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 .
Application Status
This action is written in response to applicant’s correspondence received 20 May 2026. Claims 1-5, 16-19, and 32-40 are currently pending. Claims 2-3 are withdrawn from prosecution as being drawn to non-elected subject matter. Accordingly, claims 1, 4-5, 16-19, and 32-40 are examined herein. The restriction requirement mailed 29 December 2023 is still deemed proper. Applicant's elected Group I, claims 1 and 4-19 without traverse in the reply filed 24 February 2026.
Any rejection or objection not reiterated herein has been overcome by amendment.
Applicant' s arguments have been thoroughly reviewed, but are not persuasive to place the
claims in condition for allowance for the reasons that follow.
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(s) 1, 4-5, 16-19, and 32-39 is/are rejected under 35 U.S.C. 103 as being unpatentable over Zhang (PG Pub No. US 2016/0208243 A1) in view of Tanaka (FEBS letters 271.1-2 (1990): 41-46). This rejection is maintained.
Regarding claims 1, 16-17, and 33, for the purposes of examination, the functional limitations of “wherein the engineered Cas12a nucleases exhibits a reduced rate of non-specific cleavage of single-stranded DNA (ssDNA) as compared to a reference wildtype Cas12a nuclease comprising the amino acid sequence of SEQ ID NO: 2” (see Claim 1), “wherein the engineered Cas12a nuclease exhibits a ssDNA cleavage rate that is less than 50% of the ssDNA cleavage rate of a reference wildtype Cas12a nuclease comprising the amino acid sequence of SEQ ID NO: 2” (see Claim 16), “wherein the reduced rate of non-specific cleavage of ssDNA is measured within 180 minutes of introducing the engineered Cas12a nuclease to ssDNA” (see Claim 17), and “wherein the engineered Cas12a nuclease cleaves dsDNA at a rate that is at least 50% of the cleavage rate of the cleavage rate of a reference wildtype Cas12a nuclease comprising the amino acid sequence of SEQ ID NO: 2” (see Claim 33) are interpreted as not structurally altering the claimed engineered Cas12a nuclease comprising the amino acid sequence of SEQ ID NO: 12. Accordingly, the claimed limitations are not given patentable weight.
Regarding claim 1, Zhang is drawn to an invention concerned with systems, methods, and compositions for targeting nucleic acids via Cpf1 enzymes (Abstract). Zhang teaches the use of a wild-type LbCpf1 enzyme having 100% sequence identity to the claimed SEQ ID NO: 12 except for a single amino acid mismatch at position R1138 of the sequence and a missing alanine at the C-terminus of the LbCpf1 enzyme (pg. 217; see SEQ ID NO: 109 in previously attached sequence alignment). Zhang teaches that an NLS may be attached to the C-terminal of the Cpf1 protein so that the protein may be optimally expressed and targeted to the nucleus of eukaryotic cells ([0049]). Zhang teaches that a reaction comprising the engineered Cpf1 may be incubated at 37 C for 30 minutes followed by a determination of successful cleavage of a PCR amplicon of a human Emx1 locus ([1647]-[1648]).
Zhang does not explicitly teach the use of an engineered Cas12a protein comprising the amino acid sequence of SEQ ID NO: 12 (Claim 1).
However, one of ordinary skill in the art would have further considered the teachings of Zhang and Tanaka as both references are common fields of endeavor pertaining to the use of Cpf1 proteins and NLS sequences.
Zhang further teaches that the LbCpf1 enzyme may be modified by a mutation at R1138 ([0238], [1716]). Zhang teaches that R1138 is a good target for mutagenesis as it is one of the positive charge residues of interest within a RuvC domain of the LbCpf1 enzyme and can enhance Cpf1 specificity when mutated ([1714]-[1715]). Zhang teaches that the disclosed wild-type CRISPR proteins may be modified via the substitution of an unmodified residue with a glutamic acid ([0280]).
Tanaka is drawn towards a study concerned with mechanisms of nuclear translocation (Abstract). Tanaka teaches that NLS sequences comprise a consensus sequence XXKK(R)XK(R) that is sufficient for nuclear translocation of proteins that are imported into the nucleus of a cell (pg. 42). Tanaka teaches the use of a human c-myc NLS sequence comprising the sequence AAKRVK (pg. 43; see Table II).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to try to modify the LbCpf1 sequence of Zhang such that it comprised an R1138E mutation and the C-terminal NLS sequence was substituted with a human c-myc NLS sequence, such that the resulting LbCpf1 sequence comprised the claimed SEQ ID NO: 12, as described by Tanaka. A person of ordinary skill in the art would have recognized that Zhang identified a need in the art to mutate the LbCpf1 protein at position R1138 in order to increase the specificity of the LbCpf1 protein. A person of ordinary skill in the art would have recognized that there are a finite number of amino acids that could be substituted at position R1138 and Zhang further teaches that utilizing a glutamic acid as a substitution in the wild type Cpf1 proteins was a preferred amino acid substitution. A person of ordinary skill in the art would have recognized that the known potential solutions could have been pursued because Zhang teaches that the Cpf1 proteins of the disclosure could be mutated and their editing efficiencies measured via cleavage assays. Further, a person of ordinary skill in the art would have had a reasonable expectation of success in substituting the C-terminal NLS sequence of Zhang for the NLS sequence of Tanaka because both references teach the use of NLS sequences that can localize a protein to the nucleus of a eukaryotic cell.
Regarding claims 4-5 and 34, Zhang teaches that the Cpf1 enzyme may be used in conjunction with a guide RNA (i.e., the engineered Cas12a nuclease is part of a ribonucleoprotein) ([0012]).
Regarding claim 17, Zhang teaches that a reaction comprising the engineered Cpf1 may be incubated at 37 C for 30 minutes followed by a determination of successful cleavage of a PCR amplicon of a human Emx1 locus (i.e., a cleavage assay may be performed within 180 minutes of introducing the engineered Cas12a) ([1647]-[1648]).
Regarding claims 18-19 and 32, Zhang teaches that the target nucleic acid may be double stranded DNA ([0221]). Zhang teaches that the Cpf1 may be expressed in a eukaryotic cell ([0212]). Zhang teaches that the eukaryotic cell may be a plant cell ([0218]).
Regarding claim 35, Zhang teaches that the guide RNA may be 15 to 35 nucleotides in length ([0049]).
Regarding claim 36, Zhang teaches that the Cpf1 ribonucleoprotein does not require a tracrRNA ([0186]).
Regarding claim 37, Zhang teaches that the Cpf1 enzyme may be provided via a polynucleotide encoding the enzyme ([0024]).
Regarding claims 38-39, Zhang teaches the use of a delivery system that can comprise the nucleic acid molecule encoding the Cpf1 enzyme and a nucleic acid molecule encoding at least one guide nucleic acid ([0350]-[0358]).
Response to Arguments
Insofar as Applicant’s arguments are applicable to the currently utilized Zhang reference, Applicant’s arguments have been fully considered but are not found persuasive.
Applicant alleges that Zhang provides a laundry list of all 20 standard amino acid residues, with no specific teaching why one of ordinary skill in the art would prefer one substitution over another for any purpose (Remarks; pg. 3). Thus, Applicant alleges that one of ordinary skill in the art would not have had any reason to have substituted a glutamic acid at position R1138 within the LbCpf1 amino acid sequence of Zhang (Remarks; pg. 5).
This argument is not found persuasive because MPEP 2134(I)(E) does not teach that an explicit motivation to try a particular solution is required in order to satisfy the “obvious to try” rationale. In the instant case Zhang teaches that (1) there was a need in the art to enhance the specificity of Cpf1 proteins and that there was a finite number of Cpf1 proteins (i.e. four different species: FnCpf1, AsCpf1, LbCpf1, and MbCpf1) and a finite number of sites to mutate within each respective protein, (2) that the possible amino acid substitutions at position R1138 were finite, and (3) the known potential solutions could have been pursued with a reasonable expectation of success because Zhang teaches that cleavage assays could be performed with mutated Cpf1 proteins and their cleavage activities measured. Thus, as required by MPEP 2143(I)(E), Zhang in view of Tanaka provides adequate rationale for a person of ordinary skill in the art to try to arrive at the claimed invention without an explicit motivation.
Applicant alleges that Zhang provides a massive laundry list of amino acid residues that can be modified (Remarks; pg. 3-5). Applicant alleges that Zhang provides at least 473 specific residues from at least four distinct Cpfl proteins that could be substituted with any of 19 other amino acid residues with no specific guidance regarding which residue(s) to modify or which amino acid residue to use as the substituent amino acid residue in order to generate a protein that exhibits a reduced rate of non-specific cleavage of single-stranded DNA as compared to SEQ ID NO: 2 (Remarks; pg. 5).
This argument is not found persuasive because the obvious to try rationale outlined above requires a specific selection from three generic groups: 1) LbCpf1 needs to be selected from FnCpf1, AsCpf1, LbCpf1, and MbCpf1; 2) R1138 needs to be selected from the possible mutational sites within the RuvC domain of LbCpf1; and 3) glutamic acid needs to be selected from other possible amino acids at R1138.
With regard to the selection of LbCpf1, Zhang teaches that AsCpf1 and LbCpf1 explicitly exhibited robust genome editing in HEK293FT cells compared to the other species of Cpf1 proteins ([0156], [1710]; see Figs. 101C, 101E, and 108A-C). Zhang teaches that AsCpf1’s and LbCpf1’s editing activities were compared to Cas9 and the two proteins exhibited comparable indel formation at target DNMT1 sites when compared to Cas9, with LbCpf1 explicitly exhibiting comparable indel formation across 4 different DNMT1 target sites while AsCpf1 only exhibited comparable indel formation across 3 of the 4 different DNMT1 target sites ([0156], [1710]; see Fig. 101E). A person of ordinary skill in the art would have recognized that the LbCpf1 protein of Zhang has an increased number of beneficial attributes, namely comparable indel formation to Cas9, when compared to the other species of disclosed Cpf1 proteins in the disclosure. Thus, a person of ordinary skill in the art would have been led to mutate the specific species of Cpf1 protein, LbCpf1, compared to the other disclosed Cpf1 proteins.
With regard to the selection of mutational site within LbCpf1, Zhang explicitly teaches that “primary residues for mutagenesis are preferably all positive charges residues within the RuvC domain” ([0295]). Zhang further teaches that R1138 is one of 21 different positively charged amino acids within the RuvC domain of the LbCpf1 protein that can be mutated in order to generate an LbCpf1 with enhanced specificity ([1715]). Thus, the possible amount of potential mutational sites within the LbCpf1 RuvC domain that could be used to generate an LbCpf1 with enhanced specificity is sufficiently finite and easily traversed as required by MPEP 2143.
With regard to the selection of amino acid residue substitute at the R1138 residue, it is noted that the purpose of Zhang’s teachings regarding LbCpf1 mutants is to get a change in the function of the protein (i.e., enhanced specificity) by targeting the positively charged amino acids for mutagenesis ([1715]). Therefore, a person of ordinary skill in the art would recognize that the possible amino acids that could achieve the function are not all possible 19 alternative amino acids. Rather, a person of ordinary skill in the art would have looked towards amino acids that had a different charge relative to the positively charged amino acids preferred for mutagenesis in order to affect a change in the LbCpf1 protein’s function. Thus, per [0921] of Zhang, a person of ordinary skill in the art would look to mutate the positively charged amino acids within the RuvC of the LbCpf1 protein with either glutamic acid or aspartic acid.
Applicant alleges that the examiner argues that tryptophan (W), tyrosine (Y), histidine (H), lysine (K), arginine (R), glutamic acid (E), aspartic acid (D), cysteine (C), serine (S), threonine, asparagine (N), and glutamine (Q) are equivalent to each other and are considered conservative substitutions simply because they are classified as "polar" (Remarks; pg. 6). Applicant alleges that the examiner’s position must be that that 60% (12/20) of the 20 common amino acid residues are fully interchangeable, and that this position is incongruous with the state of the art at the time of filing (Remarks; pg. 6). Applicant alleges that an R1138E substitution is not a conservative substitution as recognized in the art (Remarks; pg. 6).
This argument is not found persuasive because examiner was utilizing consistent language with the disclosure of Zhang wherein amino acids with similar chemical properties (i.e., side chains or R-groups) are termed “conservative”. Additionally, as described above, a person of ordinary skill in the art would recognize that the possible amino acids that could achieve the function are not all possible 19 alternative amino acids. Because Zhang teaches that both arginine and glutamic acids are polar amino acids (i.e., the two amino acids are recognized in the art to have similar chemical properties and side chains or R-groups), and a person of ordinary skill in the art would have looked towards amino acids that had a different charge relative to the positively charged amino acids preferred for mutagenesis in order to affect a change in the LbCpf1 protein’s function, one of ordinary skill in the art would have looked to substitute glutamic acid at position R1138 in order to affect a change in the LbCpf1’s function (i.e., namely, enhanced specificity via the mutation of a positively charged amino acid within the RuvC site of the LbCpf1 to a negatively charged amino acid).
Applicant alleges that examiner has not demonstrated why one of ordinary skill in the art would have expected SEQ ID NO: 12 to exhibit a reduced rate of non-specific cleavage activity of ssDNA as compared to SEQ ID NO: 2 by introducing an R1128E substitution (Remarks; pg. 7).
This argument is not found persuasive because, as discussed above, Zhang teaches that mutating an LbCpf1 protein at R1138 would have resulted in an LbCpf1 protein with enhanced specificity. Thus, a person of ordinary skill in the art would have expected introducing an R1138E substitution within the LbCpf1 protein of Zhang to have resulted in an LbCcpf1 protein with a reduced rate of non-specific cleavage activity of ssDNA (i.e., enhanced specificity) when compared to an unmutated LbCpf1 protein.
Applicant alleges that the examiner’s reliance on Tanaka is unclear because examiner has not demonstrated that any part of SEQ ID NO: 12 comprises the human c-myc NLS sequence AAKRVK provided by Tanaka (Remarks; pg. 7). Applicant alleges that examiner has thus failed to provide any rationale for the additional alanine present in SEQ ID NO: 12 that is absent in Zhang (Remarks; pg. 7). Applicant alleges that in order to arrive at the claimed amino acid sequence, one of ordinary skill in the art would need to select to incorporate only an alanine from the NLS of Tanaka, despite Tanaka providing no guidance to why one of ordinary skill in the art would do so (Remarks; pg. 7).
This argument is not found persuasive because the addition of Tanaka’s NLS sequence to the C-terminus of the protein sequence of Zhang, as discussed above in the maintained 35 USC 103 rejection of record, renders obvious the claimed engineered Cas12a nuclease comprising the claimed SEQ ID NO: 12, as currently claimed. MPEP 211.03 teaches that the phrase “comprising” is open-ended and does not exclude additional, unrecited elements. Because the NLS of Tanaka begins with an alanine, attaching it to the C-terminus of the Lbcpf1 protein of Zhang would result in the addition of the missing alanine at the C-terminus of the LbCpf1 of Zhang that is present within the claimed Cas12a nuclease comprising the claimed SEQ ID NO: 12.
Allowable Subject Matter
Claim 40 is objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
Regarding claim 40, the claim requires that the engineered Cas12a consists of the claimed SEQ ID NO: 12 (see Claim 40).
Regarding the closest prior art, the teachings of Zhang (PG Pub No. US 2016/0208243 A1) and Tanaka (FEBS letters 271.1-2 (1990): 41-46) are discussed above as applied to claim 1.
Neither Zhang in view of Tanaka nor the prior art teaches or suggests the addition of a single alanine to the C-terminus of the LbCpf1 nuclease of Zhang, such that the LbCpf1 nuclease consisted of the claimed SEQ ID NO: 12 (Claim 40).
Accordingly, the claimed engineered Cas12a is both novel and non-obvious in view of the closest prior art.
Additionally, Applicant has provided adequate written description support for an engineered Cas12a consisting of the claimed SEQ ID NO: 12 that exhibits a reduced rate of non-specific cleavage of single-stranded DNA (ssDNA) as compared to a reference wildtype Cas12a nuclease comprising the amino acid sequence of SEQ ID NO: 2 ([0160]-[0161]; see Example 5). Applicant has identified an R1138E substitution present in the claimed engineered Cas12a nuclease consisting of the claimed SEQ ID NO: 12 that resulted in maintained dsDNA cutting activity and a lack of ssDNase activity ([0161]; see Table 6). Therefore, Applicant has provided adequate written description support for the claimed engineered Cas12a nuclease under 35 USC 112(a).
Conclusion
THIS ACTION IS MADE FINAL. 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 KYLE T REGA whose telephone number is (571)272-2073. The examiner can normally be reached Mon-Fri, 9AM-5PM (EDT/EST).
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Neil Hammell can be reached at 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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/KYLE T REGA/Examiner, Art Unit 1636
/NEIL P HAMMELL/Supervisory Patent Examiner, Art Unit 1636
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