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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 Jube 29, 2026 has been entered.
Applicant canceled claims 6, 12-20, 22, 25-27 and 29. Claims 1-5, 7-11, 21, 23-24, 28 and 30 are currently pending and under examination. Any objection or rejection of record in the previous Office Action, which is not addressed in this action has been withdrawn in light of Applicant’s amendments and/or arguments.
Claim Objections
Claims 1, 4 and 8 are objected to because of the following informalities:
In claim 1, lines 15, “allow the modification of DNA/RNA targets” should read “allow modification of DNA/RNA targets”.
In claim 4, lines 17, “allow the modification of DNA/RNA targets” should read “allow modification of DNA/RNA targets”.
In claim 4, lines 26-27, “to the single molecule sequencing reaction” should read “to the single molecule long-read sequencing reaction”.
In claim 8, line 2-3, “proteins from the reaction” should read “proteins from the single molecule sequencing reaction”.
Appropriate correction 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 1-5, 7-11, 21, 23-24, 28 and 30 are rejected under 35 U.S.C. 101 because the claimed invention is directed to one or more judicial exceptions (i.e., product of nature, a law of nature, a natural phenomenon, or an abstract idea) without significantly more.
Every claimed invention must be examined to determine whether the claimed invention complies with 35 U.S.C. 101, particularly whether the claimed invention falls within a 35 U.S.C. 101 judicial exception of non-patentable subject matter (e.g., an abstract idea, law of nature, natural phenomenon, natural product etc.). Phenomena of nature, though just discovered, natural products, mental processes, and abstract intellectual concepts are not patentable, as they are the basic tools of scientific and technological work. See MPEP 2106. As per the “2019 Revised Subject Matter Eligibility Guidance” (Federal Register Vol. 84, No. 4, available 01-07-2019), claims drawn to a process, machine, manufacture or composition of matter are further analyzed according to a two-part process to determine if A) the claim(s) is/are “directed to” a judicial exception because the claims(s) recite(s) a judicial exception (i.e. prong one) that is not integrated into a practical application (i.e. prong two) and, if so, if B) the claim(s) provide(s) an inventive concept, i.e. recite(s) additional elements that amount to significantly more than the judicial exception.
Subject Matter Eligibility Test for Products and Processes
Step 1 - Is the Claim to a Process, Machine, Manufacture or Composition of Matter? YES
Claims 1-5, 7-11, 21, 23-24, 28 and 30 are directed to one of the statutory classes. Claims 1-5, 7-11, 21, 23-24, 28 and 30 are directed to a method for segregating a plurality of modified and non-modified polynucleotide constructs into a plurality of compartments (Process).
Step 2A, Prong One — Does the Claim Recite an Abstract Idea, Law of Nature, or Natural Phenomenon? YES
Claims 1-5, 7-11, 21, 23-24, 28 and 30 recite the abstract idea of receiving, organizing and processing data using mathematical concepts. Claims directed to nothing more than abstract ideas, natural phenomena, and laws of nature are not eligible for patent protection (see MPEP 2106.04). Abstract ideas include mathematical concepts, (mathematical formulas or equations, mathematical relationships and mathematical calculations), certain methods of organizing human activity, and mental processes (including procedures for collecting, observing, determining, evaluating, and organizing information (See MPEP 2106.04(a)(2)). In particular, these abstract ideas include:
• Detecting and counting the DNA/RNA molecules referred to in step c)i and/or c)ii, based on the sequencing results (detecting and counting may be a mental process, the human mind is capable of receiving/ collecting data, observing/evaluating, organizing information and comparing/receiving/organizing data using mathematical concepts such as mathematical formulas/equations, mathematical relationships and mathematical calculations).
• Evaluating the modifying activity of one or more nucleic acid modifying enzymes against one or more of the DNA/RNA targets using calculations and comparing modified and unmodified polynucleotides using specific formulas (mental process, human mind is capable of receiving/ collecting data, observing/evaluating/determining, organizing information as well as comparing/receiving/organizing data using mathematical concepts such as mathematical formulas/equations, mathematical relationships and mathematical calculations ).
Therefore, the claims recite elements that constitute one or more judicial exceptions
Step 2A, Prong Two — Does the Claim Recite an Additional Elements that Integrate the Judicial Exception into a Practical Application? NO.
The Supreme Court has long distinguished between principles themselves, which are not patent eligible, and the integration of those principles into practical applications, which are patent eligible. However, absent are any additional elements recited in the claim beyond the judicial exceptions which integrate the exception into a practical application of the exception. The “integration into a practical application” requires an additional element or a combination of additional elements in the claim to apply, rely on, or use the judicial exception in a manner that imposes a meaningful limit on the judicial exception, such that it is more than a drafting effort designed to monopolize the exception.
The claim analysis continues with identifying additional elements beyond the judicial exceptions that might evidence integration of the judicial exceptions into a practical application. The steps or elements in addition to the judicial exceptions are: “subjecting [the] molecules to single molecule sequencing”, “the plurality of the polynucleotide constructs encode different variants of the nucleic acid modifying enzyme, and/or different DNA or RNA targets”, “subjecting the compartments to conditions which allow in vitro expression of RNAs and proteins as well as subjecting the compartments to conditions which allow the modification of DNA/RNA targets by nucleic acid modifying enzymes which have modification activity towards said DNA or RNA targets”, which is not indicative of integration into practical application. These steps, recited at a high level of generality, comprise routine data gathering, which is considered an insignificant extra-solution activity. This data gathering is required for using the judicial exceptions. (See MPEP 2106.05(g)). There are no further/additional steps which applies either the identified judicial exception into practical application. Thus, a careful evaluation of the claim as a whole fails to reveal the practical application of the judicial exception to, e.g., effect an improvement to the functioning of a computer or other technology/technical field, effect a particular treatment or prophylaxis for a disease or medical treatment, implement a particular machine that is integral to the claim, or effect a transformation or reduction of a particular article to a different state or thing, or to apply the judicial exception in another meaningful way beyond generally linking its use to a particular technological environment. Accordingly, the claims do not integrate the judicial exception(s) into a practical application and is therefore directed to a judicial exception.
Step 2B - Does the Claim Recite Additional Elements that Amount to Significantly More than the Judicial Exception? NO.
The Supreme Court has identified a number of considerations for determining whether a claim with additional elements amounts to “significantly more” than the judicial exception(s) itself. The claims as a whole are analyzed to determine whether any additional element/step, or combination of additional elements/steps, in addition to the identified judicial exception(s) is sufficient to ensure that the claim amounts to “significantly more” than the exception(s).
The eligibility analysis proceeds with identifying any additional elements or limitations, separate from the judicial exceptions, that might potentially render the claims directed to a judicial exception patent eligible. To render the claims patent- eligible, these elements must comprise meaningful limitations that add to or transform the judicial exception to the effect that it amounts to significantly more than the natural correlation or abstract idea itself - i.e. provide an “inventive concept’. The elements that are in addition to the judicial exception comprise: subjecting the molecules to single molecule sequencing, the plurality of the polynucleotide constructs encode different variants of the nucleic acid modifying enzyme, and/or different DNA or RNA targets, subjecting the compartments to conditions which allow in vitro expression of RNAs and proteins as well as subjecting the compartments to conditions which allow the modification of DNA/RNA targets by nucleic acid modifying enzymes which have modification activity towards said DNA or RNA targets. When considered separately and in combination, these elements do not add significantly more to the judicial exception. These steps are well-understood, routine and conventional activities in the field. For example, Zhang et al (U.S. Patent Application Publication US 2020/0190487 A1, published June 18, 2020, effectively filed December 17, 2018), discloses subjecting the molecules to single molecule sequencing, the plurality of the polynucleotide constructs encode different variants of the nucleic acid modifying enzyme, and/or different DNA or RNA targets and subjecting the compartments to conditions which allow in vitro expression of RNAs and proteins as well as subjecting the compartments to conditions which allow the modification of DNA/RNA targets by nucleic acid modifying enzymes which have modification activity towards said DNA or RNA targets. The claims recite an abstract idea with additional elements. Because these elements are not inventive concepts, the claims do not integrate the abstract idea into a practical application. The judicial exception alone cannot provide that inventive concept or practical application (MPEP 2106.05). The claims therefore do not include additional elements that are sufficient to amount to significantly more than the judicial exception.
Accordingly, the claims do not qualify as patent-eligible subject matter.
For further information, please see the latest revision of MPEP 2104-2106 {Patent Subject Matter Eligibility Under 35 U.S.C. 101}, including MPEP 2106.04 {Eligibility Step 2A: Whether a Claim is Directed to a Judicial Exception} and 2106.05 {Eligibility Step 2B: Whether a Claim Amounts to Significantly More}, as well as the guidance on Subject Matter Eligibility, including the 2019 Guidance issued Jan. 7, 2019, and the October 2019 Update, provided on the USPTO website at https:/Awww.uspto.gov/patent/laws-and-regulations/examination-policy/subject-matter- eligibility.
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.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1-4, 7-10, 20-21, 23-24, 28 and 30 are rejected under 35 U.S.C. 102 (a)(1) and (a)(2) as being anticipated by Zhang et al (U.S. Patent Application Publication US 2020/0190487 A1, published June 18, 2020, effectively filed December 17, 2018). This is a new rejection as necessitated by amendments.
Regarding claim 1, Zhang teaches a method comprising the steps of: a) segregating a plurality of polynucleotide constructs into compartments (Page 1, [0008], Page 16, [0177], Page 22, [0207], Page 81, [0794], Page 100, [0912]-[0913], Page 161, [1242] and Page 163, [1286]). Zhang teaches each compartment comprises a single polynucleotide construct, and each polynucleotide construct comprises a first polynucleotide sequence encoding a nucleic acid modifying enzyme or a variant thereof, operably linked to a first promoter (Page 1, [0008], Page 12, [0012]). Zhang teaches a second polynucleotide sequence comprising a DNA target or a DNA template encoding an RNA target (Page 14, [0161], Page 53, [0526], Page 54, [0540] and Pages 68, [0634]). Zhang teaches when the second polynucleotide sequence comprises a DNA template encoding an RNA target, said RNA target is co-expressed contiguously with the nucleic acid modifying enzyme as a single RNA transcript, driven by the first promoter (Page 1, [0008], Page 12, [0012], Page 16, [0177], Page 17, [0181], Page 67, [0630], Page 77, [0764], Page 80, [0785], Page 161, [1242] and Page 163, [1286]). Zhang teaches the plurality of the polynucleotide constructs encode different variants of the nucleic acid modifying enzyme, and/or different DNA or RNA targets (Page 12, [0141], Page 26, [0271] and [0276], Page 45, [0435], Page 67, [0630], Page 77, [0766], Page 93, [0867] and Page 95, [0880]). Zhang teaches subjecting the compartments to conditions which allow the modification of DNA and/or RNA targets by RNA-guided nucleic acid modifying enzymes which have functional activity towards said DNA or RNA targets in the presence of a gRNA (Page 14, [0161], Pages 17-18, [0182], Pages 19-20, [0189]-[0191], Page 26, [0275] and Page 53, [0562]). Zhang teaches producing a population of DNA/RNA molecules that comprises polynucleotide constructs and/or RNA transcripts or fragments thereof that have been modified by the nucleic acid modifying enzymes (Page 14, [0161], Pages 17-18, [0182], Pages 19-20, [0189]-[0191], Page 26, [0275] and Page 53, [0562]). Zhang teaches polynucleotide constructs and/or RNA transcripts which have not been modified by the nucleic acid modifying enzyme(s) (Pages 19-20, [0189]-[0191] and Page 33, [0329]). Zhang teaches harvesting the population of DNA/RNA molecules and subjecting the same to single molecule long-read sequencing (Page 246, [1434]). Zhang teaches the harvested population of DNA/RNA molecules is not subjected to ligation and amplification before being subjected to the single molecule sequencing reaction (Page 246, [1434] and Figs. 45A-45C, and 72). Zhang teaches detecting and counting the DNA/RNA molecules (Page 84, [0819], Page 85, [0824] and Page 100, [0913]).
Regarding claim 2, Zhang teaches the nucleic acid modifying enzyme is an RNA-guided nucleic acid modifying enzyme and each compartment further comprises a guide RNA or a nucleotide template encoding the same (Pages 1, [0008], Page 2, [0012], Page 11, [0135] and Page 16, [0174]).
Regarding claim 3, Zhang teaches the nucleic acid modifying enzyme is an RNA-guided nucleic acid modifying enzyme, each polynucleotide further comprises a third polynucleotide sequence encoding a variant guide RNA (gRNA) (Page 3, [0040], 26, [0276] and Page 161, [1243]). Zhang teaches the plurality of the polynucleotide constructs encode different variants of the nucleic acid modifying enzyme, and/or different DNA or RNA targets, and/or different gRNAs (Page 12, [0141], Page 26, [0271] and [0276], Page 45, [0435], Page 67, [0630], Page 77, [0766], Page 93, [0867] and Page 95, [0880]).
Regarding claim 4, Zhang teaches a method comprising the steps of: a) segregating a plurality of polynucleotide constructs into compartments (Page 1, [0008], Page 16, [0177], Page 22, [0207], Page 81, [0794], Page 100, [0912]-[0913], Page 161, [1242] and Page 163, [1286]). Zhang teaches each compartment comprises a single polynucleotide construct, and each polynucleotide construct comprises a first polynucleotide sequence encoding a guide RNA (gRNA) operably linked to a first promoter (Page 1, [0008], Page 12, [0012], Page 16, [0177], Page 17, [0181], Page 77, [0764], Page 161, [1242] and Page 163, [1286]). Zhang teaches a second polynucleotide sequence comprising a DNA target or a DNA template encoding an RNA target (Page 14, [0161], Page 53, [0526], Page 54, [0540] and Pages 68, [0634]). Zhang teaches the second polynucleotide sequence comprises a DNA template encoding an RNA target (Page 14, [0161], Page 53, [0526], Page 54, [0540] and Pages 68, [0634]). Zhang teaches RNA target is co-expressed contiguously with the gRNA as a single RNA transcript, driven by the first promoter (Page 1, [0008], Page 12, [0012], Page 16, [0177], Page 17, [0181], Page 67, [0630], Page 77, [0764], Page 80, [0785], Page 161, [1242] and Page 163, [1286]). Zhang teaches the plurality of the polynucleotide constructs encode different gRNAs, and/or different DNA or RNA targets (Page 26, [0271] and [0276], Page 45, [0435], Page 67, [0630], Page 93, [0867] and Page 95, [0880]). Zhang teaches each compartment further comprises an RNA- guided nucleic acid modifying enzyme or a variant thereof or a nucleotide template encoding the same (Page 1, [0008], Page 16, [0177], Page 22, [0207], Page 81, [0794], Page 100, [0912]-[0913], Page 161, [1242] and Page 163, [1286]). Zhang teaches subjecting the compartments to conditions which allow in vitro transcription and/or translation of RNAs and proteins (Pages 39-40, [0380]-[0384] and Page 43, [0403]). Zhang teaches subjecting the compartments to conditions which allow the modification of DNA and/or RNA targets by RNA-guided nucleic acid modifying enzymes which have functional activity towards said DNA or RNA targets in the presence of a gRNA (Page 14, [0161], Pages 17-18, [0182], Pages 19-20, [0189]-[0191], Page 26, [0275] and Page 53, [0562]). Zhang teaches producing a population of DNA/RNA molecules that comprises polynucleotide constructs and/or RNA transcripts or fragments thereof that have been modified by the nucleic acid modifying enzymes (Page 14, [0161], Pages 17-18, [0182], Pages 19-20, [0189]-[0191], Page 26, [0275] and Page 53, [0562]). Zhang teaches polynucleotide constructs and/or RNA transcripts which have not been modified by the nucleic acid modifying enzyme(s) (Pages 19-20, [0189]-[0191] and Page 33, [0329]). Zhang teaches harvesting the population of DNA/RNA molecules and subjecting the same to single molecule long-read sequencing (Page 246, [1434]). Zhang teaches the harvested population of DNA/RNA molecules is not subjected to ligation and amplification before being subjected to the single molecule sequencing reaction (Page 246, [1434] and Figs. 45A-45C, and 72). Zhang teaches detecting and counting the DNA/RNA molecules (Page 84, [0819], Page 85, [0824] and Page 100, [0913]).
Regarding claim 7, Zhang teaches breaking the compartments by physical or chemical methods (Page 22, [0207]).
Regarding claim 8, Zhang teaches purifying the harvested DNA/RNA molecules to remove excess DNA, RNA and/or proteins from the reaction (Page 65, [0612] and Page 176, [1385]).
Regarding claim 9, Zhang teaches the harvested population of DNA/RNA molecules are not subjected to further modifications before being subjected to the single molecule sequencing reaction, except for modifications required of the single molecule sequencing (Page 84, [0819], Page 85, [0824] Page 100, [0913] and Page 246, [1434]).
Regarding claim 10, Zhang teaches the detection and counting of the DNA/RNA molecules which have been modified by the nucleic acid modifying enzymes is based only on data generated during the single molecule sequencing and does not require further modifications or processing of the DNA/RNA molecules (Page 84, [0819], Page 85, [0824] Page 100, [0913] and Page 246, [1434]).
Regarding claim 20, Zhang teaches the first and second polynucleotide constructs (Pages 10-11, [0131] and Page 13, [0147]-[0150]).
Regarding claim 21, Zhang teaches the DNA or RNA target comprises a protospacer that is at least partially complementary to the guide RNA and the DNA target also comprises a proximal Protospacer Adjacent Motif (PAM) sequence (Page 16, [0174]-[0175] and Page 21, [0202]).
Regarding claim 23, Zhang teaches when the polynucleotide construct comprises a DNA template encoding an RNA target, the RNA target further comprises a proximal Protospacer Flanking Sequence (PFS) (Page 16, [0174]-[0175] and Page 21, [0202]).
Regarding claim 24, Zhang teaches the nucleic acid modifying enzyme is a CRISPR-associated protein (Cas) (Page 1, [0008]-[0009] and Pages 17-18, [0182]).
Regarding claim 28, Zhang teaches the compartments are emulsion droplets (Page 175, [1378]).
Regarding claim 30, Zhang teaches the segregation is achieved using microfluidics (Page 175, [1378] and Pages 164, [1288]).
Zhang teaches each and every limitation of claims 1-4, 7-10, 20-21, 23-24, 28 and 30 and therefore Zhang anticipates claims 1-4, 7-10, 20-21, 23-24, 28 and 30.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al (U.S. Patent Application Publication US 2020/0190487 A1, published June 18, 2020, effectively filed December 17, 2018), as applied to claims 1-4, 7-10, 20-21, 23-24, 28 and 30 above, in view of in view of Ryan et al. (“Improving CRISPR-Cas specificity with chemical modifications in single-guide RNA’s”, Nucleic Acid Research, Vol. 46, No. 2, published December 04, 2017), previously cited in the September 29, 2025 Office Action. This is a new rejection as necessitated by amendments.
Regarding claim 5, Zhang teaches evaluating the modifying activity of one or more nucleic acid modifying enzymes against one or more of the DNA/RNA targets (Page 14, [0161], Pages 17-18, [0182], Pages 19-20, [0189]-[0191], Page 26, [0275] and Page 53, [0562]). Zhang teaches analyzing enzymatic activity (Page 10, [0127]).
Zhang does not teach or suggest evaluating the modifying activity of one or more nucleic acid modifying enzymes against one or more of the DNA/RNA targets, by calculating the number of polynucleotide constructs and/or RNA transcripts that have been modified by the nucleic acid modifying enzyme, and comparing it against the number of polynucleotide constructs and/or RNA transcripts that have not been modified by the nucleic acid modifying enzymes, or against the total number of polynucleotide constructs and/or RNA transcripts. Zhang does not teach or suggest the enzymatic activity is represented by a value calculated using any one of the following formulas:
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Ryan teaches evaluating the modifying activity, specifically cleavage activity, of one or more CRISPR-Cas9 activity against one or more of the guide RNA targets, by calculating the number of polynucleotide constructs and/or RNA transcripts that have been modified by the nucleic acid modifying enzyme, and comparing it against the number of polynucleotide constructs and/or RNA transcripts that have not been modified by the nucleic acid modifying enzymes, or against the total number of polynucleotide constructs and/or RNA transcripts (Abstract and Page 793, Right Column, First Full Paragraph). Ryan teaches the enzymatic activity is represented by a value calculated using any one of the following formulas:
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(Page 793, Right Column, First Full Paragraph). Ryan teaches mapping reads across a window including a cleavage site, and discarding reads that fail to map across the window fully (Page 794, Left Column, Last Paragraph). Ryan teaches that chemical modifications at specific sites in guide sequences can dramatically improve the specificity and flexibility of CRISPR–Cas systems for precise editing of genes and genomes (Page 802, Left Column, First Full Paragraph).
It would have been obvious to one having ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Zhang, to include the teachings of Ryan, to evaluate cleavage activity by calculating the enzyme activity by comparing the number of polynucleotide constructs and/or RNA transcripts that have been modified by the nucleic acid modifying enzyme, and comparing it against the total number of polynucleotide constructs and/or RNA transcripts, using the formula:
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Using these methods, including that chemical modifications at specific sites in guide sequences, dramatically improve the specificity and flexibility of CRISPR–Cas systems for precise editing of genes and genomes as taught by Ryan (Page 802, Left Column, First Full Paragraph).
Claim 11 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al (U.S. Patent Application Publication US 2020/0190487 A1, published June 18, 2020, effectively filed December 17, 2018) and Ryan et al. (“Improving CRISPR-Cas specificity with chemical modifications in single-guide RNA’s”, Nucleic Acid Research, Vol. 46, No. 2, published December 04, 2017), previously cited in the September 29, 2025 Office Action, as applied to claim 5 above, in view of Pinello et al. (“Analyzing CRISPR genome editing experiments with CRISPResso” Nat Biotechnol. 34(7), published July 12, 2016), previously cited in the September 29, 2025 Office Action. This is a new rejection as necessitated by amendments.
Regarding claim 11, Zhang teaches the modification activity is cleavage activity (Pages 17-18, [0182] and Page 126, [1033]).
Zhang and Ryan do not teach or suggest the detection and calculation of modified and unmodified polynucleotide constructs or RNA transcripts are achieved by aligning sequencing reads of the DNA/RNA molecules against a reference sequence which contains a window of cleavage sites for the nucleic acid modifying enzymes. Zhang and Ryan do not teach or suggest, when the sequencing read of a DNA/RNA molecule is mapped across both 5' and 3' of the window of cleavage sites, the DNA/RNA molecule is an unmodified polynucleotide construct or RNA target. Zhang and Ryan do not teach or suggest when the end of the sequencing read of a DNA/RNA molecule is mapped to a region within the window of cleavage sites, the DNA/RNA molecule is a modified polynucleotide construct or RNA target. Zhang and Ryan do not teach or suggest when the end of the sequencing read of a DNA/RNA molecule does not map within the window of cleavage sites, the DNA/RNA molecule is non-informative and is not used for the measurement of modification activity.
Pinello teaches a polynucleotide construct as well as an RNA transcript (Page 2, First-Third Paragraph). Pinello teaches enumerating/quantification and visualizing specific CRISPR-Cas9 cleavage events and selected off-target sites (Page 2, First Paragraph). Pinello teaches sequencing the polynucleotide constructs and/or RNA transcripts which have and have not been modified by the nucleic acid modifying enzymes (Page 2, Last Paragraph—Page 3, Second Paragraph and Fig. 1). Pinello teaches the first and second polynucleotide sequences are fully or partially overlapping (Fig. 1). Pinello teaches the detection and calculation of modified and unmodified polynucleotide constructs or RNA transcripts are achieved by aligning sequencing reads of the DNA/RNA molecules against a reference sequence which contains a window of cleavage sites for the nucleic acid modifying enzymes (Page 2, Second Paragraph, Page 3, Second Paragraph and Fig. 1). Pinello teaches when the sequencing read of a DNA/RNA molecule is mapped across both 5' and 3' of the window of cleavage sites, the DNA/RNA molecule is an unmodified polynucleotide construct or RNA target (Page 2, Second Paragraph, Page 3, Second Paragraph and Fig. 1). Pinello teaches when the end of the sequencing read of a DNA/RNA molecule is mapped to a region within the window of cleavage sites, the DNA/RNA molecule is a modified polynucleotide construct or RNA target (Page 2, Second Paragraph, Page 3, Second Paragraph and Fig. 1). Pinello teaches when the end of the sequencing read of a DNA/RNA molecule does not map within the window of cleavage sites, the DNA/RNA molecule is non-informative and is not used for the measurement of modification activity (Page 2, Second Paragraph, Page 3, Second Paragraph and Fig. 1). Pinello teaches using the disclosed methods allows for accurate quantification and visualization of CRISPR-Cas9 outcomes, as well as comprehensive evaluation of effects on coding sequences, noncoding elements and selected off-target sites (Page 2, First Paragraph).
It would have been obvious to one having ordinary skill in the art before the effective filing date of the invention to have modified the teachings of Zhang and Ryan to include the teachings of Pinello, sequencing the polynucleotide constructs and/or RNA transcripts which have and have not been modified as well as using modified and unmodified polynucleotide constructs or RNA transcripts achieved by aligning sequencing reads of the DNA/RNA molecules against a reference sequence which contains a window of cleavage sites for the nucleic acid modifying enzymes. Using these methods allows for accurate quantification and visualization of CRISPR-Cas9 outcomes, as well as comprehensive evaluation of effects on coding sequences, noncoding elements and selected off-target sites as taught by Pinello (Page 2, First Paragraph).
Response to Arguments
Applicant’s arguments and amendments, filed June 29, 2026 regarding the rejections of under 35 U.S.C. § 102 and 103 have been fully considered and are deemed to be persuasive. Therefore, these rejections are withdrawn.
However, upon further consideration, new grounds of rejection are made in view of Applicant’s amendment.
As discussed above, newly cited Zhang discloses methods directed to modified and unmodified polynucleotide constructs and/or RNA transcripts or fragments thereof , harvesting the population of DNA/RNA molecules and subjecting the same to single molecule sequencing as well as the harvested population not being subjected to ligation and amplification before being subjected to the single molecule sequencing reaction.
Therefore, for all these reasons, and those listed above, Zhang, Zhang in view of Ryan and Zhang in view of Pinello are deemed to render the instant invention obvious.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JESSICA DANIELLE PARISI whose telephone number is (571)272-8025. The examiner can normally be reached Mon - Friday 7:30-5:00 Eastern with alternate Fridays off.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Heather Calamita can be reached at 571-272-2876. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/JESSICA D PARISI/Examiner, Art Unit 1684
/HEATHER CALAMITA/Supervisory Patent Examiner, Art Unit 1684
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