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 .
Office Action: Notice
Any objection or rejection of record in the previous Office Action, mailed 10/2/2025,
which is not addressed in this action has been withdrawn in light of Applicants' amendments
and/or arguments. This action is FINAL
Claim Status
Claims 1, 4-6, 12 and 19 have been amended (12/24/2025). No new matter was added. Thus, claims 1-21 are under examination (12/24/2025).
Priority
Claims 1-21 receive a priority date of 12/20/2019, the effective filing date of European Patent Application No. EP19218832.4. All priority documents have been received.
Information Disclosure Statement
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
The Information Disclosure Statement filed 12/24/2025 has been considered.
Objections Withdrawn
Specification:
The objections to the specification due to the use of a trademark or tradenames are withdrawn in view of Applicant’s amendments.
The objections to the specification due to the use of a hyperlink or browser-executable code are withdrawn in view of Applicant’s amendments.
Claims:
The objections to claims 12 and 19 for minor clerical and/or formatting issues, are withdrawn due to Applicant’s amendments.
Rejections Withdrawn
Claim Rejections - 35 USC § 112(b)
The rejections of claims 1-21 under 35 U.S.C. 112(b) or pre-AIA 35 U.S.C. 112, 2nd paragraph, are withdrawn in view of Applicant’s amendments of claims 1 to address and further clarify indefiniteness.
Claim Rejections - 35 USC § 101
The rejection of claims 1-21 under 35 U.S.C. 101 is withdrawn in view of Applicant’s amendments of claim 1, as well as Applicant’s clarification of the intended invention. Specifically, the amended instant claims are no longer directed to a naturally occurring product, but a man-made adapter and a multi-step laboratory process that does not exist in nature. Amended independent claim 1 now recites a double-stranded adapter with a protelomerase recognition sequence and a staggered end, which constitutes a structural modification conferring markedly different characteristics from any naturally occurring nucleic acid. Further, the Applicant clarified that claims 5-20 are method claims that recite a specific sequence of human-directed laboratory steps (ligation, protelomerase-mediated closing, targeted cleavage, and amplification), thereby integrating any natural components into a practical application. When considered as a whole, the claims are directed to patent-eligible subject matter under Step 2A/2B because they recite significantly more than a judicial exception and are not merely isolated products of nature.
Claim Rejections - 35 USC § 102
The rejection of claims 1-21 under 35 U.S.C. 102 (a)(1) and (a)(2) as being anticipated by Arakawa et al., (WO 2017/081097 A1, published 5/18/2017) are withdrawn in view of Applicant’s amendments, specifically relating to increased limitations of the protelomerase recognition sequence.
New Rejections
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
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(s) 1-21 are rejected under 35 U.S.C. 103 as being unpatentable over Arakawa et al., (WO 2017/081097 A1, published 5/18/2017), and in further view of Ooi et al. (“Recombineering linear DNA that replicate stably in E. coli”, Plasmid, published 1/2008).
Regarding claims 1-3, Arakawa teaches a method to construct a gRNA library by molecular biological techniques (i.e., adapters), without relying on bioinformatics, and which allows forward genetics screening of any species, independent of their genetic characterization and it is possible to create guide or identifier sequences even from unknown genetic information (Figure 2; p. 4, lines 5-15). Further, Arakawa teaches that one synthesizes cDNA from the mRNA sequence using a semi-random primer containing a complementary sequence to the PAM and then cuts out the 20-mer adjacent to the PAM using type IIS and type III restriction enzymes to create a gRNA library (p. 4, lines 10-15). Further, Arakawa teaches that the second strand of cDNA was synthesized by primer extension using a primer that annealed at the 5' SMART tag sequence with advantage 2 PCR polymerase, which generated A-overhang or staggered ends at the 3' terminus, where this A-overhang was ligated with 3' linker I, which contains EcoP15I and Acul sites for cutting out the guide sequence afterwards (p. 39, lines 20-30).
Also, Arakawa teaches that a further object of the invention is a method for obtaining a guide sequence comprising the following steps: a) DNA synthesis from an RNA or a DNA using a semi-random primer, b) generation of guide sequences by molecular biological methods where the guide sequence is preferably generated from mass RNA or DNA by molecular biological methods including cDNA synthesis and/or restriction digest and/or DNA ligation and/or PCR (p. 5, lines 5-10). Arakawa teaches that said guide sequence is preferably generated cutting the synthetized DNA to obtain a guide sequence consisting of 20 base pairs and the cutting is preferably carried out with at least one type III restriction enzyme and/or a type IIS restriction enzyme (p. 5, lines 10-15).
Arakawa teaches that the previously described method of library construction via nucleic acid adapters or guide sequences can be applied to a library containing 73,000 sgRNAs was used to generate knockout collections and performed screens in two human cell lines, where a screen for resistance to the nucleotide analog 6-thioguanine identified all expected members of the DNA mismatch repair pathway, whereas another for the DNA topoisomerase II (TOP2A) or TeIN protelomerase poison etoposide identified TOP2A, as expected, and also cyclin-dependent kinase 6, CDK6 (p. 2, lines 20-25).
Regarding claims 4-7, Arakawa teaches that the previously described method of library construction via nucleic acid adapters or guide sequences includes using a semi-random primer (NCCNNN) that contained the PAM-complementary CCN, cDNA was reverse-transcribed from poly(A) RNA of the chicken B cell line DT40crel (II, 12l (Figure le), where the 5' SMART tag sequence containing the EcoP15I site was added onto the 5' side by the switching mechanism at RNA transcript (SMART) method (p. 39, lines 20-25). Further, Arakawa teaches that the second strand of cDNA was synthesized by primer extension using a primer that annealed at the 5' SMART tag sequence with advantage 2 PCR polymerase, which generated A-overhang or staggered ends at the 3' terminus, where this A-overhang was ligated with 3' linker I, which contains EcoP15I and Acul sites for cutting out the guide sequence afterwards (p. 39, lines 20-30). Arakawa also teaches that the dscDNA was digested with EcoP15I to remove the 5' SMART tag sequence and was ligated with 5' linker I that included a BsmBI site, a cloning site for the gRNA expression vector followed by the DNA then being digested with Bglll to destroy the 5' SMART tag backbone and amplified by PCR (Figure Id; p. 39, lines 25-30).
Arakawa teaches that the previously described method of library construction via nucleic acid adapters or guide sequences includes Type IIS or type III restriction enzymes cleave sequences separated from their recognition sequences where the type III restriction enzyme, EcoP15I, cleaves 25/27 bp away from its recognition site but requires a pair of inversely-oriented recognition sites for efficient cleavage) and the type IIS restriction enzyme, Acul, cleaves 13/15 bp away from its recognition site (p. 39, lines 10-15).
Arakawa teaches that the previously described method of library construction via nucleic acid adapters or guide sequences can be applied to a library containing 73,000 sgRNAs was used to generate knockout collections and performed screens in two human cell lines, where a screen for resistance to the nucleotide analog 6-thioguanine identified all expected members of the DNA mismatch repair pathway, whereas another for the DNA topoisomerase II (TOP2A) or TeIN protelomerase poison etoposide identified TOP2A, as expected, and also cyclin-dependent kinase 6, CDK6 (p. 2, lines 20-25).
Regarding claims 8-9, Arakawa teaches that the previously described method of library construction via nucleic acid adapters or guide sequences includes Type IIS or type III restriction enzymes cleave sequences separated from their recognition sequences where the type III restriction enzyme, EcoP15I, cleaves 25/27 bp away from its recognition site but requires a pair of inversely-oriented recognition sites for efficient cleavage) and the type IIS restriction enzyme, Acul, cleaves 13/15 bp away from its recognition site (p. 39, lines 10-15). Additionally, Arakawa teaches that the semi-random primer within the previously described method can potentially target any NGG on mRNA, generating a highly complex gRNA library that covers more than 90% of the annotated genes (Fig. 4B) and the method described here could be applied to CRISPR systems in organisms other than S. pyogenes by customizing the semi-random primer (p. 43, lines 5-15).
Regarding claims 10-11, Arakawa teaches that the previously described method of library construction via nucleic acid adapters or guide sequences includes an additional step where the guide sequence fragment is purified from the digested DNA and ligated with a further linker sequence at the 3' end comprising a restriction site which is a cloning site for the gRNA expression vector and optionally a ninth restriction site, preferably Aatll restriction site (p. 7, lines 15-20).
Arakawa teaches that the previously described method of library construction via nucleic acid adapters or guide sequences includes using a semi-random primer (NCCNNN) that contained the PAM-complementary CCN, cDNA was reverse-transcribed from poly(A) RNA of the chicken B cell line DT40crel (II, 12l (Figure le), where the 5' SMART tag sequence containing the EcoP15I site was added onto the 5' side by the switching mechanism at RNA transcript (SMART) method (p. 39, lines 20-25). Further, Arakawa teaches that the second strand of cDNA was synthesized by primer extension using a primer that annealed at the 5' SMART tag sequence with advantage 2 PCR polymerase, which generated A-overhang or staggered ends at the 3' terminus, where this A-overhang was ligated with 3' linker I, which contains EcoP15I and Acul sites for cutting out the guide sequence afterwards (p. 39, lines 20-30). Arakawa also teaches that the dscDNA was digested with EcoP15I to remove the 5' SMART tag sequence and was ligated with 5' linker I that included a BsmBI site, a cloning site for the gRNA expression vector followed by the DNA then being digested with Bglll to destroy the 5' SMART tag backbone and amplified by PCR (Figure Id; p. 39, lines 25-30).
Regarding claims 12-16, Arakawa teaches that the previously described method of library construction via nucleic acid adapters or guide sequences includes contacting a population of cells with a composition comprising a vector system comprising one or more packaged vectors comprising a) a first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence that targets a DNA molecule encoding a gene product, wherein the polynucleotide sequence comprises (a) a guide sequence capable of hybridizing to a target sequence, (b) a tracr mate sequence, and (c) a tracr sequence, and b) a second regulatory element operably linked to a Cas protein and a selection marker, wherein components (a) and (b) are located on same or different vectors of the system, wherein each cell is transduced or transfected with a single packaged vector, selecting for successfully transduced cells, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in the genomic loci of the DNA molecule encoding the gene product, wherein the CRISPR complex comprises a CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence, wherein the guide sequence is selected from the library of the invention, wherein the guide sequence targets the genomic loci of the DNA molecule encoding the gene product and the CRISPR enzyme cleaves the genomic loci of the DNA molecule encoding the gene product and whereby each cell in the population of cells has a unique gene knocked out in parallel (p. 10, lines 5-25).
Further, Arakawa teaches that the CRISPR enzyme described above is truncated, and/or comprised of less than one thousand amino acids or less than four thousand amino acids, and/or is a nuclease (i.e., exonuclease) or nickase, and/or is codon optimized, and/or comprises one or more mutations, and/or comprises a chimeric CRISPR enzyme, and/or the other options as herein discussed (p. 16, lines 10-15)
Arakawa also teaches that the previously described methods and uses may be carried out in any kind of cells or organisms (p. 10, lines 25-30).
Regarding claim 17, Arakawa teaches that the previously described method of library construction via nucleic acid adapters or guide sequences can be applied to a library containing 73,000 sgRNAs was used to generate knockout collections and performed screens in two human cell lines, where a screen for resistance to the nucleotide analog 6-thioguanine identified all expected members of the DNA mismatch repair pathway, whereas another for the DNA topoisomerase II (TOP2A) or TeIN protelomerase poison etoposide identified TOP2A, as expected, and also cyclin-dependent kinase 6, CDK6 (p. 2, lines 20-25). Further, Arakawa teaches that the previously described repair identification method can incorporate a negative selection screen for identifying essential genes via numerous gene sets corresponding to fundamental processes and sgRNA efficiency associated with specific sequence motifs, enabling the prediction of more effective sgRNAs and therefore, these results establish Cas9/sgRNA screens as a powerful tool for systematic genetic analysis in mammalian cells (p. 2, lines 25-30).
Regarding claims 18-19, Arakawa teaches that the previously described method of library construction via nucleic acid adapters or guide sequences includes using a semi-random primer (NCCNNN) that contained the PAM-complementary CCN, cDNA was reverse-transcribed from poly(A) RNA of the chicken B cell line DT40crel (II, 12l (Figure le), where the 5' SMART tag sequence containing the EcoP15I site was added onto the 5' side by the switching mechanism at RNA transcript (SMART) method (p. 39, lines 20-25). Further, Arakawa teaches that the second strand of cDNA was synthesized by primer extension using a primer that annealed at the 5' SMART tag sequence with advantage 2 PCR polymerase, which generated A-overhang or staggered ends at the 3' terminus, where this A-overhang was ligated with 3' linker I, which contains EcoP15I and Acul sites for cutting out the guide sequence afterwards (p. 39, lines 20-30). Arakawa also teaches that the dscDNA was digested with EcoP15I to remove the 5' SMART tag sequence and was ligated with 5' linker I that included a BsmBI site, a cloning site for the gRNA expression vector followed by the DNA then being digested with Bglll to destroy the 5' SMART tag backbone and amplified by PCR (Figure Id; p. 39, lines 25-30).
Additionally, Arakawa teaches a CRISPR-Cas system sgRNA library obtainable by the above defined method (p. 8, lines 5-10).
Regarding claim 20, Arakawa teaches that the previously described method of library construction via nucleic acid adapters or guide sequences further comprises extracting DNA and determining the depletion or enrichment of the guide sequences by deep sequencing (p. 12, lines 10-15).
Regarding claim 21, Arakawa teaches kits comprising a panel comprising a selection of unique CRISPR-Cas system guide RNAs comprising guide sequences from the library of the invention, wherein the selection is indicative of a particular physiological condition; or in other embodiments a panel of target sequences is focused on a relevant or desirable pathway, such as an immune pathway or cell division (p. 9, lines 10-20).
Arakawa does not teach or suggest using a double-stranded protelomerase recognition sequence, specifically, as a cleavage tool for use as an adapter in a library workflow.
Ooi teaches a novel application of recombineering to linearize DNA by capping their ends with individual telomeres derived from bacteriophage N15, which exists as a linear prophage in E. coli and where the N15 telomerase occupancy site was recombined into circular DNA and resolved into individual telomeres by the phage N15 protelomerase enzyme (Abstract). Further, Ooi teaches that in order to further advance recombineering technology they developed a novel method to purify high-quality linear DNA directly from E. coli without restriction digestion or gel purification where linearization is achieved in E. coli by capping of DNA ends with individual telomeres derived from the bacteriophage N15 telomerase occupancy site (Introduction: Paragraphs 3-5). Ooi also teaches that he protelomerase gene (TelN) was recombineered into the chromosome of the host DH10B to develop a specific strain expressing the TelN enzyme for tos resolution (Fig. 1a) and one application of this linearization system is for building artificial chromosome vectors for studying DNA requirements for centromere formation using linear constructs (Discussion: Paragraphs 1-4).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify Arakawa’s library construction method to incorporate the protelomerase recognition and processing system taught by Ooi, because both references address the manipulation and processing of double-stranded DNA fragments in E. coli, and Ooi expressly teaches a reliable enzymatic mechanism for the site-specific cleavage and end-processing of linear dsDNA. Incorporating Ooi’s protelomerase-based cleavage system into Arakawa’s workflow would have been a predictable substitution for known restriction-based processing steps to achieve controlled DNA fragment processing and stabilization. The motivation to combine arises from the recognized need in the art for precise, efficient, and stable processing of linear DNA fragments during library construction, particularly when generating large or complex DNA populations, as addressed by Ooi’s linear DNA recombineering system. Applying Ooi’s teachings to Arakawa’s library construction method would have been a logical design choice to improve handling and processing of double-stranded DNA intermediates without altering the fundamental purpose of Arakawa’s gRNA library generation.
A person of ordinary skill in the art would have had a reasonable expectation of success in making this combination because Ooi demonstrates successful implementation of bacteriophage-derived protelomerase activity in E. coli using standard recombineering techniques, and Arakawa already employs conventional enzymatic DNA synthesis, ligation, and digestion steps in similar host systems. The combination merely applies a known, well-characterized DNA processing mechanism to an analogous DNA manipulation context, yielding predictable results consistent with established molecular biology principles.
Applicant’s Response: The Applicant argues that Arakawa does not teach using a double-stranded protelomerase recognition sequence or bacteriophage-derived protelomerase as a cleavage or adapter element in a library construction workflow, asserting that Arakawa relies on restriction enzymes and conventional adapters. The Applicant further asserts that Arakawa contains no disclosure of adapters, protelomerase recognition sequences, or their use in producing a nucleic acid library, and therefore cannot anticipate the claimed subject matter.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully and fully considered and are found to be partially persuasive, as discussed below.
Although the newly amended claim set was found to not be anticipated, in that every limitation of the claims under 35 USC 102 was met, the claims remain unpatentable under 35 USC 103. Notably, Arakawa teaches the core guide-library construction workflow using adapters, PCR, and enzymatic cleavage, while Ooi teaches the use of a bacteriophage N15 protelomerase (TeIN) and its double stranded recognition site to cleave and covalently process DNA ends in a predictable and controlled manner. A person of ordinary skill in the art would have been motivated to combine these teachings to substitute TeIN-mediated end processing for conventional restriction-enzyme-based cleavage in Arakawa’s library preparation, in order to achieve precise end formation and improved control over DNA termini. Further, Ooi demonstrates that TeIN reliably processes engineered recognition sites with a reasonable expectation of success, rendering the claimed combination obvious despite the absence of an express TeIN disclosure in Arakawa.
Conclusions
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any extension fee 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 date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ELIZABETH ROSE LAFAVE whose telephone number is (703)756-4747. The examiner can normally be reached Compressed Bi-Week: M-F 7:30-4:30.
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/ELIZABETH ROSE LAFAVE/Examiner, Art Unit 1684
/HEATHER CALAMITA/Supervisory Patent Examiner, Art Unit 1684