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 .
Nucleotide and/or Amino Acid Sequence Disclosures
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiencies and the required response to this Office Action are as follows:
Specific deficiency – Nucleotide and/or amino acid sequences appearing in the drawings are not identified by sequence identifiers in accordance with 37 CFR 1.821(d). Sequence identifiers for nucleotide and/or amino acid sequences must appear either in the drawings or in the Brief Description of the Drawings. FIG5E, FIG 8, FIG 15B, FIG 16B and FIG 20B show nucleotide sequences without SEQ ID NOs and which are also not identified by SEQ ID NO in the specification on pages 7-13.
Required response – Applicant must provide:
• Replacement and annotated drawings in accordance with 37 CFR 1.121(d) inserting the required sequence identifiers;
AND/OR
• A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required sequence identifiers into the Brief Description of the Drawings, consisting of:
o A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
o A copy of the amended specification without markings (clean version); and
o A statement that the substitute specification contains no new matter.
Application Status
This action is written in response to applicant’s correspondence received on 06/03/2026. Claims 1-17, 19, 21, 23 are currently pending. Claims 1-11, 13-17, 19, 21 are withdrawn from prosecution as being drawn to nonelected subject matter. Accordingly, claims 12 and 23 are examined herein. The restriction requirement mailed on 02/23/2026 is still deemed proper. Applicant's elected Group III and species 6) AtCas9 without traverse in the reply filed on 06/03/2026.
Election/Restrictions
Applicant's election without traverse of Group III and species AtCas9 in the reply filed on 06/03/2026 is acknowledged.
Claims 1-11, 13-17, 19, 21 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected Groups I & II and species 1)-5), there being no allowable generic or linking claim. Claims 12 and 23 are examined on the merit herein.
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.
Priority
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. This application is a 371 of PCT/CN2022/077097 filed on 02/21/2022.
Acknowledgment is made of applicant's claim for foreign priority based on an application filed in PCT/CN2021/076930 filed on 02/19/2021.
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Drawings
The drawing is objected to because 37 CFR 1.84 (u)(1) states “View numbers must be preceded by the abbreviation "FIG.”.
In the current case, the view number for Figure 1 is preceded by the word "Figure" instead of
the abbreviation "FIG.".
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
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.
Claim 12 is rejected under 35 U.S.C. 102(a)(1) as being anticipated by Tsui (ACS Synth Biol. 2017 Jun 16;6(6):1103-1113).
Tsui teaches a method, comprising contacting the target nucleic acid with a CRISPR-Cas system comprising: a Cas protein with a corresponding protospacer adjacent motif (PAM) required for targeting a linear double stranded DNA, and a guide RNA comprising a guide sequence that hybridizes to a target sequence in the target nucleic acid, adjacent to a target PAM sequence, wherein the target sequence has an underwound topology and the target PAM sequence is not the corresponding PAM of the Cas protein.
Tsui demonstrates the method above in Figure 6 (Page1109): see below. The CRISPR-Cas system shown comprises: a Cas protein, AceCas9, with a corresponding PAM, PAMWT 5’-NNNCC-3’ (Figure 6 legends (A)), required for targeting a linear double stranded DNA, the ccdB plasmid that has a unique protospacer sequence and either a PAMWT, or a double mutant thereof, PAMC(4,5)T 5’-NNNTT-3’ that is not the corresponding PAMWT (Figure 6, legends (A)). A single guide RNA (sgRNA) with either a 20nt, 24nt, or 26nt spacer targeting the cytotoxic ccdB gene was used to complete the method. Tsui further teaches that plasmids have negative supercoil structure, i.e. underwound topology, given that the instant application teaches that “Naturally existing underwound DNA such as negative supercoil or Z-form dsDNA have smaller torque, …” (Page 47, ¶[0175], lines 8-10), and that negative supercoiling in the plasmid specifically enhances the editing tolerance for mutations in the spacer and the proximal nucleotides in the PAM (Page 1109, last ¶).
Although the recitation “for editing a target nucleic acid” is intended use and does not involve additional active step that distinguishes the claim from the prior art, Tsui nevertheless demonstrates effective editing of the cytotoxic ccdB gene induced under Arabinose treatment using 26nt sgRNA with the mutant PAMC(4,5)T, resulting in cell survival (Figure 6, panel C).
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Claim 12 is rejected under 35 U.S.C. 102(a)(1) as being anticipated by Ivanov (Proc Natl Acad Sci U S A. 2020 Mar 17;117(11):5853-5860).
Ivanov (2020) teaches a method, comprising contacting the target nucleic acid with a CRISPR-Cas system comprising: a Cas protein, Streptococcus pyogenes dCas9 (Page 5854, left column, 5th ¶) or Cas9 (Page 5857, last ¶), with a corresponding protospacer adjacent motif (PAM; TGG, Page 5855, Fig. 1, legend, line 3) required for targeting a linear double stranded DNA, and a guide RNA comprising a guide sequence that hybridizes to a target sequence in the target nucleic acid, adjacent to a target PAM sequence, wherein the target sequence has an underwound topology and the target PAM sequence is not the corresponding PAM of the Cas protein by comparing the R-loop formation states using PAM-distal and PAM-proximal mismatches, i.e. λ1 DNA target site mutated PAM (TCG, instead of the SpyCas9 corresponding PAM, TGG; Table S3, page 15 of Ivanov Supplementary Information, included in the same Ivanov pdf attached and listed in PTO-892, page 23/24), and evaluating the impact of target DNA topology on the targeted gene editing effect using Cas9-based cleavage assays (Page 5859, last ¶).
Ivanov further teaches that “we observed a high level of Cas9 promiscuity at target sites with many PAM-distal mismatches under physiological levels of negative supercoiling” (Page 5858, right column, second ¶, lines 1-3). And it further teaches “In the presence of PAM-distal mismatches, we observe that dCas9 can achieve stable binding …. Mechanical stabilization of the complete R-loop in negatively supercoiled substrates can lead to cleavage even in the presence of extensive mismatches.” (Page 5854, left column, 4th ¶, lines 9-19). As discussed in the §102 rejection above, negative supercoil is one form of underwound topology. Hence, Ivanov teaches that underwound topology allows Cas9 to bind with mutated PAM sites with substantial tolerance for both distal and proximal mismatches in target DNA PAM sites, thereby supporting target-specific gene editing wherein the target PAM sequence is not the corresponding PAM of the Cas protein.
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 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 12 is rejected under 35 U.S.C. 103 as being unpatentable over Jinek (Science. 2012 Aug 17;337(6096):816-21) in view of Tsui (2017; full citation above) or Ivanov (2020; full citation above).
Jinek (2012) teaches a method for editing a target nucleic acid, comprising contacting the target nucleic acid with a CRISPR-Cas system comprising: a Cas9 protein with a corresponding protospacer adjacent motif (PAM) required for targeting a linear double stranded DNA, and a single guide RNA chimera comprising a guide sequence that hybridizes to a target sequence in the target nucleic acid, adjacent to a target PAM sequence (Page 820, Fig. 5A-D).
Jinek does not teach the target sequence has an underwound topology and that the target PAM sequence is not the corresponding PAM of the Cas protein.
The teachings of Tsui (2017) and Ivanov (2020) have been discussed above and applied herein.
It would have been obvious to one with ordinary skills in the art (OSITAs) before the effective filing date of the claimed invention to have modified the PAM requirement of known Cas proteins in the art based on target DNA topology in search of strategies to achieve PAM-less genome-wide targeting. By modifying the methods of Jinek (2012) under the conditions of underwound target nucleic acid created by Tsui (2017) or Ivanov (2020), OSITAs would have substituted target sites with sites adjacent to different/mutated PAM motifs, under conditions of underwound target DNAs, which would have merely amounted to a simple substitution of prior art elements according to known methods to yield predictable results. One would have been motivated to do so because the discovery of Tsui (2017) or Ivanov (2020) that underwound target DNA topology reduces the stringency of corresponding PAM sequences for well-known Cas proteins. One would have reasonable expectation of success because both Tsui (2017) and Ivanov (2020) demonstrate, independently, that negative supercoils can lead to promiscuous interactions between the Cas protein and the alternative PAM motifs, (Page 6/7, FIG. 4C).
Claim 23 is rejected under 35 U.S.C. 103 as being unpatentable over Jinek (2012), in view of Tsui (2017; full citation above) or Ivanov (2020; full citation above), further in view of Haft (PLoS Comput Biol. 2005 Nov;1(6):e60), NCBI RefSeq WP_058095017.1 (2015, NCBI RefSeq_WP_058095017.1_2015.pdf listed in PTO-892; Cited on IDS filed on 08/17/2023), and Walton (Science. 2020;368 (6488): 290-296).
The teachings of Jinek (2012), Tsui (2017), and Ivanov (2020) have been discussed above and applied herein. None of Jinek, Tsui, or Ivanov teaches AtCas9, or its corresponding PAM.
However, Haft (2005; Page 0476, Table 1, HMM TIGR01865) deposited the NCBI RefSeq WP_058095017.1 (2015) for a type II CRISPR RNA-guided endonuclease Cas9 derived from Alicyclobacillus tengchongensis, later became named as AtCas9, as evidenced by Shi (Mol Cell. 2022 Nov 3;82(21):4160-4175.e6).
Neither Haft nor NCBI RefSeq WP_058095017.1 teaches the corresponding PAM sequences of the Cas9 derived from Alicyclobacillus tengchongensis.
However, Walton (2020) teaches a high-throughput PAM determination assay (HT-PAMDA) involving a library of PAMs that cover all possible sequences to comprehensively profile and screen the PAM preferences of a large number of SpCas9 variants (Page 1 of 7; Right column, second ¶, lines 3-6; Fig. S2A, see below). Because the HT-PAMDA assay involves predesigned PAM libraries, Walton further teaches that “The complexity of each library was estimated to be greater than 105 unique PAMs based on the number of transformants.” (Supplementary Data, Page 2, the last 3 lines).
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It would have been obvious to one with ordinary skills in the art (OSITAs) before the effective filing date of the claimed invention to have modified the PAM-target DNA topology relationships of all known Cas proteins in the art, including the Cas9 derived from Alicyclobacillus tengchongensis before it was even named AtCas9, in search of those that can be exploited for PAM-less genome-wide targeting. Motivated by the discovery of Tsui (2017) or Ivanov (2020) that underwound target DNA topology reduces the stringency of corresponding PAM sequences for well-known Cas proteins, expanding the target range in a genome. To modify the methods of Tsui (2017) or Ivanov (2020) by replacing the Cas protein to the Cas9 derived from Alicyclobacillus tengchongensis, taught by Haft (2005) and NCBI RefSeq WP_058095017.1, it would have merely amounted to a simple substitution of prior art elements according to known methods to yield predictable results. One would have been motivated to do so because Walton (2020) teaches that the PAM sequence requirements of altered targeting stringency can be determined efficiently for any Cas protein under any targeting conditions using known, finite number of solutions to address known problems, and providing predictable outcomes, forming an “obvious to try” rationale. One would have reasonable expectation of success because Walton describes successful and efficient PAM identification for multiple variants that lack the canonical PAM motifs (Page 6/7, FIG. 4C).
Based on Tsui’s teaching that “a Cas9-specific 3−8 nucleotides (nts) adjacent to the protospacer known as PAM” (Page 1103, right column, 2nd ¶), a 4 nucleotide PAM library of 44 = 256 PAMs could have been tested first to ensure quality control and proper work flow, and it would have yielded some embodiments that would have included CNNN and RNNA, wherein each N is independently A, T, C or G, and R is A or G, thereby arriving at the claimed inventions. A more comprehensive HT-PAMDA assay would have included a total of 87,360 PAM motifs for HT-PAMDA screen (Total = 44 + 45 + 46 + 47 + 4⁸ = 87,360), and OSITAs would have identified the canonical corresponding PAM motifs of AtCas9 because at least some embodiments of 87,360 randomly designed 8nt PAM candidates would have included CNNN and RNNA, wherein each N is independently A, T, C or G, and R is A or G, thereby arriving at the claimed inventions. PAM candidates that are not either CNNN or RNNA would also have been identified.
Conclusion
No claims are allowable.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Delphinus D. Yu whose telephone number (571) 272-1576. The examiner can normally be reached Mon-Thr 7:30am to 4:30pm Fri 10am to 2pm ET.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Neil P Hammell can be reached on (571) 270-5919. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/DELPHINUS DOU YI YU/Examiner, Art Unit 1636
/NEIL P HAMMELL/Supervisory Patent Examiner, Art Unit 1636
Prosecution Insights