17413802
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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 05/18/2026 has been entered.
Election/Restrictions
Applicant’s election without traverse of Group I (encompassing claim 1, as amended and claims 115-132 in the reply filed on 06/11/2025) is acknowledged.
Claim 51 is withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to nonelected group II, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 06/11/25.
Claims Status
Claims 1, 51, 116-118,120 and 122-132 are pending.
Claim 51 is withdrawn.
Claims 1, 116-118,120 and 122-132 are currently under examination.
Priority
This application is a 371 of PCT/US20/60333 filed on 11/13/2020 which claims benefit of 62/935,705 filed on 11/15/2019. Accordingly, the priority date of instant claims is determined to be 11/15/2019., the filing date of 62/935,705.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1, 116-118,120 and 122-132 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
The claims are broadly drawn to two or more detection oligonucleotides, wherein each detection oligonucleotide comprises in order from 5' to 3':(i) a first target binding sequence,(ii) a linking region, and (iii) a second target binding sequence, wherein the linking region comprises a guide RNA binding site, wherein, when the first target binding sequence and the second target binding sequence bind a target sequence, the 5' and 3' terminal nucleotides of the detection oligonucleotide are disposed immediately adjacent to one another or are separated by a gap region comprising between 1 and 100 nucleotides, wherein each detection oligonucleotide binds a unique target sequence of interest in the same genome, and wherein each detection oligonucleotide comprises the same guide RNA binding site.
Relevant to the lack of particular structural limitations in the rejected claims drawn to detection oligonucleotides, MPEP 2163 states:
The claimed invention as a whole may not be adequately described if the claims require an essential or critical feature which is not adequately described in the specification and which is not conventional in the art or known to one of ordinary skill in the art.
In the case of the instant claims, the functionality of (i) a first target binding sequence,(ii) a linking region, and (iii) a second target binding sequence, wherein the linking region comprises a guide RNA binding site, wherein, the first target binding sequence and the second target binding sequence bind a target sequence, such as binding target, linking, guide RNA binding as critical features of the claimed system.
The specification teaches “proximity dependent probes, each set comprising two or more
Proximity dependent probes, each proximity dependent probe comprising a guide polynucleotide recognition sequence and a target binding sequence” (Para. 7). However, the specification does not teach the structure of detection oligonucleotides that are capable of binding a target sequence and guide RNA and linking regions.
While the skilled artisan may be capable of developing detection oligonucleotides that bind a target sequence and guide RNA and link regions, possession may not be shown by merely describing how to obtain possession of members of the claimed genus or how to identify their common structural features. See University of Rochester, 358 F.3d at 927, 69 USPQ2d at 1895.
Further, while a particular agent may bind a target sequence and guide RNA and link regions, for example, there is no indication, teaching, guidance, or expectation that ALL detection oligonucleotides bind a target sequence and guide RNA and link regions as is broadly claimed.
The claims encompass a genus of structurally undefined detection oligonucleotides which require a specific functionality. However, the specification fails to teach how to distinguish members of the claimed genus of nucleic acid molecules which possess the claimed functionality: detection oligonucleotides that bind targets and guide RNA, and link regions as broadly claimed, from nonmembers.
In analysis of the claims for compliance with the written description requirement of 35 U.S.C. 112, first paragraph, and particularly for claims drawn to a genus, the written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species. A "representative number of species" means that the species which are adequately described are representative of the entire genus. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus. See AbbVie Deutschland GmbH & Co., KG v. Janssen Biotech, Inc., 759 F.3d 1285, 1300, 111 USPQ2d 1780, 1790 (Fed. Cir. 2014) (Claims directed to a functionally defined genus of antibodies were not supported by a disclosure that "only describe[d] one type of structurally similar antibodies" that "are not representative of the full variety or scope of the genus."). The disclosure of only one species encompassed within a genus adequately describes a claim directed to that genus only if the disclosure "indicates that the patentee has invented species sufficient to constitute the gen[us]." See Enzo Biochem, 323 F.3d at 966, 63 USPQ2d at 1615.
Further, University of California v. Eli Lilly and Co., 43 USPQ2d 1398, 1404, 1405 held that:
To fulfill the written description requirement, a patent specification must describe an invention and do so in sufficient detail that one skilled in the art can clearly conclude that "the inventor invented the claimed invention." Lockwood v. American Airlines, Inc., 107 F.3d 1565, 1572, 41 USPQ2d 1961, 1966 (1997); In re Gosteli, 872 F.2d 1008, 1012, 10 USPQ2d 1614, 1618 (Fed. Cir. 1989) (" [T]he description must clearly allow persons of ordinary skill in the art to recognize that [the inventor] invented what is claimed."). Thus, an applicant complies with the written description requirement "by describing the invention, with all its claimed limitations, not that which makes it obvious," and by using "such descriptive means as words, structures, figures, diagrams, formulas, etc., that set forth the claimed invention." Lockwood, 107 F.3d at 1572, 41 USPQ2d at 1966.
An adequate written description of a DNA, such as the cDNA of the recombinant plasmids and microorganisms of the '525 patent, "requires a precise definition, such as by structure, formula, chemical name, or physical properties," not a mere wish or plan for obtaining the claimed chemical invention. Fiers v. Revel, 984 F.2d 1164, 1171, 25 USPQ2d 1601, 1606 (Fed. Cir. 1993). Accordingly, "an adequate written description of a DNA requires more than a mere statement that it is part of the invention and reference to a potential method for isolating it; what is required is a description of the DNA itself." Id. at 1170, 25 USPQ2d at 1606.
Thus, considering the breadth of the detection oligonucleotides required by the claims, their specific required functionalities, and the teachings of the instant specification, it is the conclusion that the specification does not provide an adequate written description of the broadly claimed subject matter. So one of skill in the art cannot envision the detailed chemical structure of the detection oligonucleotides thereof encompassed by the claimed.
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 1, 116-118,120 and 122-132 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 1, 130 and 131 are indefinite over the limitations “binding sequence”, “binding site”, “bind a target sequence”, and “binds a unique target sequence”. It is unclear as to whether the intended binding is direct sequence specific binding (for example hybridization) or any molecular mechanism of indirect binding. Claims 116-118, 120 and 122-129 depend on claim 1. Claim 132 depends on claim 131.
Claim 1 is indefinite over the limitations “a guide RNA comprising a spacer sequence capable of hybridizing to the guide RNA binding site”. It is unclear whether the “guide RNA binding site”(ln 15-16) is designed to hybridize to the target DNA sequence, as traditional guide sequences, or if the guide sequence is specific to only the “linking region” (ln 8) of the detection oligonucleotide. Claims 116-118, 120 and 122-129 depend on claim 1. Furthermore, the structural relationship of the guide RNA to target in regards to the structural relationship of guide RNA to the detection oligonucleotide is not clear, especially in the context of any molecular mechanism of indirect binding comprising “detection oligonucleotide”.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1, 116-118, 120 and 122-129 are rejected under 35 U.S.C. 103 as being unpatentable over Smargon et al. (“Smargon”; US Patent App. Pub. No. US 20170211142 A1, July 27, 2017) in view of Zhang et al. (“Zhang”; (2018). Direct visualization of single-nucleotide variation in mtDNA using a CRISPR/Cas9-mediated proximity ligation assay. Journal of the American Chemical Society, 140(36), 11293-11301., August 20, 2018).
Claim interpretations: The claims are subject to the following interpretation: The claims are drawn to a “system.” The specification recites a “system” wherein the “system” is defined in terms of structural limitations. In addition, the claims recite structural limitations of the “system.” Thus, the “system” is interpreted to encompass any collection of reagents and parts used together that are not necessarily part of a completely integrated single unitary device. Any further interpretation of the word is considered an “intended use” and does not impart any further structural limitation on the claimed subject matter.
Smargon discloses systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered DNA or RNA-targeting systems comprising a novel DNA or RNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA. (Abstract)
Regarding claim 1, Smargon teaches system “comprising i) a Type VI-B CRISPR-Cas effector protein, and ii) a Type VI-B CRISPR-Cas crRNA, wherein the crRNA comprises a) a guide sequence that is capable of hybridizing to a target RNA sequence, and b) a direct repeat sequence. The Type VI-B CRISPR-Cas effector protein forms a complex with the crRNA, and the guide sequence directs sequence-specific binding of the complex to the target RNA sequence, whereby there is formed a CRISPR complex comprising the Type VI-B CRISPR-Cas effector protein complexed with the guide sequence that is hybridized to the target RNA sequence” (Para. 14). Thus, Smargon suggests a nucleic acid detection system comprising: a CRISPR-Cas protein, and a CRISPR-Cas guide RNA sequence comprising a spacer capable of hybridizing to the guide RNA binding site and comprising a sequence capable of forming a complex with the CRISPR-Cas protein.
However, Smargon does not explicitly teach a two or more detection oligonucleotides, wherein each detection oligonucleotide comprises in order from 5' to 3': (i) a first target binding sequences (ii) a linking region, and(ii) a second target binding sequence, wherein the linking region comprises a guide RNA binding site, wherein, when the first target binding sequence and the second target binding sequence bind a target sequence, the 5' and 3' terminal nucleotides of the detection oligonucleotide are disposed immediately adjacent to one another or are separated by a gap region comprising between 1 and 100 nucleotides, wherein each detection oligonucleotide binds a unique target sequence of interest in the same genome, and wherein each detection oligonucleotide comprises the same guide RNA binding site.
Zhang discloses CRISPR/Cas9- mediated proximity ligation assay (CasPLA) to image SNV in mtDNA at single-molecule resolution. This method uses two Cas9 probes to target a specific mtDNA sequence, followed by proximity ligation and in situ rolling circle amplification (RCA) to reveal the spatial localization of individual wild-type and mutated mtDNAs in single cells. (Pg. 11293, Introduction, Col. 2, Para. 2)
Regarding claim 1, Zhang teaches a system comprising “DNA proximity probes” (Pg. 11294, Col. 1, Para. 1; Scheme 1 see below) and “two distinct circularized DNAs for RCA (Figure 5a)” (Pg. 1297, Col. 1, Para. 2; Fig. 5a). Zhang teaches a system as illustrated in Scheme 1 that depicts a proximity ligation probe, with a first target binding sequence at the 5’ end, a linking region, and a second target binding sequence at the 3’ end disposed adjacent separated by a gap (see annotation of interpretation on Scheme 1); the 5’ and 3’ terminal sequence of the proximity probe indirectly bind the target genome through direct interaction with other probes and the interaction of the other probes with sgRNA and the interaction of the sgRNA with the target genome.
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The “proximity ligation probes” reads on one or more detection oligonucleotides and the MPEP states "Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." (MPEP 2144.05). It would be obvious to the ordinary artisan to optimize the system to use two detection oligonucleotides such as in Fig. 5a. Zhang teaches a system as illustrated in Scheme 1 that depicts the linking region comprising a region interacting with guide RNA, thus reads on a guide RNA binding site of the detection oligonucleotide. The linking region reads on any region that may link the 5’ end to the 3, thus the region highlighted in Scheme 1 reads on a linking region. Zhang teaches a system comprising “CasPLA for Nuclear Genome Imaging… The following CasPLA procedure was the same with CasPLA for mtDNA imaging” (Pg. 11299, Col. 2, Para. 2) and “CasPLA probes targeting mutated KRAS gene, followed by proximity ligation and RCA” (Pg.11298, Col. 1 Para. 3). Hence, it would be obvious to the skilled artisan to detect a sequence of the genome using the detection oligonucleotide. Zhang teaches a system as illustrated in Fig. 5a of simultaneous imaging of wild-type (A) and mutant (G) sequences, where each proximity probe binds a unique target sequence of interest in the same cells. (Fig. 5a, see below). Zhang teaches a system as illustrated in Fig. 5a where each proximity probe appears to comprise a similar guide RNA site on the left side that interacts with guide RNA.
Thus, Smargon and Zhang suggest a system comprising detection oligonucleotides, wherein each detection oligonucleotide comprises in order from 5' to 3': (i) a first target binding sequences (ii) a linking region, and(iii) a second target binding sequence, wherein the linking region comprises a guide RNA binding site, wherein, when the first target binding sequence and the second target binding sequence bind a target sequence, the 5' and 3' terminal nucleotides of the detection oligonucleotide are disposed immediately adjacent to one another or are separated by a gap region comprising between 1 and 100 nucleotides, wherein each detection oligonucleotide binds a unique target sequence of interest in the same genome, and wherein each detection oligonucleotide comprises the same guide RNA binding site.
Smargon and Zhang are both considered to be analogous to the claimed invention because they are in the same field of targeting nucleic acids with CRISPR effector proteins and guide sequences. Smargon suggests that “numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention” (Para. 1239). Therefore, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to have modified the nucleic acid detection system comprising CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA as taught by Smargon to incorporate the system comprising CRISPR/Cas-9 (Class 2) system, detection oligonucleotides comprising a first target binding sequence, a linking region, and a second target binding sequence, and guide RNA to target a specific sequence(s) as taught by Zhang and provide a system comprising a CRISPR-Cas protein, detection oligonucleotides comprising a first target binding sequence, a linking sequence, and a second target binding sequence, and a guide RNA. These claim elements were known in the art and one of skill in the art could have combined these elements by known methods with no change in their respective functions, and the combination would have yielded the predictable outcome according to the limitations of claim 1. Doing so would allow for increased signal to noise detection of probes and efficient identification and specific recognition of CRISPR-Cas system for target sequence(s).
The teachings of Smargon and Zhang are documented above in the rejection of claim 1 under 35 U.S.C. 103. Claim 116, 118, 120, 122, 125, 127-129 depends on claim 1. Claim 117 depends claim 116, which depends on claim 1. Claim 124 depends claim 118, which depends on claim 1. Claim 123 depends claim 122, which depends on claim 1. Claim 126 depends claim 125, which depends on claim 1.
Regarding claim 116, Smargon teaches a system wherein “the crRNA library consists of targets that tile the entire RNA viral genome one base pair at a time” (Para. 1202). Smargon teaches a system wherein “Targeting of putative control elements on the other hand (e.g. by tiling the region of the putative control element” (Para. 867). Thus, Smargon and Zhang suggest a system wherein the target binding sequences of the plurality of detection oligonucleotides are designed to tile across distinct regions of the target sequence.
Regarding claim 117, Smargon teaches a system wherein “comprises two or more Type VI-B CRISPR-Cas crRNAs” (Para. 17). Two or more crRNAs is interpreted as comprising 2 to 50, 2 to 200, or 2 to 1000 detection oligonucleotides. Thus, Smargon and Zhang suggest a system wherein the plurality of detection oligonucleotides comprise 2 to 50 detection oligonucleotides, 2 to 200 detection oligonucleotides, or 2 to 1000 detection oligonucleotides.
Regarding claim 118, Smargon teaches a system wherein “the crRNA library consists of targets that tile the entire RNA viral genome one base pair at a time” (Para. 1202).
Regarding claim 119, Smargon teaches a system wherein “the crRNA library consists of targets that tile the entire RNA viral genome one base pair at a time” (Para. 1202). Smargon teaches a system wherein “wherein the crRNA comprises a) a guide sequence that is capable of hybridizing to a target RNA sequence, and b) a direct repeat sequence” (Para. 14). Smargon teaches a system wherein “the terms “CRISPR repeat,” “direct repeat,” “repeat sequence,” or “repeat” have the conventional meaning as used in the art, i.e., multiple short direct repeating sequences, which show very little or no sequence variation within a given CRISPR array” (Para. 223). Thus, Smargon and Zhang suggest a system wherein the detection oligonucleotides are designed to bind target sequences tiled across a region of the genome.
Regarding claim 120, Zhang teaches a system wherein “we designed a CRISPR/Cas9 mediated proximity ligation assay” (Pg. 11293, Col. 2, Para. 2; Scheme 1 see below) Thus, Smargon and Zhang suggest a system wherein the detection oligonucleotide is circularized by ligation, splinted ligation, hybridization, or proximity extension.
Regarding claim 122, Smargon teaches a system wherein “the repeats are short elements that occur in clusters that are regularly spaced by unique intervening sequences” (Para. 787). “The mRNA contained in the extracted nucleic acid sample is then detected by amplification procedures” (Para. 808). Smargon teaches a system wherein “amplification means any method employing a primer and a polymerase capable of replicating a target sequence with reasonable fidelity” (Para. 809). “Amplification procedures” are interpreted as general and comprising Forward and Reverse primers and polymerase to bind to regions of the sequence to amplify the region of interest. Thus, Smargon and Zhang suggest a system wherein the linking region further comprises a forward primer binding sequence, a reverse primer binding sequence, an RNA polymerase binding sequence, and/or a barcode.
Regarding claim 123, Smargon teaches a system wherein “the expression of the guide sequence is under the control of the T7 promoter and is driven by the expression of T7 polymerase” (Para. 836). Thus, Smargon and Zhang suggest a system wherein the intervening sequences comprise a T7 promoter, wherein the T7 promoter is oriented to express the CRISPR-Cas guide RNA binding site of the detection oligonucleotide.
Regarding claim 124, Smargon teaches a system wherein “target antibiotic resistance gene” (Para.1212). Thus, Smargon and Zhang suggest a system wherein the region of the genome comprises an antibiotic resistance gene, a repetitive genetic element, a genomic region conserved across one or more genus or species, or a species-specific genomic region.
Regarding claims 125 and 126, Smargon teaches a system wherein “In an embodiment of the invention, the single effector protein comprises a Class 2 Type VI-B effector protein. Class 2 Type VI-B effector proteins include two subgroups, Type VI-Bl and Type VI-B2, which are also referred to as Group 29 proteins and Group 30 proteins” (Para. 10) and “Group 29 and group 30 systems comprise a large single effector… termed Cas13b (Para.11). Thus, Smargon and Zhang suggest a system wherein the CRISPR-Cas protein is a Cas13 or Cas 12 and wherein the CRISPR-Cas is a Cas13 selected from Cas13a, Cas13b, Cas13c, and Cas13d; or wherein the CRISPR-Cas is a Cas12 selected from Cas12a, Cas12b, and Cas12c.
Regarding claim 127, Smargon teaches a system wherein " amplification means any method employing a primer and a polymerase capable of replicating a target sequence with reasonable fidelity. Amplification may be carried out by natural or recombinant DNA polymerases such as TaqGold™, T7 DNA polymerase, Klenow fragment of E. coli DNA polymerase, and reverse transcriptase. A preferred amplification method is PCR” (Para.773; Para. 809; Para. 811). Thus, Smargon and Zhang suggest a system comprising: amplification reagents for amplifying the detection oligonucleotide; amplification reagents selected from Polymerase Chain Reaction (PCR) reagents, Recombinase Polymerase Amplification (RPA) reagents, Rolling Circle Amplification (RCA) reagents, and/or Multiple Displacement Amplification (MDA) reagents; DNA methylation enrichment agents; and/or size selection reagents to enrich for cell free DNA (cfDNA).
Regarding claim 128, Smargon teaches a system wherein “isolate and/or purify the RNA” (Para. 338) and “The mRNA contained in the extracted nucleic acid sample is then detected by amplification procedures” (Para. 808). Thus, Smargon and Zhang suggest a system wherein the target sequences of interest are in cell free nucleic acids.
Regarding claim 129, Smargon teaches a system wherein “The methods and uses as described herein … may target particular cells… target cells may for instance be …cells infected by a specific (e.g. viral) pathogen, etc. (Para. 329). Thus, Smargon and Zhang suggest a system wherein the genome is the genome of a pathogen.
Response to Arguments
Applicants’ arguments filed 05/18/2026 (Pg. 9-11) with respect to claims 1, 116-118, 120, 122-129 have been considered but are not persuasive. To clarify some instances argued in the response filed 05/18/2026 see responses to each argument made by Applicant below:
Applicants’ argument: “Zhang fails to teach or suggest any detection oligonucleotide containing a guide RNA binding site, much less a guide RNA containing a spacer capable of hybridizing to the guide RNA binding site.” (Pg. 9) and “Zhang specifies that the spacer sequences of each sgRNA binds mtDNA and, therefore, cannot possibly hybridize with any part of any proximity ligation probe.” (Pg. 9)
Response: Applicant' s arguments have been fully considered and found unpersuasive because as stated above in the Non-final office action and highlighted in Scheme 1, Smargon and Zhang do suggest a detection oligonucleotide containing a guide RNA binding site and a guide RNA containing a spacer capable of hybridizing to the guide RNA binding site. Furthermore, as stated above it is unclear whether the claim limitations are directed to direct binding or indirect binding. Also, it is unclear if the guide binding site is drawn to the nucleic acid target or detection oligonucleotide.
Applicants’ argument: “Scheme I of Zhang nowhere depicts any such "linking region" falling between two target binding sequences as contacting any sgRNA. Zhang only discloses a terminal region of a proximity ligation probe binding a stem-loop of a sgRNA.” (Pg. 9)
Response: Applicant' s arguments have been fully considered and found unpersuasive because as stated above in the Non-final office action and highlighted in Scheme 1, the linking region reads on any region that may link the 5’ end to the 3’ end. Therefore, as highlighted in the Scheme 1, the region that sits between the 5’ end to the 3’ end also indirectly binds two target sequences. Thus, Smargon and Zhang do suggest a linking region falling between two target binding sequences. Furthermore, the indicated linking region is also illustrated in Scheme 1 as indirectly interacting with guide RNA (sgRNA). Thus, Smargon and Zhang do suggest a linking region falling between two target binding sequences as contacting any sgRNA.
Applicants’ argument: “The sgRNAs of Zhang would not function as intended if modified such that their spacers bind a guide RNA binding site in a linking region of a proximity ligation probe as recited in the present claims because the proximity ligation assay of Zhang requires that each sgRNA spacer is complementary to a target sequence in mtDNA” (Pg. 9)
Response: Applicant' s arguments have been fully considered and found unpersuasive because as stated above in the non-final office action the structural relationship of the guide RNA in relation to the detection oligonucleotide and target is unclear, thus the intended function is unclear. Furthermore, a recitation of the intended use of the claimed invention must result in a structural difference between the claimed invention and the prior art in order to patentably distinguish the claimed invention from the prior art. If the prior art structure is capable of performing the intended use, then it meets the claim.
Applicants’ argument: “Zhang also fails to teach or suggest detection oligonucleotides binding a unique target sequence of interest in the same genome. The proximity ligation probes of Zhang, as depicted in Scheme 1, bind to sgRNA molecules and not to any genome (e.g., mtDNA).” (Pg. 11) and “Zhang does not and cannot teach or suggest proximity ligation probes binding a target site in a genome.” (Pg. 11)
Response: Applicant' s arguments have been fully considered and found unpersuasive because as stated above in the non-final office action Zhang teaches simultaneous imaging of wild-type (A) and mutant (G) sequences, where each proximity probe binds a unique target sequence of interest in the same cells. (Figure 5a) Furthermore, Zhang illustrates Scheme 1 that depicts a proximity ligation probe, with a first target binding sequence at the 5’ end, a linking region, and a second target binding sequence at the 3’ end disposed adjacent separated by a gap (see annotation of interpretation on Scheme 1); the 5’ and 3’ terminal sequence of the proximity probe indirectly bind the target genome through direct interaction with other probes and the interaction of the other probes with sgRNA and the interaction of the sgRNA with the target genome. Thus Smargon and Zhang suggest binding of the detection oligonucleotide with the target sequence in the same genome.
Claim 130 is rejected under 35 U.S.C. 103 as being unpatentable over Smargon et al. (“Smargon”; US Patent App. Pub. No. US 20170211142 A1, July 27, 2017) in view of Zhang et al. (“Zhang”; (2018). Direct visualization of single-nucleotide variation in mtDNA using a CRISPR/Cas9-mediated proximity ligation assay. Journal of the American Chemical Society, 140(36), 11293-11301., August 20, 2018).
Smargon discloses systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered DNA or RNA-targeting systems comprising a novel DNA or RNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA. (Abstract)
Regarding claim 130, Smargon teaches system “comprising i) a Type VI-B CRISPR-Cas effector protein, and ii) a Type VI-B CRISPR-Cas crRNA, wherein the crRNA comprises a) a guide sequence that is capable of hybridizing to a target RNA sequence, and b) a direct repeat sequence. Smargon also suggests a system in which oligonucleotides are used in combination with Cas effector protein and guide RNA to detect target nucleic acids. The Type VI-B CRISPR-Cas effector protein forms a complex with the crRNA, and the guide sequence directs sequence-specific binding of the complex to the target RNA sequence, whereby there is formed a CRISPR complex comprising the Type VI-B CRISPR-Cas effector protein complexed with the guide sequence that is hybridized to the target RNA sequence” (Para. 14). Thus, Smargon suggests a nucleic acid detection system comprising: a CRISPR-Cas protein; and a guide RNA comprising a sequence capable of hybridizing to the guide RNA binding site and comprising a sequence capable of forming a complex with the CRISPR-Cas protein.
Smargon teaches a system wherein “the repeats are short elements that occur in clusters that are regularly spaced by unique intervening sequences” (Para. 787). “The mRNA contained in the extracted nucleic acid sample is then detected by amplification procedures” (Para. 808). Smargon teaches a system wherein “amplification means any method employing a primer and a polymerase capable of replicating a target sequence with reasonable fidelity” (Para. 809). “Amplification procedures” are interpreted as general and comprising Forward and Reverse primers and polymerase to bind to regions of the sequence to amplify the region of interest. Thus, Smargon suggests a system wherein the linking region of the detection oligonucleotide further comprises a forward primer binding sequence, a reverse primer binding sequence, an RNA polymerase binding sequence, and/or a barcode.
Smargon does not explicitly teach a system wherein the B) two or more detection oligonucleotides, wherein each detection oligonucleotide comprises in order from 5' to 3': (i) a first target binding sequence, (ii) a linking region, and iii) a second target binding sequence, wherein the linking region comprises: a forward primer binding sequence, a reverse primer binding sequence, an RNA polymerase binding sequence, and a guide RNA binding site, wherein, when the first target binding sequence and the second target binding sequence bind a target sequence, the 5' and 3' terminal nucleotides of the detection oligonucleotide are disposed immediately adjacent to one another or are separated by a gap region comprising between 1 and 100 nucleotides, wherein each detection oligonucleotide binds a unique target sequence of interest in the same genome, wherein each detection oligonucleotide comprises the same guide RNA binding site, and wherein the detection oligonucleotides are circularized by ligation, splinted ligation, hybridization, or proximity extension;
Zhang discloses CRISPR/Cas9- mediated proximity ligation assay (CasPLA) to image SNV in mtDNA at single-molecule resolution. This method uses two Cas9 probes to target a specific mtDNA sequence, followed by proximity ligation and in situ rolling circle amplification (RCA) to reveal the spatial localization of individual wild-type and mutated mtDNAs in single cells. (Pg. 11293, Introduction, Col. 2, Para. 2)
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Regarding claim 130, Zhang teaches a system comprising “CRISPR/Cas9 mediated proximity ligation assay” (Pg. 11293, Col. 2, Para. 2; Scheme 1 see below). Zhang teaches a system comprising “When the paired CasPLA probes bind in close proximity to each other, they can then guide the subsequently added linear oligonucleotides to form a circular structure, which can be covalently joined by enzymatic DNA ligation”(Pg. 11294, Col. 1, Para. 1). Thus, Smargon and Zhang suggest a system wherein the detection oligonucleotide is circularized by ligation, splinted ligation, hybridization, or proximity extension.
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Regarding claim 130, Zhang teaches a system comprising “DNA proximity probes” (Pg. 11294, Col. 1, Para. 1; Scheme 1 see below) and “two distinct circularized DNAs for RCA (Figure 5a)” (Pg. 1297, Col. 1, Para. 2; Fig. 5a). Zhang teaches a system as illustrated in Scheme 1 that depicts a proximity ligation probe, with a first target binding sequence at the 5’ end, a linking region, and a second target binding sequence at the 3’ end disposed adjacent separated by a gap. (see annotation of interpretation on Scheme 1); the 5’ and 3’ terminal sequence of the proximity probe indirectly bind the target genome through direct interaction with other probes and the interaction of the other probes with sgRNA and the interaction of the sgRNA with the target genome. The “proximity ligation probes” reads on one or more detection oligonucleotides and the MPEP states "Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." (MPEP 2144.05). It would be obvious to the ordinary artisan to optimize the system to use two detection oligonucleotides such as in Fig. 5a. Zhang teaches a system as illustrated in Scheme 1 that depicts the linking region comprising a region interacting with guide RNA, thus reads on a guide RNA binding site of the detection oligonucleotide. The linking region reads on any region that may link the 5’ end to the 3, thus the region highlighted in Scheme 1 reads on a linking region. Zhang teaches a system comprising “CasPLA for Nuclear Genome Imaging… The following CasPLA procedure was the same with CasPLA for mtDNA imaging” (Pg. 11299, Col. 2, Para. 2) and “CasPLA probes targeting mutated KRAS gene, followed by proximity ligation and RCA” (Pg.11298, Col. 1 Para. 3). Hence, it would be obvious to the skilled artisan to detect a sequence of the genome using the detection oligonucleotides. Zhang teaches a system as illustrated in Fig. 5a of simultaneous imaging of wild-type (A) and mutant (G) sequences, where each proximity probe binds a unique target sequence of interest in the same cells. (Fig. 5a, see above). Zhang teaches a system as illustrated in Fig. 5a where each proximity probe appears to comprise a similar guide RNA site on the left side that interacts with guide RNA.
Thus, Smargon and Zhang suggest a system wherein the target binding sequence of the detection oligonucleotide comprises a first target binding sequence and a second target binding sequence that are separated by intervening sequences: wherein the first target binding sequence and the second target binding sequence hybridize upon the target sequence directly adjacent to one another; or wherein the first target binding sequence and the second target binding sequence hybridize upon the target sequence such that there is at least a single nucleotide gap region between the first target binding sequence and the second target binding sequence.
Accordingly, Smargon and Zhang suggest a system comprising the limitations of claim 130.
Smargon and Zhang are both considered to be analogous to the claimed invention because they are in the same field of targeting nucleic acids with CRISPR effector proteins and guide sequences. Smargon suggests that “numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention” (Para. 1239). Therefore, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to have modified the nucleic acid detection system comprising CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA as suggested by Smargon to incorporate the system comprising a CRISPR/Cas system, two or more detection oligonucleotides comprising a first target binding sequence, a linking region, and a second target binding sequence that mediate proximity ligation, and guide RNA to target a specific genomic sequence as suggested by Zhang and provide a system comprising a CRISPR-Cas proteins, two or more detection oligonucleotides comprising a first target binding sequence, a linking sequence, and a second target binding sequence, and a guide RNA. Furthermore, it would also be obvious before the effective filling date, that the linking region as suggested by Zhang, would have the amplification means employing a primer and a polymerase capable of replicating a target sequence as suggested by Smargon to conduct RCA as suggested by Zhang. These claim elements were known in the art and one of skill in the art could have combined these elements by known methods with no change in their respective functions, and the combination would have yielded the predictable outcome according to the limitations of claim 130. Doing so would allow for increased signal to noise detection of probes and efficient identification and specific recognition of CRISPR-Cas system for target sequence(s).
Response to Arguments
Applicants’ arguments filed 05/18/2026 (Pg. 9-11) with respect to claim 130 have been considered but are not persuasive as discussed above.
Claims 131-132 are rejected under 35 U.S.C. 103 as being unpatentable over Smargon et al. (“Smargon”; US Patent App. Pub. No. US 20170211142 A1, July 27, 2017) in view of Zhang et al. (“Zhang”; (2018). Direct visualization of single-nucleotide variation in mtDNA using a CRISPR/Cas9-mediated proximity ligation assay. Journal of the American Chemical Society, 140(36), 11293-11301., August 20, 2018).
Smargon discloses systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered DNA or RNA-targeting systems comprising a novel DNA or RNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA. (Abstract)
Regarding claims 131-132, Smargon teaches system “comprising i) a Type VI-B CRISPR-Cas effector protein, and ii) a Type VI-B CRISPR-Cas crRNA, wherein the crRNA comprises a) a guide sequence that is capable of hybridizing to a target RNA sequence, and b) a direct repeat sequence. The Type VI-B CRISPR-Cas effector protein forms a complex with the crRNA, and the guide sequence directs sequence-specific binding of the complex to the target RNA sequence, whereby there is formed a CRISPR complex comprising the Type VI-B CRISPR-Cas effector protein complexed with the guide sequence that is hybridized to the target RNA sequence” (Para. 14). “Type VI-B” is considered Cas 13b. Smargon also teaches a system comprising “two or more Type VI-B CRISPR-Cas crRNAs” (Para. 17). Smargon suggests a system comprising “independent guide RNAs targeting the same gene” (Para. 949). The MPEP states "Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." (MPEP 2144.05). It would be obvious to the ordinary artisan to optimize the system to use two or more CRISPR-Cas proteins, and two or more guide RNAs. Thus, Smargon suggests a nucleic acid detection system comprising: two or more CRISPR-Cas proteins, and two or more guide RNAs, comprising a sequence capable of hybridizing to the guide RNA binding site; and wherein the two or more CRISPR-Cas proteins are Cas 13, Cas 12, or a combination thereof.
Smargon does not explicitly teach a system comprising two or more sets of detection oligonucleotides, each set comprising a plurality of detection oligonucleotides, wherein each detection oligonucleotide in a set comprises:(i)a first target binding sequence,(ii) a linking region, and(ii) a second target binding sequence, wherein the linking region comprises a guide RNA binding site, wherein, when the first target binding sequence and the second target binding sequence bind a target sequence, the 5' and 3' terminal nucleotides of the detection oligonucleotide are disposed immediately adjacent to one another or are separated by a gap region comprising between 1 and 100 nucleotides, wherein each detection oligonucleotide in a set binds a unique target sequence of interest in the same genome, and wherein each detection oligonucleotide within a set of detection oligonucleotides comprises the same guide RNA binding site that identifies the set of detection oligonucleotide.
Zhang discloses CRISPR/Cas9- mediated proximity ligation assay (CasPLA) to image SNV in mtDNA at single-molecule resolution. This method uses two Cas9 probes to target a specific mtDNA sequence, followed by proximity ligation and in situ rolling circle amplification (RCA) to reveal the spatial localization of individual wild-type and mutated mtDNAs in single cells. (Pg. 11293, Introduction, Col. 2, Para. 2)
Regarding claims 131-132, Zhang teaches a system as illustrated in Scheme 1 and Fig. 5a that depicts two Cas enzymes (Cas9) and two guide RNAs (sgRNA). Thus Zhang also suggests a system comprising two or more CRISPR-Cas protein and two or more guide RNAs.
Regarding claims 131-132, Zhang teaches a system comprising “DNA proximity probes” (Pg. 11294, Col. 1, Para. 1; Scheme 1 see below) and “two distinct circularized DNAs for RCA (Figure 5a)” (Pg. 1297, Col. 1, Para. 2; Fig. 5a, see below). Zhang teaches a system as illustrated in Scheme 1 that depicts a proximity ligation probe, with a first target binding sequence at the 5’ end, a linking region, and a second target binding sequence at the 3’ end disposed adjacent separated by a gap. (see annotation of interpretation on Scheme 1); the 5’ and 3’ terminal sequence of the proximity probe indirectly bind the target genome through direct interaction with other probes and the interaction of the other probes with sgRNA and the interaction of the sgRNA with the target genome. The “proximity ligation probes” reads on one or more detection oligonucleotides and the MPEP states "Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." (MPEP 2144.05). It would be obvious to the ordinary artisan
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to optimize the system to use two detection oligonucleotides such as in Fig. 5a.
Zhang teaches a system as illustrated in Scheme 1 that depicts the linking region comprising a region interacting with guide RNA, thus reads on a guide RNA binding site of the detection oligonucleotide. The linking region reads on any region that may link the 5’ end to the 3, thus the region highlighted in Scheme 1 reads on a linking region. Zhang teaches a system comprising “CasPLA for Nuclear Genome Imaging… The following CasPLA procedure was the same with CasPLA for mtDNA imaging” (Pg. 11299, Col. 2, Para. 2) and “CasPLA probes targeting mutated KRAS gene, followed by proximity ligation and RCA” (Pg.11298, Col. 1 Para. 3). Hence, it would be obvious to the skilled artisan to detect a sequence of the genome using the detection oligonucleotide. Zhang teaches a system as illustrated in Fig. 5a of simultaneous imaging of wild-type (A) and mutant (G) sequences, where each proximity probe binds a unique target sequence of interest in the same cells. (Fig. 5a, see below). Zhang teaches a system as illustrated in Fig. 5a where each proximity probe appears to comprise a similar guide RNA site on the left side that interacts with guide RNA.
Thus, Smargon and Zhang suggest a system comprising two or more sets of detection oligonucleotides, each set comprising a plurality of detection oligonucleotides, wherein each detection oligonucleotide in a set comprises:(i)a first target binding sequence,(ii) a linking region, and (ii) a second target binding sequence, wherein the linking region comprises a guide RNA binding site, wherein, when the first target binding sequence and the second target binding sequence bind a target sequence, the 5' and 3' terminal nucleotides of the detection oligonucleotide are disposed immediately adjacent to one another or are separated by a gap region comprising between 1 and 100 nucleotides, wherein each detection oligonucleotide in a set binds a unique target sequence of interest in the same genome, and wherein each detection oligonucleotide within a set of detection oligonucleotides comprises the same guide RNA binding site that identifies the set of detection oligonucleotide.
Accordingly, Smargon and Zhang suggest the limitations of claims 131-132.
Smargon and Zhang are both considered to be analogous to the claimed invention because they are in the same field of targeting nucleic acids with CRISPR effector proteins and guide sequences. Smargon suggests that “numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention” (Para. 1239). Therefore, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to have modified the nucleic acid detection system comprising CRISPR effector protein(s) and guide RNA(s); and CRISPR-Cas proteins are Cas 13 as suggested by Smargon to incorporate the system comprising two or more CRISPR-Cas proteins, two or more sets of detection oligonucleotides comprising a first target binding sequence, a linking region, and a second target binding sequence that mediate proximity ligation, and guide RNA to target a specific region of the genome, and two or more guide RNAs as suggested by Zhang and provide a system comprising two or more CRISPR-Cas protein, two or more detection oligonucleotides comprising a first target binding sequence, a linking sequence, and a second target binding sequence, and two or more guide RNA. The MPEP states, "Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." (MPEP 2144.05). These claim elements were known in the art and one of skill in the art could have combined these elements by known methods with no change in their respective functions, and the combination would have yielded the predictable outcome according to the limitations of claim 131. Doing so would allow for increased signal to noise detection of probes and efficient identification and specific recognition of CRISPR-Cas system for target sequence(s).
Response to Arguments
Applicants’ arguments filed 05/18/2026 (Pg. 9-11) with respect to claim 130 have been considered but are not persuasive as discussed above.
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure. Carsten-Peter Carstens (“Carstens”; US Patent App. Pub. No. US 20150211058 A1, Jul. 30, 2015) (Para. 12-circulaor probes or templates)
No claims are in condition for allowance.
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/KENDRA R VANN-OJUEKAIYE/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
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