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 .
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Any objection or rejection of record in the previous Office Action, which is not addressed in this action has been withdrawn in light of Applicant’s amendments and/or arguments. This action is Final.
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.
Claim 4 is rejected under 35 U.S.C. 102 (a)(1) and (a)(2) as being anticipated by Steinberg et al (WIPO International Application Publication WO 2018/11898 A1, published June 28, 2018), cited on the IDS filed April 12, 2022. This rejection is maintained and modified as necessitated by amendments.
Regarding claim 4, Steinberg teaches a method comprising the steps of: a) segregating a plurality of polynucleotide constructs into compartments (Page 1, [0003]-[0004] and Page 17, [0062]). Steinberg 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 13-14, [0048]). Steinberg teaches a second polynucleotide sequence comprising a DNA target or a DNA template encoding an RNA target (Page 45, [0169], Page 49, [0178], Pages 1-16, [0063], Page 19, [0067], Page 4, [0016] and Page 9, [0034]). Steinberg teaches when the second polynucleotide sequence comprises a DNA template encoding an RNA target, and RNA target is co-expressed contiguously with the gRNA as a single RNA transcript, driven by the first promoter (Pages 17-18, [0063], Page 19, [0067], Pages 19-20, [0069], Page 4, [0016] and Page 9, [0034]). Steinberg teaches the plurality of the polynucleotide constructs encode different gRNAs, and/or different DNA or RNA targets (Page 18, [0064], Pages 20-21, [0071] and Page 30, [0103]). Steinberg teaches each compartment further comprises an RNA- guided nucleic acid modifying enzyme or a variant thereof or a nucleotide template encoding the same (Page 10, [0037]). Steinberg teaches subjecting the compartments to conditions which allow in vitro transcription and/or translation of RNAs and proteins (Page 1, [0003], Pages 22-23, [0073] and Pages 17-18, [0063]). Steinberg 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 3-4, [0014]-[0016]). Steinberg 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 3, [0014]-[0015]). Steinberg teaches polynucleotide constructs and/or RNA transcripts which have not been modified by the nucleic acid modifying enzyme(s) (Page 8, [0031], Page 9, [0038] and Pages 12-13, [0045]). Steinberg teaches harvesting the population of DNA/RNA molecules produced in step (c) and subjecting the same to single molecule long-read sequencing (Page 25, [0082] and [0084]). Steinberg teaches detecting and counting the DNA/RNA molecules referred to in step c)i based on the sequencing results (Page 25, [0082] and [0084]).
Steinberg teaches each and every limitation of claim 4 and therefore Steinberg anticipates claim 4.
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.
Claims 1-3, 7-11, 21, 23-24, 28 and 30 are rejected under 35 U.S.C. 103 as being unpatentable over Steinberg et al (WIPO International Application Publication WO 2018/11898 A1, published June 28, 2018), cited on the IDS filed April 12, 2022, 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 rejection is maintained and modified as necessitated by amendments.
Regarding claim 1, Steinberg teaches a method comprising the steps of segregating a plurality of polynucleotide constructs into compartments (Page 1, [0003]-[0004] and Page 17, [0062]). Steinberg teaches each compartment comprises a single polynucleotide construct, wherein each polynucleotide construct comprises a first polynucleotide sequence encoding a nucleic acid modifying enzyme or a variant thereof, operably linked to a first promoter (Pages 3-4, [0015]-[0016]). Steinberg teaches a second polynucleotide sequence comprising a DNA target or a DNA template encoding an RNA target (Page 45, [0169], Page 49, [0178], Pages 1-16, [0063], Page 19, [0067], Page 4, [0016] and Page 9, [0034]). Steinberg 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 (Pages 17-18, [0063], Page 19, [0067], Pages 19-20, [0069], Page 4, [0016] and Page 9, [0034]). Steinberg teaches the plurality of the polynucleotide constructs encode different variants of the nucleic acid modifying enzyme, and/or different DNA or RNA targets (Page 18, [0064], Pages 20-21, [0071] and Page 30, [0103]). Steinberg teaches subjecting the compartments to conditions which allow in vitro expression of RNAs and proteins (Page 1, [0003], Pages 22-23, [0073] and Pages 17-18, [0063] ). Steinberg teaches subjecting the plurality of 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 (Page 3, [0014]-[0015]). Steinberg 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 3, [0014]-[0015]). Steinberg teaches polynucleotide constructs and/or RNA transcripts which have not been modified by the nucleic acid modifying enzymes (Page 8, [0031], Page 9, [0038] and Pages 12-13, [0045]). Steinberg teaches harvesting the population of DNA/RNA molecules produced in step (c) and subjecting the same to single molecule sequencing (Page 25, [0082] and [0084]). Steinberg teaches detecting and counting the DNA/RNA molecules referred to in step c)i based on the sequencing results (Page 25, [0082] and [0084]).
Regarding claim 2, Steinberg 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 1-2, [0004] and [0007]).
Regarding claim 3, Steinberg 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) (Pages 1-2, [0004] and [0007]). Steinberg 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 18, [0064], Pages 20-21, [0071] and Page 30, [0103]).
Regarding claim 7, Steinberg teaches breaking the compartments by physical or chemical methods Page 2, [0005]).
Regarding claim 8, Steinberg teaches purifying the harvested DNA/RNA molecules to remove excess DNA, RNA and/or proteins from the reaction (Page 57, [0201] and Page 59, [0205]).
Regarding claim 9, Steinberg 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 46, [0175]).
Regarding claim 10, Steinberg 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 25, [0082] and [0084] and Page 46, [0175]).
Regarding claim 11, Steinberg teaches the modification activity is cleavage activity (Page 2, [0004]).
Regarding claim 20, Steinberg teaches the first and second polynucleotide constructs (Pages 3-4, [0015]-[0016]).
Regarding claim 21, Steinberg 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 (Pages 42-44, [0159]-[0164]).
Regarding claim 23, Steinberg 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 20, [0071]).
Regarding claim 24, Steinberg teaches the nucleic acid modifying enzyme is a CRISPR-associated protein (Cas) (Page 42, [0157] and [0159] and Page 51, [0183].
Regarding claim 28, Steinberg teaches each of the compartments further comprises in vitro transcription and translation (IVTT) reagents, said IVTT reagents enable the in vitro transcription and/or translation of proteins and/or RNAs (Page 1, [0003], Pages 22-23, [0073] and Pages 17-18, [0063]). Steinberg teaches the compartments are emulsion droplets (Page 7, [0028]-[0029] and Page 17, [0062]).
Regarding claim 30, Steinberg teaches the segregation is achieved using microfluidics (Pages 22-23, [0073]).
Steinberg does not teach or suggest the first and second polynucleotide sequences are fully or partially overlapping. Steinberg does 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. Steinberg does 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. Steinberg does 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. Steinberg does 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 Steinberg 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).
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Steinberg et al (WIPO International Application Publication WO 2018/11898 A1, published June 28, 2018), cited on the IDS filed April 12, 2022, and 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, as applied to claims 1-3, 7-11, 20-21, 23-24, 28 and 30 above, 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 rejection is maintained and modified as necessitated by amendments.
Regarding claim 5, Steinberg teaches evaluating the modifying activity of one or more nucleic acid modifying enzymes against one or more of the DNA/RNA targets (Page 4, [0016]). Steinberg teaches analyzing enzymatic activity (Abstract and Pages 22-21, [0073]).
Steinberg and Pinello do 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. Steinberg and Pinello do 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 Steinberg and Pinello, 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).
Response to Arguments
Applicant’s arguments and amendments, filed December 22, 2025 regarding the rejections of claims 6 and 20 under 35 U.S.C. § 112(b) have been fully considered and these rejections are rendered moot due to the cancellation of claims 6 and 20. Therefore, these rejections are withdrawn.
Applicant’s arguments and amendments, filed December 22, 2025 regarding the rejections of claims 1-3, 5, 7-11, 21, 23-24 and 28 under 35 U.S.C. § 112(b) have been fully considered and are deemed to be persuasive. Therefore, these rejections are withdrawn.
Applicant’s arguments and amendments, filed December 22, 2025 regarding the rejections of claims 6, 13, 15-16 and 20 under 35 U.S.C. § 102 and 103 have been fully considered and these rejections are rendered moot due to the cancellation of claims 6, 13, 15-16 and 20. Therefore, these rejections are withdrawn.
Applicant’s arguments and amendments, filed December 22, 2025 regarding the rejections of claim 4 under 35 U.S.C. § 102 and 103 have been fully considered but they are not persuasive.
Applicant asserts “that claims 1and 4 differs from Steinberg at least insofar that an intermediate step of ligation/amplification with a capture probe is not needed, allowing evaluation of modified and unmodified nucleic acid fragments, which is single molecule sequencing”.
However, it is noted that “an intermediate step of ligation/amplification with a capture probe is not needed” is not a claim limitation, and the claims do not make any mention of lacking amplification/ligation or capture probe and it is not proper to import claim limitations from the specification into the claims. “Though understanding the claim language may be aided by explanations contained in the written description, it is important not to import into a claim limitations that are not part of the claim. For example, a particular embodiment appearing in the written description may not be read into a claim when the language is broader than the embodiment.” Superguide Corp. v. DirecTV Enterprises, Inc., 358 F.3d 870, 875, 69 USPQ2d 1865, 1868 (Fed. Cir. 2004).
Additionally, claim 4 and 1 do not require “allowing evaluation of modified and unmodified nucleic acid fragments”. Claims 1 and 4 as amended require in step c) “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, thereby producing a population of DNA/RNA molecules that comprises: i. polynucleotide constructs and/or RNA transcripts or fragments thereof that have been modified by the nucleic acid modifying enzyme(s); and/OR
ii. polynucleotide constructs and/or RNA transcripts which have not been modified by the nucleic acid modifying enzyme(s)”. The and/or of the claims, allows for either both steps i and ii, OR just i, OR just ii to be required by the claim. Therefore, claims 1 and 4 as amended does not require allowing evaluation of modified and unmodified nucleic acid fragments. Additionally, Steinberg discloses “sequencing the cleaved nucleic acid template… any sequencing method can be used” therefore reading on the claim limitation including “single molecule sequencing”(Page 25, [0082] and [0084]).
Moreover, the instant specification also defines that “modification activity is cleavage activity (Page 28, [0090]), therefor polynucleotide constructs and/or RNA transcripts which have not been modified by the nucleic acid modifying enzyme, using the broadest reasonable interpretation, may be interpreted as polynucleotide constructs and/or RNA transcripts which have not been cleaved (uncleaved). As discussed above, Steinberg does in fact disclose a population of unmodified/uncleaved nucleic acid templates (Page 8, [0031], Pages 10-11, [0038], Pages 12-13, [0045]).
Therefore, for all these reasons, and those listed above, Steinberg, Steinberg and Pinello, Steinberg and Pinello in view of Ryan are deemed to render the instant invention obvious.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to 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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/JESSICA D PARISI/Examiner, Art Unit 1684
/HEATHER CALAMITA/Supervisory Patent Examiner, Art Unit 1684