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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 June 19, 2026 has been entered.
Application Status
Applicant’s remarks and amendments to the claims filed June 19, 2026 are acknowledged. Claims 1, 4, 8, and 14 were amended, and claims 2-3, 7, 9, and 15-19 were cancelled. Claims 1, 4, 8, 10-14, and 20 are pending and under consideration herein.
Notice Regarding Evidence Submitted in the Remarks to Traverse Rejections
Applicant’s remarks include evidence submitted to traverse the § 103 rejections raised in the prior action, where the evidence is not otherwise provided for in the disclosure. See the figures and associated description on pgs. 7-9 of the remarks.
37 CFR § 1.132 states that “When any claim of an application or a patent under reexamination is rejected or objected to, any evidence submitted to traverse the rejection or objection on a basis not otherwise provided for must be by way of an oath or declaration under this section.” Applicant has not submitted the evidence traversing the rejections through the proper avenue. The evidence currently presented in the remarks, and any additional evidence which Applicant wishes to have considered in an effort to traverse rejections, must be submitted in the form of an affidavit or declaration under 37 CFR § 1.132. See MPEP 716. The evidence above which is presented in the remarks will not be considered, accordingly.
Withdrawn Rejections
Applicant’s amendments to the claims are sufficient to overcome the § 112(b) and § 112(d) rejections raised in the prior action. Applicant’s amendments to the claims are also sufficient to overcome the § 102 rejections over Choudhary, and § 103 rejections over primary reference Choudhary, in view of Chatterjee, Young, Reddington, and/or PDB_5B43 raised in the prior action. These rejections are withdrawn, accordingly.
Applicant’s remarks and amendments to the claims have been thoroughly reviewed, but are not found persuasive to place the claims in condition for allowance for the reasons that follow. Any rejection or objection not reiterated herein has been overcome by amendment.
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 - The incorporation by reference paragraph required by 37 CFR 1.821(c)(1) is defective. See item 1) a) or 1) b) above. USPTO records indicate that the size of the sequence listing of record is “63,429 bytes,” rather than “61,900 bytes” as recited in the incorporation by reference paragraph.
Required response – Applicant must provide:
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required incorporation-by-reference paragraph, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
Priority
Acknowledgment is made of Applicant's claim for priority under 35 U.S.C. 119(a)-(d) or (f), 365(a) or (b), or 386(a), and claim for benefit under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c). The certified copy of Application No. CN201911092554.8, filed November 11, 2019, has been received. However, Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date as follows:
The disclosure of the prior-filed foreign application, Application No. CN201911092554.8, fails to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application. Specifically, the aforementioned application does not appear to set forth the CRISPR family proteins Cas φ or Cas12g recited in instant claim 4. The first disclosure of the aforementioned CRISPR family proteins is in Application No. PCT/CN2020/127992 (see pg. 6). Accordingly, claims 4, 8, 10-14, and 20 are not entitled to the filing date of the foreign priority document, and the effective filing date of claims 4, 8, 10-14, and 20 is November 11, 2020. Claim 1 under examination finds support in the prior-filed foreign application. The effective filing date of claim 1 is November 11, 2019.
Claim Objections
Claims 4 and 8 are objected to because of the following informalities:
Claim 4 recites “but retaining binding activity. wherein the unnatural amino acid is AeF….” The premature period in this phrase should be deleted.
Claim 4 recites “wherein the unnatural amino acid is AeF.” It is clear that the unnatural amino acid must be AeF. Thus, the earlier recited phrase “wherein the unnatural amino acid bears an azido, an alkynyl, a cyclopropenyl or a tetrazine group” should preferably be deleted.
Claim 4 also recites “wherein the CRISPR family protein is SpCas9 mutated at position G1367 relative to SEQ ID NO. 1; or wherein the CRISPR family protein is AsCas12a mutated at position M806 relative to SEQ ID NO. 3.” It is clear that one of the recited CRISPR family proteins (i.e., “SpCas9 mutated at position G1367…,” “AsCas12a mutated at position M806…”) is required. The earlier recited phrase “the CRISPR family protein comprises Cas9 protein, Cas12a protein, CasX protein, Cas φ protein, Cas12g protein from different species or genera, or an inactivated counterpart thereof having no cleavage activity but retaining binding activity” should preferably be deleted.
Claim 8 recites “selecting a position G1367 relative to SEQ ID NO. 1… or a position M806 relative to SEQ ID NO. 3.” It is clear that the recited positions are required. The phrase “one or more specific amino acid sites in the amino acid sequence of the CRISPR family protein” should preferably be deleted.
Claim 8 also recites “mutating the codon encoding the amino acid at the one or more sites selected in step (1).” As stated above, it is clear that one of the positions above must be selected (“selecting a position G1367 relative to SEQ ID NO. 1… or a position M806 relative to SEQ ID NO. 3”). The claim should preferably be amended to recite “mutating the codon encoding the amino acid at theposition selected in step (1).”
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1, 4, 8, 10-14, and 20 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention. The rejections that follow are new and necessitated by Applicant’s amendments to the claims.
Claim 1 recites “mutating the CRISPR family protein site-specifically with an unnatural amino acid… wherein the unnatural amino acid is AeF.” The claim also recites that the CRISPR family protein is “SpCas9 mutated at position G1367 by one or more unnatural amino acids at one or more of: positions K3, D39, H41… or… AsCas12a mutated at position M806 by one or more unnatural amino acids at one or more of: positions M806, L834, S835….” It is clear that the CRISPR family protein must be site-specifically mutated with the unnatural amino acid AeF. However, the latter recited phrase makes the structure of the CRISPR family proteins used and produced by the method unclear. It is not clear whether the latter recited phrase intends to limit the position at which the mutation with AeF must occur, or whether the latter recited phrase is reciting characteristics of the CRISPR family protein which is used in the method (i.e., that it is already mutated with one or more unnatural amino acids at one or more of the recited positions). It is also not clear which positions must be mutated in the CRISPR family protein, or what mutations are encompassed because of the phrase “mutated at position [G1367 or M806] by one or more unnatural amino acids at one or more of: positions….” Because neither the structure of the CRISPR family protein used in the method, nor the resulting CRISPR family protein are clear, the claim is indefinite.
Claim 4 recites “A CRISPR family protein site-specifically mutated by an unnatural amino acid… wherein the unnatural amino acid is AeF.” The claim also recites that the CRISPR family protein is “SpCas9 mutated at position G1367… or… AsCas12a mutated at position M806….” It is clear that the CRISPR family protein must be site-specifically mutated with the unnatural amino acid AeF. However, the latter recited phrase makes the structure of the CRISPR family proteins used and produced by the method unclear. It is not clear whether the latter recited phrase intends to limit the position at which the mutation with AeF must occur, or whether the latter recited phrase is reciting characteristics of the CRISPR family protein which is used in the method (i.e., that it is already mutated at the recited position). Because neither the structure of the CRISPR family protein used in the method, nor the resulting CRISPR family protein are clear, the claim is indefinite.
Claims 8, 10-14, and 20 are rejected for depending from claim 4 and failing to remedy the indefiniteness.
Response to Remarks - 35 USC § 112(b)
Applicant’s remarks regarding the § 112(b) rejections raised in the prior action have been reviewed. The remarks are moot, because they do not pertain to the new rejections necessitated by Applicant’s amendments above.
Claim Interpretation
In view of the indefiniteness described above, and in accordance with Applicant’s intended scope based on the remarks, for the purposes of prior art, claims 1 and 4 are interpreted hereinafter as being drawn to:
1. A method for site-specific conjugation of a nucleic acid to a CRISPR family protein, comprising:
(a) mutating SpCas9 at position G1367 relative to SEQ ID NO: 1, or AsCas12a at position M806 relative to SEQ ID NO: 3,, wherein the unnatural amino acid is AeF; and
(b) conjugating the nucleic acid to the CRISPR family protein site-specifically mutated by the unnatural amino acid obtained in step (a), thereby obtaining a site-specific nucleic acid conjugate of the CRISPR family protein
4. A CRISPR family protein site-specifically mutated by an unnatural amino acid, wherein the CRISPR family protein is SpCas9 mutated at position G1367 relative to SEQ ID NO: 1 with the unnatural amino acid, or AsCas12a mutated at position M806 relative to SEQ ID NO: 3 with the unnatural amino acid, and wherein the unnatural amino acid is AeF
Claim Rejections - 35 USC § 102 – Ling
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.
Claims 4, 8, 10-14, and 20 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Ling (Ling et al., 8 April 2020, Science Advances, 6: eaaz0051, pg. 1-8 and Supplemental Information; of record). The rejections that follow are maintained and modified as necessitated by Applicant’s amendments to the claims.
As stated above in paragraph 7, due to the recitation of Casφ protein and Cas12g protein in claim 4, the effective filing date of claims 4, 8, 10-14, and 20 is November 11, 2020.
The phrases “relative to SEQ ID NO: 1” and “relative to SEQ ID NO: 3” are not interpreted as requiring either SEQ ID NO: 1 or 3. The phrases are interpreted as references for the amino acid positions in an SpCas9 or an AsCas12a amino acid sequence. A recitation in the prior art of the recited position in the recited CRISPR family protein is interpreted as meeting these limitations.
Regarding claim 4, Ling teaches a CRISPR family protein, which is an SpCas9 protein, and which is site-specifically mutated by the unnatural amino acid AeF at position G1367 (“On the basis of the structure of Streptococcus pyogenes Cas9 (SpyCas9), a total of 11 amino acids were selected as AeF mutation sites (Fig. 1A):… G1367… The codons of these residues were mutated to the TAG codon, and the mutant proteins were expressed…,” pg. 2, left col.; “K1151-AeF and G1367-AeF showed the highest expression yields and quality,” pg. 2, right col.; “we prepared and assessed two AeF-modified Cas9 D576-AeF-G1367-AeF in cell with double-adaptor conjugates,” pg. 4, right col.).
Regarding claim 8, Ling teaches a method for preparing an SpCas9 site-specifically mutated by the unnatural amino acid AeF (pgs. 2-3; Fig. 1-2). Ling teaches the method comprises:
(1) selecting position G1367 in the amino acid sequence of SpCas9 based on structural information (“On the basis of the structure of Streptococcus pyogenes Cas9 (SpyCas9), a total of 11 amino acids were selected as AeF mutation sites (Fig. 1A):… G1367…,” pg. 2, left col.);
(2) mutating the codon encoding the amino acid at the site selected in step (1) to codon TAG by a genetic engineering method, to obtain a mutated CRISPR protein gene (“The codons of these residues were mutated to the TAG codon,” pg. 2, left col.; “Mutagenesis PCR was conducted using KOD-One PCR Master Mix,” pg. 6, left col.);
(3) ligating the mutated CRISPR protein gene obtained by in step (2) to an expression plasmid by means of molecular cloning, to obtain an expression vector plasmid comprising a mutant sequence (“All plasmids were constructed using Gibson assembly… Mutagenesis PCR was conducted using KOD-One PCR Master Mix,” pg. 6, left col.); and
(4) co-transfecting the expression vector plasmid obtained in (3) and a tool plasmid for unnatural amino acid insertion into a host cell, culturing the host cell in a culture solution supplemented with the unnatural amino acid, and inducing the expression of the mutated protein in the host cell (“E. coli BL21 cells transformed with pET28a-Cas9-mutants and pUltra-MjPolyRS were cultivated in 2YT medium… AeF was added,” pg. 6, left col.; “the mutant proteins were expressed in Escherichia coli BL21 cells with a previously developed polyspecific Methanococcus jannacschii tyrosyl-tRNA synthetase (MjPolyRS)/tRNACUA pair,” pg. 2, left col.).
Ling teaches the tool plasmid encodes tRNA and an aminoacyl-tRNA synthetase that recognizes TAG codon and inserts the unnatural amino acid, AeF (“Methanococcus jannacschii tyrosyl-tRNA synthetase (MjPolyRS)/tRNACUA pair,” pg. 2, left col.).
Regarding claims 10-11, and 20, Ling teaches a vector comprising a nucleic acid sequence encoding the CRISPR family protein site-specifically mutated with the unnatural amino acid (“All plasmids were constructed using Gibson assembly… Mutagenesis PCR was conducted using KOD-One PCR Master Mix,” pg. 6, left col.; “the mutant proteins were expressed in Escherichia coli BL21 cells…,” pg. 2, left col.; “E. coli BL21 cells transformed with pET28a-Cas9-mutants,” pg. 6, left col.). As stated above, the codon corresponding to the unnatural amino acid is TAG (“The codons of these residues were mutated to the TAG codon,” pg. 2, left col.).
Regarding claims 12-13, Ling teaches a site-specific nucleic acid conjugate of the CRISPR family protein, wherein the nucleic acid conjugate is a donor DNA (“ssODN conjugate,” at least pgs. 2-3; Fig. 2A).
Regarding claim 14, Ling teaches a method for improving gene editing efficiency and/or reducing off-target effects comprising transfecting a host cell with a gRNA and the CRISPR family protein site-specifically mutated by the unnatural amino acid, and a site-specific nucleic acid conjugate of the CRISPR family protein (“As shown in Fig. 2C, cells cotransfected with the G1367-AeF RNP and DBCO-modified ssODN had a 1.6 times HDR increase as compared to other groups, indicating that covalent conjugation of ssODN donor DNA to Cas9 can enhance HDR efficiency,” pg. 3).
Response to Remarks - 35 USC § 102
Applicant’s remarks state that Ling is not prior art because the claims have been amended to remove the terms which conferred the effective filing date of November 11, 2020. The terms are still recited in claim 4, and therefore, the effective filing date of claims 4, 8, 10-14, and 20 is November 11, 2020. Ling remains prior art, and anticipates claims 4, 8, 10-14, and 20 for the reasons described above.
Allowable Subject Matter
Claim 1 would be allowable if amended as described in paragraph 15, so as to overcome the rejection(s) under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), 2nd paragraph, set forth in this Office action.
The prior art generally teaches modifying CRISPR family proteins with an unnatural amino acid for the purposes of conjugating a nucleic acid thereto (see Choudhary; of record). The prior art also provides evidence that means to site-specifically mutate proteins of interest with unnatural amino acids were known, as were means to optimize the position at which the unnatural amino acid is incorporated (see Reddington; of record). However, based on the prior art reviewed during examination, there is no teaching or suggestion of a method of preparing a site-specific nucleic acid conjugate of a CRISPR family protein site-specifically mutated with the unnatural amino acid AeF, wherein the CRISPR family protein is either SpCas9 or AsCas12a, and wherein AeF is at position G1367 or M806, respectively.
In order to arrive at the claimed method based on the available prior art, the skilled artisan would have needed to engage in a vast screen of various known CRISPR family proteins (e.g., Cas9s, Cas12s, etc.), incorporate one or more of the various known unnatural amino acids at one or more sites in the CRISPR family proteins, and then test the resulting proteins to see which CRISPR family proteins functioned. Such a screen would not have had a “finite” number of possible solutions from which the instantly claimed method is selected, owing to the many CRISPR family proteins known in the relevant prior art, and positions at which the various unnatural amino acids known in the prior art could be incorporated, and because none of the prior art reviewed during the search provides guidance to the skilled artisan which would have sufficiently narrowed the possible solutions to a “finite” number.
For example, while Choudhary and others teach the organization and function of various residues in specific CRISPR family proteins, neither Choudhary, Reddington, nor the remaining prior art reviewed, direct the skilled artisan to any specific domains, or a range of positions, at which to make mutations with unnatural amino acids. Furthermore, even when informed by structural information, the results of such a screen would not have been reasonably predictable. For example, Reddington concludes based on unnatural amino acid incorporation in a different protein (i.e., sfGFP) that “efficacy is dependent on the site modified and the nature of the incoming group,” and that “it is clear that the choice of residue is important in terms of efficiency of modification and the influence on function” (pg. 8420, right col. to pg. 8421).
Reddington’s proposed solution to determine suitable incorporation sites is scanning mutagenesis, which, as described above is within the artisan’s technical grasp, but does not allow the skilled artisan to reasonably predict which incorporation sites are suitable, and with which unnatural amino acid(s), or which resultant CRISPR family proteins will function. Indeed, Reddington states that “the prediction of the suitability of a modification site [is] difficult,” because proteins “are highly complex and dynamic molecules with each residue sampling a unique local environment,” and “effects, such as accessibility, local charge and ultimately residue reactivity, vary not only from protein to protein but even site to site within the same protein” (pg. 8419, left col.). As such, based on the prior art reviewed during examination, the subject matter of claim 1 is free of the prior art.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JENNA L PERSONS whose telephone number is (703)756-1334. The examiner can normally be reached M-F: 9-5pm.
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/JENNA L PERSONS/Examiner, Art Unit 1637
/Soren Harward/Primary Examiner, TC 1600
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