molecules blocked at the 3' termini, a polymerase and a CRISPR-Cas system, not process(es).
Applicant’s arguments that Hu et al. do not teach: (i) nucleic acid reporter molecules distinct from the target nucleic acid is also not persuasive as these claims (excluding claim 29) omit any requirement of the underlined portion of limitation.
The Office only partially agrees with Applicant’s Remarks of 12/30/2025, pg 11, 1st paragraph that the system of Hu et al. do not teach reporter molecules blocked at the 3' termini, particularly because of the following.
Concerning the poly (AT-TA) tail of the reporter nucleic acid substrate of Hu et al., Applicant is reminded that the instant claims omit any limitation that recite what type of 3’ termini block is being required for the reporter molecules, and/or what the role for the 3’ block is.
Hu et al. does teach reporter molecules blocked at the 5' termini with a biotin moiety and a 3’ poly AT-TA) tail. The biotin moiety certainly prevents nucleic acid degradation from 5’ termini.
Hu et al. also does teach detection of uncleaved 5’-biotinylated DNA substrates that retain a pre-incorporated poly(AT-TA) segment as illustrated by scheme 1, since only the biotinylated DNA substrates are captured on bead and only DNA substrates with poly (AT-TA) tail can promote fluorescence labeling with CuNPs to generate fluorescence signaling.
In contrast to Applicant’s arguments (see Remarks of 12/30/2025, pg 11, 4th paragraph of section 4), none of the instant claims 1-9, 11, 14-17, 25-29 recite any limitations directed at a system comprising a CRISPR-Cas cleaved reporter molecules, the cleaved reporter comprising of a polynucleotide tail by generated incorporation of dTTP nucleotides by a terminal deoxynucleotidyl transferase, said tail further comprising copper nanoparticles chelated at metal binding sites located on the polynucleotide tail.
None of the instant claims 1-9, 11, 14-17 and 25-29 recite any limitations relating to fluorescence signal generation by copper chelates of polynucleotide tails on the CRISPR-Cas cleaved reporter molecules, only after CRISPR-dependent cleavage of 3’ termini blocked nucleic acid reporter molecules, wherein CRISPR-Cas cleavage of 3’ termini blocked nucleic acid reporter molecules is followed by formation of a polynucleotide tail on the cleaved nucleic acid reporter fragment generated by a terminal deoxynucleotidyl transferase incorporation of dTTP to generate a poly(dT) polynucleotide tail, wherein the polynucleotide tails comprise copper metal binding sites.
Concerning the rejections under 35 U.S.C. 103 citing Li et al in view of Luo et al. and Polansky, Applicant’s argues that Li et al. do not teach a detection system providing a polymerase that generates a polynucleotide tail on a cleaved reporter molecule (see Remarks of 12/30/2025, pg 13, 2nd and 3rd paragraphs) and that it is not obvious to combine Luo et al. with Li et al. as Luo et al. is directed to a detection system for detecting DNase I activity, based on poly-T-templated copper nanoparticle formation and signal generation in Luo et al. is conditional on the presence and activity of DNase I while Li et al is directed to sequence-specific nucleic acid detection using CRISPR-Cas12a and require target pre-amplification.
Applicant’s arguments are not persuasive because since the rejection(s) under 35 U.S.C. 103 citing Li et al in view of Luo et al. and Polansky are not providing a rationale for obviousness based on bodily incorporation of one reference into the others but rather provides a rational for obviousness simply based on what the combination of known teachings from the references makes obvious to one of ordinary skill in the pertinent art. (see In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981).
Based on the noted teachings from each one of the references cited in the rejection(s) under 35 U.S.C. 103, it would have been prima facie obvious to the ordinary skilled artisan, before the effective filing date of the instant invention, to generate a modified nucleic acid detection system that detects the presence of a target sequence via the combination of CRISPR-Cas nuclease activity on a 3’ termini blocked nucleic acid reporter molecule to form an unblocked nucleic acid reporter molecule fragment, and poly(dT) extension of the unblocked nucleic acid fragment generated by CRISPR-Cas cleavage by terimanl deoxynucleotidyl transferase activity of a polymerase, and the final use of fluorescent copper nanoparticles templating on the poly(dT) tail.
Luo et al. already teach it a matter of routine practice in the art to generate a nucleic acid detection system that detects by DNase I nuclease activity via the combination of DNAse I cleavage of a 3’ termini blocked nucleic acid reporter molecule so as to form unblocked nucleic acid reporter molecule and then provides poly(dT) extension by terminal transferase activity of the cleaved reporter nucleic acid fragment generated via DNase I cleavage and copper templating on poly(dT) tail generated by terminal transferase activity; while Li et al. teach that it was already known to provide CRISPR-Cas for cleavage of a 3’ termini blocked nucleic acid reporter molecule.
Specification
The specification is objected to as failing to provide proper antecedent basis for the claimed subject matter of claim 21. Claim 21 recites the limitation “wherein the detection of the target nucleic acid is performed in a subject”. The specification does not have any disclosure of the method claimed by claim 21, i.e. a method according to claim 18, wherein the method further comprises detecting a detectable cluster performed in a subject. See 37 CFR 1.75(d)(1) and MPEP § 608.01(o). Correction of the following is required.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 1-9, 11, 14, 15-17, 25-26 and 28-29 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a judicial exception without significantly more.
Concerning claims 1-9, 11, 14, 25-26 and 28-29
Claims 1-9, 11, 25-26 and 28-29 are construed as being a collection of natural products, thereby rendering claims 1-9, 11, 14 and 25-26 and 28-29 as falling within the law of nature exception.
Specifically, the instant collection or system comprises: CRISPR-Cas comprising an effector protein and one or more guide RNAs and nucleic acid reporter molecules having a 3’ termini block (e.g. circular nucleic acids) and a polymerase.
Furthermore, claims 1-9, 11, 14 and 25-26 and 28-29 do not include additional elements that are sufficient to amount to significantly more than the judicial exception.
The following inquiries are used to determine whether a claim is drawn to patent-eligible subject matter:
Step 1. Is the claim directed to a process, machine, article of manufacture, or composition of matter?
Yes- claims 1-9, 11 and 25-26 and 28-29 are clearly directed to a composition comprising of products that are not distinguishable from their natural counterparts.
Step 2A, prong one. Does the claim recite a law of nature, a natural phenomenon, or an abstract idea (recognized judicial exceptions)?
Yes- claims 1-9, 11 and 25-26 and 28-29 are directed to a collection/composition comprising of products that are not distinguishable from their natural counterparts.
The claimed CRISPR-Cas complex comprising an effector protein and one or more guide RNAs; nucleic acid reporter molecules having a 3’ termini block (e.g. circular nucleic acids) and a polymerase of the instant composition(s)/collection(s)/system have no functional differences relative to their naturally occurring counterparts.
MPEP 2106.04(b)(i) identifies nucleic acid sequence(s) having no structural or functional differences from naturally occurring nucleic acids as an example of a patent-ineligible natural product.
In addition, as discussed in MPEP 2016.04(b)(II), “[P]roduc-t of nature exceptions include both naturally occurring products and non-naturally occurring products that lack markedly different characteristics from any naturally occurring counterpart.” See Ambry Genetics, 774 F.3d at 760, 113 USPQ2d at 1244.
Step 2A, prong two. Does the claim as a whole integrate the recited judicial exception into a practical application of the exception?
No - This evaluation is performed by identifying whether there are any additional elements recited in the claim beyond the judicial exception, and evaluating those additional elements individually and in combination to determine whether the claim as a whole integrates the exception into a practical application.
In the instant case, claims 1-9, 11 and 25-26 and 28-29 do not appear to recite any additional elements beyond those that may reasonably be consider naturally occurring elements i.e. the claimed nucleic acid reporters having a 3’ termini block encompass circular nucleic acids, which are not required to include non-naturally occurring elements or non-naturally occurring nucleotides, None of claims 1-9, 11 and 25-26 and 28-29 require the instant composition/system to comprise cleavable nucleic acids that are end capped at the ‘3 hydroxy termini with a non-naturally occurring element selected from dideoxynucleotide (ddNTP), inverted dNTP, C3 spacer, or amino, nor does the claims recite instant composition/system with one or more elements of the system having functions not possessed by naturally occurring counterparts.
Step 2B. Does the claim recite additional elements that amount to significantly more than the judicial exception?
No- This part of the eligibility analysis evaluates whether the claim as a whole amount to significantly more than the recited exception, i.e., whether any additional element, or combination of additional elements, adds an inventive concept to the claim. MPEP 2106.05.
In the instant case, as detailed above, the claims are directed to a collection/system that comprise CRISPR-Cas complex comprising an effector protein and one or more guide RNAs; nucleic acid reporters having a 3’ termini block which encompass circular nucleic acids, and a polymerase. Each element of the instant collection/system of claims 1-9, 11 and 25-26 and 28-29 resemble and are indistinguishable from their naturally occurring counterparts.
Further, the CRISPR-Cas complex comprising an effector protein and one or more guide RNAs; and a polymerase and 3’ termini blocked nucleic acids that are termed reporter molecules in the composition/systems of claims 1-9, 11 and 25-26 and 28-29 do not have any markedly different characteristic as a collection than each of these elements have individually.
Claim 14
Further regarding claim 14, which is directed to a diagnostic device, this claim fails to recite any elements such that claim 14 is distinguishable from claim 1. The body of claim 14 does not recite any device or features that would be associated with an article of manufacture, Claim 14 recites the limitation “a source of electromagnetic radiation” which is not an article of manufacture/device and which encompasses a naturally occurring source such as the sun. Claim 14 therefore is rejected for the same reasons that claim 1 is rejected under 35 U.S.C. 101.
In view of the foregoing, claims 1-9, 11, 14 and 25-26 and 28-29 are rejected under 35 U.S.C. 101 for being directed to ineligible subject matter.
Concerning claims 15-17
Claims 15-17 are construed as being a collection of naturally occurring products separated into three containers (A)-(C), thereby rendering claims 15-17 as falling within the law of nature exception.
Specifically, the instant kit comprises: a first container comprising CRISPR-Cas comprising an effector protein and one or more guide RNAs and nucleic acid reporters having a 3’ termini block which encompass circular nucleic acids, and a polymerase, a second container comprising a detectable compound (e.g. a radioisotope, or a metal, or a polynucleotide) and a third container comprising a reductant (e.g. ascorbate or EDTA).
Furthermore, claims 15-17 do not include additional elements that are sufficient to amount to significantly more than the judicial exception.
The following inquiries are used to determine whether a claim is drawn to patent-eligible subject matter:
Step 1. Is the claim directed to a process, machine, article of manufacture, or composition of matter?
Yes- claims 15-17 are clearly directed to a composition comprising of products that are not distinguishable from their natural counterparts.
Step 2A, prong one. Does the claim recite a law of nature, a natural phenomenon, or an abstract idea (recognized judicial exceptions)?
Yes- claims 15-17 are directed to a collection/composition comprising of products that are not distinguishable from their natural counterparts.
The claimed CRISPR-Cas complex comprising an effector protein and one or more guide RNAs and nucleic acid reporters having a 3’ termini block which encompass circular nucleic acids, and a polymerase of the instant kit have no functional difference relative to their naturally occurring counterpart. The claimed detectable compound of the instant kit and the claimed reductant each broadly encompasses naturally occurring products.
MPEP 2106.04(b)(i) identifies nucleic acid sequence(s) having no structural or functional differences from naturally occurring nucleic acids as an example of a patent-ineligible natural product.
In addition, as discussed in MPEP 2016.04(b)(II), “[P]roduct of nature exceptions include both naturally occurring products and non-naturally occurring products that lack markedly different characteristics from any naturally occurring counterpart.” See Ambry Genetics, 774 F.3d at 760, 113 USPQ2d at 1244.
Step 2A, prong two. Does the claim as a whole integrate the recited judicial exception into a practical application of the exception?
Step 2A, prong two. Does the claim as a whole integrate the recited judicial exception into a practical application of the exception?
No - This evaluation is performed by identifying whether there are any additional elements recited in the claim beyond the judicial exception, and evaluating those additional elements individually and in combination to determine whether the claim as a whole integrates the exception into a practical application.
In the instant case, claims 15-17 do not appear to recite any additional elements that are distinguishable as non-naturally occurring and the claimed kit do not comprise at least a mixture composition that includes a non-naturally occurring element e.g. the CRISPR-Cas in container A is not recited to be in a composition/mixture together with a 3’ termini blocked-reporter nucleic acids, said blocker positioned at the 3’ termini is a non-naturally occurring element selected from dideoxynucleotide (ddNTP), inverted dNTP, C3 spacer, or amino. None of claims 15-17 recite a kit comprising a composition/mixture having one or more elements of the mixture having functions not possessed by a naturally occurring counterpart.
Claims 15-17 do not recite any feature/elements that amount to more than the judicial exception (i.e. the claims appear to be directed to a composition of matter that contains only elements that are not distinguishable as non-naturally occurring) and there are no additional elements to integrate the judicial exceptions into a practical application.
In view of the foregoing, claims 15-17 are rejected under 35 U.S.C. 101 for being directed to ineligible subject matter.
Claim Interpretation
Prior to analysis of the art, the claims must be construed. As noted in MPEP 2111, citing Phillips v. AWH Corp., 415 F.3d l303, 75 USPQ2d l321 (Fed. Cir. 2005), "During patent examination, the pending claims must be 'given their broadest reasonable interpretation consistent with the specification.' ".
Claim 18 recites the limitation of “a method for detecting a target nucleic acid using the CRISPR- Cas system as defined in claim 1, nucleic acid reporter molecules as defined in claim 1, nucleotides, and detectable compounds;
the method comprising:
i) targeting the effector protein with the guide sequence to the target sequence of the target nucleic acid, thereby activating the nucleic acid cleavage activity of the effector protein;
ii) cleaving the nucleic acid reporter molecules with the activated effector protein to generate nucleic acid fragments;
iii) adding the polymerase and the nucleotides to form a polynucleotide tail attached to the nucleic acid fragments; and
iv) binding the detectable compounds to the polynucleotide tail, thereby forming a detectable cluster; and
v) detecting the presence of the target nucleic acid by detecting the detectable cluster.
For the purposes of search, examination and in the interest of compact prosecution, claim 18 is construed as being directed to a method for detecting a target nucleic acid, the method comprising:
i) contacting (a) nucleic acid reporter molecules blocked at the 3' termini, and (b) a CRISPR-Cas system comprising an effector protein and one or more guide RNAs having a guide sequence, the guide sequence being capable of targeting the effector protein to a target sequence of a target nucleic acid, and the effector protein exhibiting target-activated nucleic acid cleavage activity capable of cleaving said nucleic acid reporter molecules to generate nucleic acid fragments;
wherein said contacting comprises targeting of the target sequence of the target nucleic acid by the effector protein with the guide sequence, thereby activating the nucleic acid cleavage activity of the effector protein; and cleaving of the nucleic acid reporter molecules by the activated effector protein to generate nucleic acid fragments;
ii) providing (c) nucleotides and (d) a polymerase exhibiting catalytic activity capable of transferring the nucleotides to the nucleic acid fragments to form polynucleotide tails attached to the generated nucleic acid fragments;
iii) binding detectable compounds to the polynucleotide tails attached to the generated nucleic acid fragments, thereby forming a detectable cluster; and
iv) detecting the presence of the target nucleic acid by detecting the detectable cluster.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 18-21 and 23-24 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.
The preamble of claim 18 is directed to a method for detecting target nucleic acid using the CRISPR-Cas system as defined in claim 1, nucleic acid reporter molecules as defined in claim 1.
The limitations “as defined in claim 1” in the preamble of claim 18 are confusing as claim 1 relates to a detection system and provides structural limitations for this system.
To obviate this rejection, Applicant may amend the process step(s) of claim 18 by reciting the structural limitations intended for the CRISPR-Cas system and nucleic acid reporters of claim 18 and how these features are to be used in one or in a series of active process steps for the purpose of detecting the presence of a target nucleic acid (see e.g. in the claim interpretation paragraph above).
Claims 19-21 and 23-24 are further rejected as they depend from claim 18.
Claim Rejections - 35 USC § 102
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 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.
Claims 1-2 and 4-7 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Carpenter et al. (WO2016/100955A2, pub. June 23, 2016: newly cited).
Regarding claims 1 and 4, Carpenter et al. teach a detection system comprising:
(i) nucleic acid reporter molecules, each molecule blocked at its 3’ termini,
(ii) a CRISPR-Cas system comprising an effector protein (e.g. Cas 9, a class 2 type) and one or more guide RNAs having a guide sequence, wherein the guide sequence is capable of targeting the effector protein to a target sequence of a target nucleic acid;
(iii) a polymerase exhibiting catalytic activity capable of transferring nucleotides to nucleic acid
fragments to form polynucleotide tails attached to the generated nucleic acid fragments.
Specifically, regarding claims 1-2, 4 and 6-7, Carpenter et al. teach:
a detection system comprising:
(i) nucleic acid reporter molecules, each molecule blocked at its 3’ termini (i.e. a library of DNA fragments tailed with poly-dG tails, said tail being resistant to exonuclease BAL-31 treatment: see para [0198]);
(ii) terminal deoxynucleotidyl transferase, provided for attaching the poly-dG tails (see para [0198]); and
(iii) CRISPR/Cas system provided for degrading DNA not of interest, and generating for intact DNA fragments having target sequence of interest, the intact fragments become substrates for further exonuclease BAL-31 treatment (see para [0198]; see also para [0148], [0152]); and
(iv) exonuclease BAL-31 unable to digest poly-dG tails, but does digest poly-dA, poly-dC, poly-dT tails (see para [0198]).
Regarding claim 5, Carpenter et al. teach RNA polymerase (para [0145]).
Accordingly, the instant claims 1-2 and 4-7 are anticipated by Carpenter et al.
Claims 1-2, 4-7, 11 and 14 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Wang et al. (August 28, 2019, Analytical chemistry, 91(19), pp.12156-12161: newly cited).
Regarding claims 1-2 and 4-7, Wang et al. teach a detection system (see pg 12157, left col., 3rd paragraph and pg 12157, right col, “Results and Discussion” and pg 12158 paragraphs below Fig. 2 of left col),
the system comprising:
(i) nucleic acid reporter molecules, each reporter molecule blocked at its 3’ termini (see ssDNA reporter comprising a fluorophore and quencher (FRET) pair, wherein the 3’ end of reporter nucleic acid is labeled with quencher: pg 12158, see abstract’s illustration; see also ssDNA disclosed on pg 12157, left col., Materials paragraph of section entitled “Experimental Section” and 200 nM quenched fluorescent ssDNA reporter disclosed on pg 12157, right col., section entitled “Cas12a-Mediated Cleavage Assay” and section entitled “Cas12aVDet Method”);
(ii) a CRISPR-Cas system comprising an effector protein (e.g. Cas 12a, a class 2 type), and one or more guide RNAs having a guide sequence, wherein the guide sequence is capable of targeting the effector protein to a target sequence of a target nucleic acid, and the effector protein exhibiting target-activated nucleic acid cleavage activity capable of cleaving said nucleic acid reporter molecules to generate nucleic acid fragments;
(pg 12157, right col., sections entitled “Cas12a-Mediated Cleavage Assay” and “Cas12aVDet Method”); Wang et al. teach crRNA and Cas12a capable of cleaving blocked nucleic acid reporter molecules (pg 12157, section entitled “Cas12a-Mediated Cleavage Assay”);
(iii) a polymerase that preamplifies target nucleic acids prior to CRISPR-Cas cleavage (see enzyme pellet disclosed in sections of pg 12157, right col., entitled “Standard RPA Reaction Assay” and “Cas12aVDet Method”; T7 RNA polymerase member of TwistAmp pellet, see pg 12157, section entitled “Standard RPA Reaction assay” and section entitled “Cas12aVDet Method”).
The polymerase of Wang et al. which is present in the one-pot detection system taught by Wang et al., is capable of transferring nucleotides to nucleic acid fragments of the 3’-termini blocked reporter molecules to form polynucleotide tails attached to the generated nucleic acid fragments
Regarding claims 1-2, Wang et al. further teach an alternative DETECTR detection assay which provides a CRISPR-Cas system, nucleic acid reporter molecules, each reporter molecule blocked at its 3’ termini and a polymerase capable of transferring nucleotides to CRISPR/Cas system generated nucleic acid fragments to form polynucleotide tails attached to CRISPR/Cas system generated nucleic acid fragments (pg 12159, last para of left col and all text of right col of page 12159).
Wang et al. teach comparison of fluorescence-based readout to turbidity-based readout. Turbidity arises in the presence of long poly(dT) or poly(U) oligonucleotides (pg 12159, right col., 2nd para).
Regarding claims 5-7, Wang et al. teach RPA providing nucleotides and RNA polymerase (member of TwistAmp pellet, see pg 12157, section entitled “Standard RPA Reaction assay” and section entitled “Cas12aVDet Method”).
Regarding claim 11, Wang et al. teach a microbial target nucleic acid (pg 12157, right col., section entitled “Detection of mycoplasma contamination in cell culture with Cas12aVDt”).
Regarding claim 14, Wang et al. teach a Blue Light Gel imager device (source of target nucleic acid) and its’ use for providing blue light and the observing of bright green fluorescence signal by naked eye under the blue light (pg 12156, abstract and pg 12157, right col., Cas12aVDet Method).
Accordingly, the instant claims 1-2, 4-7, 11 and 14 are anticipated by Wang et al.
Claim 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 1-9, 11, 14-17, 25 and 28-29 are rejected under 35 U.S.C. 103 as being unpatentable over Li et al. (April 24, 2018, Cell discovery, 4(20), pp. 1-4: previously cited) in view of Luo et al. (2017, Talanta, 169, pp.57-63: previously cited) and Polansky (US2004/0023207: previously cited).
Li et al. (claims 1-7, 11-12, 14-15, 17, 25 and 27-29)
Regarding claims 1 and 4, Li et al. teach a nucleic acid detection system (see pg 3, legend of Figs. 1a and 1b). The detection system of Li et al. comprises:
(a) a CRISPR-Cas system comprising an effector protein (i.e. Cas9 endonuclease) and one or more guide RNAs having a guide sequence (i.e. sgRNA), the guide sequence being capable of targeting the effector protein to a target sequence of a target nucleic acid, and the effector protein exhibiting target-activated nucleic acid cleavage activity capable of cleaving nucleic acid reporter molecules to generate nucleic acid fragments (see pg 3, legend of Figs. 1a and 1b and see pg 1, last para of left col and pg 1, all text of right col.; Li et al. teach their detection system comprising Cas12a, crRNA (crRNA-T1), target DNA (pUC18-T1) and quenched fluorescent ssDNA (HEX-N12-BHQ1).
(b) The detection system of Li et al. teach nucleic acid reporter molecules that are blocked at the 3' termini (i.e. quenched fluorescent ssDNA reporter (e.g., HEX-N12-BHQ1) which comprises a BHQ1 3’ termini block that prevents polymerase extension).
(c) The detection system of Li et al. further comprises a polymerase (see pg 3, legend of Fig. 1a which discloses a PCR or isothermal amplification).
More specifically, regarding claims 1, 3-4 and 25 and 28-29, Li et al. teach a method termed HOLMES (one-HOur Low-cost Multipurpose highly Efficient System), used for fast detection of target DNA as well as target RNA. The method exploits the property of Cas12a, a class 2 type V-A CRISPR-Cas to cleave non-targeted ssDNAs (in their case, quenched fluorescent ssDNA reporter (e.g., HEX-N12-BHQ1) provided as a probe is cleaved) upon the formation of the Cas12a/crRNA/target DNA ternary complex.
It is noted here that Cas12a taught by Li et al. corresponds to the instant effector protein which has the claimed functional abilities recited in claim 1, that the cRNA taught by Li et al. corresponds to the instant one or more guide RNA, and that the non-targeted quenched fluorescent ssDNA reporter taught by Li et al. corresponds to the instant nucleic acid reporter molecules which is susceptible to cleavage in a manner as recited by claim 1 (pg 1, last para of left col and pg 1, all text of right col.).
Regarding claims 1 and 3 and 28-29, Li et al. teach “in HOLMES, if a target DNA exists in the reaction system, the Cas12a/crRNA binary complex forms a ternary complex with the target DNA, which will then trans-cleave non-targeted ssDNA reporter in the system, illuminating the HEX fluorescence (or any other fluorescence)” (pg 1, last para of left col and pg 1, all text of right col.).
Regarding claims 1 and 5-7, Li et al. teach a polymerase that is capable of transferring nucleotides to cleave nucleic acid fragments so as to form nucleic acid fragments comprising of polynucleotide tails (see pg 1, right col., 2nd para, see PCR that occurs after HOLMES).
Li et al. teach DNA polymerase and/or RNA polymerase via PCR that occurs after HOLMES: see pg 1, right col., 2nd para). The PCR requires a dNTP mix therefore Li et al. teach a system comprising nucleotides and dTTP.
Regarding claim 14, Li et al. teach a diagnostic device (see Supplementary document of Li et al. on pg 2, last para of the page, which discloses that fluorescence emission was excited at 535 nm and detected at 556 nM using a Varioskan Flash from Thermo Fisher Scientific).
Regarding claim 15, Li et al. teach:
(i) a CRISPR-Cas12a system in a first container (see pg 3, legend of Figs. 1a-1b);
(ii) a detectable compound in a second container (see pg 3, legend of Figs. 1a-1b which discloses quenched fluorescent ssDNA (HEX-N12-BHQ1); and
(iv) a source of electromagnetic radiation (Supplementary document of Li et al. on pg 2, last para of the page teach that fluorescence emission was excited at 535 nm and detected at 556 nM using a Varioskan Flash from Thermo Fisher Scientific).
Regarding claims 2 and 17, Li et al. teach the detectable compound comprises a polynucleotide (see pg 3, legend of Figs. 1a-1b which discloses quenched fluorescent ssDNA (HEX-N12-BHQ1: a detectably labeled polynucleotide probe capable of hybridizing to polynucleotides that are fully or partially complementary).
Regarding claim 11, Li et al. teach target nucleic acid is a microbial nucleic acid (detecting DNA viruses (e.g., pseudorabies virus (PRV) and RNA viruses (e.g., Japanese encephalitis virus (JEV): pg 2, last para of left col and pg 3, 1st para of the right col below the legend of Fig. 1).
Omitted from Li et al. (claims 8-9, 15-17)
Regarding claims 8-9, Li et al. do not teach polynucleotide tails comprising of metal-binding sites capable of chelating metal, wherein the metal is copper.
Regarding claims 15-17, Li et al. do not teach a kit in parts.
Regarding claim 15 and 16, Li et al. do not teach:
(iii) a reductant in a third container or (iii) a detectable compound comprising a metal, respectively.
Luo et al. (claims 1-3, 5-9, 12, 14-17, 25, 27, 29)
Regarding claims 1-3 and 5-9, Luo et al. is directed to a label-free and ultrasensitive assay method that combines the special functions of DNase I and terminal deoxynucleotidyl transferase (TdT) to detect the presence of DNase I activity in diluted serum (see abstract and pg 58, left col., last para and pg 58, right col., 1st para and pg 58, right col, section 2.2).
Accordingly, Luo et al. teach a detection system comprising:
(b) a polymerase that is terminal deoxynucleotidyl transferase (TdT), which is able to add multiple random nucleotides to the 3'-hydroxyl terminus of a DNA strand in a template-independent manner (abstract and pg 58, right col., section 2.3).
Regarding claims 1-3 and 6-9 and 25 and 27, the system of Luo et al. further comprises ‘3-phosphorylated DNA primer, blocked from TdT polymerization until the presence of DNase I, which digests the primer to release ‘3-hydroxylated fragments (3-phosphorylated DNA primer is construed as being equivalent to the instant nucleic acid reporter molecules as claimed by claim 12) (see abstract and pg 58, left col., last para and pg 58, right col., 1st para and pg 58, right col, section 2.2).
Luo et al. teach the tailing of digested primer by TdT in a dTTP pool to generate superlong poly(T) tails attached to cleaved ssDNA primer, which become template for CuNPs formation since poly T-CuNPs form through chemical reduce of thymine-complexed Cu2+ to Cu° by reducing agents along the poly T DNA scaffold (abstract and pg 58, right col, section 2.3 and pg 58, left col., 2nd and 3rd paragraphs).
Regarding claim 3, Luo et al. teach a fluorescence detection system (see pg 59, left col., section 2.4 and pg 59, right col., Fig. 1) as the fluorescence of the generated superlong poly(T)-CuNPs are measured.
Regarding claim 14, Luo et al. teach providing a diagnostic device for assay (see pg 59, left col., section 2.4: wherein Luo et al. teach QuantaMaster™ fluorescence spectrophotometer (PTI, Canada), which excites sample at 350 nm. Luo et al. teach emission wavelength range of 500–690 nm and a slit width of 5 nm. Luo et al. further teach fluorescent images were acquired by a WD-9403 imaging system (Shangai China) under UV irradiation).
Regarding claims 15-16, Luo et al. teach:
(ii) copper nanoparticles (CuNPs) in a container (pg 59, left col., section 2.4); and
(iii) sodium ascorbate as reductant in a container (pg 59, left col., section 2.4) as well
(iv) the optional source of electromagnetic radiation (pg 59, left col., section 2.4: wherein Luo et al. teach QuantaMaster™ fluorescence spectrophotometer (PTI, Canada), which excites sample at 350 nm. Luo et al. teach emission wavelength range of 500–690 nm and a slit width of 5 nm. Luo et al. further teach fluorescent images were acquired by a WD-9403 imaging system (Shangai China) under UV irradiation).
Regarding claims 2, 12, 17, 25, 27, Luo et al. teach the detectable compound comprises a polynucleotide (see pg 58, right col., section 2.1 for the nucleotide sequence of Luo’s primer DNA comprising a ‘3-PO4 block; AND see pg 59, left col., section 2.4 which teach super long poly(T)-CuNPs comprising the primer DNAs).
Omitted from Li et al. and Luo et al. (claims 15-17)
Regarding claims 1 and 8-9 and 15-17, Luo et al. do not teach their system for cleaving off-target single stranded primer reporter molecules as a CRISPR-Cas system and therefore do not teach a first container comprising (i) a CRISPR-Cas system.
Instead, Luo et al. teach the use of a DNAse-I based system for cleaving single stranded primer reporter molecules.
Regarding claims 15-17, Li et al. and Luo et al. do not teach any of elements (i)-(iv) noted above, as members of a kit in parts.
Polansky (claims 15-17)
Polansky (US2004/0023207) taught (paragraph [0919]): “Well known advantages of commercial kits include convenience and reproducibility due to manufacturing standardization, quality control and validation procedures.”
It would have been prima facie obvious to an ordinary skilled artisan, wanting to detect nucleic acids via a CRISPR-Cas based detection system, before the effective filing date of the instant invention, to apply the teachings of Li et al., to the detection method as taught by Luo et al., thereby modify the detection method as taught by Luo et al. which is directed at signal generation associated with DNase I cleavage activity into a method that is directed at signal generation associated with CRISPR-Cas cleavage system.
The ordinary skilled artisan would have been readily privy to the fact that DNAse-I and CRISPR-Cas both play a functionally equivalent role as elements useful to cleave an ssDNA (off-target) or nucleic acid fragment substrates and generate unblocked fragments that would be available for further extension and would have expected the substitution CRISPR-Cas for DNase-I to have a reasonable expectation of success.
Furthermore, the ordinary skilled artisan would have been motivated before the effective filing date of the instant invention, to combine into various containers, each container comprising one element useful for performing the modified CRISPR-Cas based detection method of Li et al. and Luo et al. including a CRISPR-Cas12a system, CuNPs/CuSO4, sodium ascorbate/ascorbic acid for benefits taught or suggested by Polansky e.g. convenience of having elements of practicing a detection method in one place.
In view of the combined teachings of all of the cited reference(s), the instant claims 1-9, 11, 14-17, 25 and 27-29 are prima facie obvious.
Claims 18-21 and 23-24 are rejected under pre-AIA 35 U.S.C. 103(a) as being unpatentable over Li et al. (April 24, 2018, Cell discovery, 4(20), pp. 1-4: previously cited) in view of Luo et al. (2017, Talanta, 169, pp.57-63: previously cited) as applied for claim 1 above.
The teachings of Li et al. and Luo et al. as it relates to claim 1 are noted in the rejection above and are further applied here.
Li et al. (claims 18, 21 and 23-24)
Regarding claim 18, Li et al. teach a method for detecting a target nucleic acid using a CRISPR-Cas system having an effector protein (i.e. Cas12a) and sgRNA as the guide RNA; using nucleic acid reporter molecules that are detectable compounds (i.e. HEX-N12-BHQ1), nucleotides (dNTP) and a polymerase.
The method of Li et al. comprise:
i) targeting the effector protein with the guide sequence to the target sequence of the target nucleic acid, thereby activating the nucleic acid cleavage activity of the effector protein (pg 3, text of the legend of Figs. 1a-1b);
ii) cleaving the nucleic acid reporter molecules with the activated effector protein to generate nucleic acid fragments (pg 3, text of the legend of Figs. 1a-1b).
Regarding claim 21, Li et al. teach a method for detecting and/or monitoring the target nucleic acid in a sample from a subject (pg 3, left col., 2nd para and last para: wherein the method is used to DNA virus, pseudorabies virus (PRV) and RNA virus, Japanese encephalitis virus (JEV)).
Regarding claim 23, Li et al. teach target nucleic acids is obtained from a biological sample or an environmental sample (see Supplementary document on pg 1 which teach DNA extraction from HEK293T cells).
Regarding claim 24, Li et al. teach presence of the target nucleic acid is diagnostic for a disease state (pg 3, left col., last para: which suggests their detection method are for concluding the presence of pseudorabies and Japanese encephalitis).
Luo et al. (claims 18-20, 23)
Regarding claim 18, Luo et al. teach a method for detecting a target nucleic acid using a DNase-I based system; nucleic acid reporter molecules (single stranded primer DNA having a ‘3 PO4 blocker), detectable compounds (i.e. copper nanoparticles, CuNPs), nucleotides (dTTP) and a polymerase specifically, terminal deoxynucleotidyl transferase (TdT), which is able to add multiple random nucleotides to the 3'-hydroxyl terminus of a DNA strand in a template-independent manner.
The method of Luo et al. comprise:
i) activating the nucleic acid cleavage activity of DNase-I (by providing DNase-I to ‘3-blocked primers to cleave block from the primers) (pg 58, right col., section 2.2; and pg 58, left col., last para and pg 58, right col, 1st para);
ii) cleaving the nucleic acid reporter molecules (by removing the block on the single stranded primer DNA having a ‘3 PO4 blocker and generating nucleic acid fragments) (pg 58, right col., section 2.2; and pg 58, left col., last para and pg 58, right col, 1st para);
iii) adding the polymerase (TdT) and the nucleotides (a pool of dTTP) to generate a polynucleotide tail attached to the nucleic acid fragments (generating primer fragment with poly(T)tails) (pg 58, right col., section 2.3 and pg 59, left col., section 2.4; and pg 58, left col., last para and pg 58, right col, 1st para); and
iv) binding the detectable compounds to the polynucleotide tail, thereby forming a detectable cluster (adding copper nanoparticles in the presence of sodium ascorbate to generate superlong poly(T)-CuNPs)(pg 59, left col., section 2.4; and pg 58, left col., last para and pg 58, right col, 1st para).
Regarding claims 19-20, Luo et al. teach a detectable metal cluster (i.e. their superlong poly(T)-CuNPs) (see pg 59, right col., Fig. 1 and pg 59, left col., section 2.4) and/or a detectable hybrid cluster comprising a labelled polynucleotide (pg 59, left col., section 2.4).
Regarding claim 23, Luo et al. teach target nucleic acids obtained from an environmental sample (pg 58, right col., section 2.1).
It would have been prima facie obvious to an ordinary skilled artisan, wanting to detect nucleic acids using a CRISPR-Cas based detection system, before the effective filing date of the instant to apply the teachings of Li et al. to the detection method as taught by Luo et al., thereby modify the detection method as taught by Luo et al. into a label-free method that provides a CRISPR-Cas system rather than a DNAse-I based detection system.
The ordinary skilled artisan would have been readily privy to the fact that DNAse-I and CRISPR-Cas both play a functionally equivalent role as elements useful to cleave an ssDNA (off-target) and would have expected the substitution of CRISPR-Cas for DNase-I to have a reasonable expectation of success.
In view of the combined teachings of all of the cited reference(s), the instant claims 18-21 and 23-24 are prima facie obvious.
Conclusion
No claims are currently allowed.
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.
Correspondence
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OLAYINKA A. OYEYEMI
Examiner
Art Unit 1681
/OLAYINKA A OYEYEMI/Examiner, Art Unit 1681
/GARY BENZION/Supervisory Patent Examiner, Art Unit 1681
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