NEW ENGINEERED HIGH FIDELITY CAS9

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 August 2026. Claims 1-2, 5, 8-13, 15-21, and 28-44 are currently pending. Claims 13 and 15-21 are withdrawn from prosecution as being drawn to non-elected subject matter. Accordingly, claims 1-2, 5, 8-12, and 28-44 are examined herein. The restriction requirement mailed 12 December 2024 is still deemed proper. Applicant's elected Group I, claims 1-2, 5, and 8-12, alongside the species of K929T, H930A, and SEQ ID NO: 22 without traverse in the reply filed 12 June 2025. 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) 1-2, 5, 8-12, and 28-44 is/are rejected under 35 U.S.C. 103 as being unpatentable over Liu (PG Pub No. US 2020/0208129 A1, filed 10 September 2018) in view of Miyata ("Two types of amino acid substitutions in protein evolution." Journal of molecular evolution 12.3 (1979): 219-236). Regarding claims 1-2 and 41 Liu is drawn to an invention concerned with variant SpCas9 proteins (Abstract). Liu teaches the use of an SpCas9 protein variant that can comprise N588A/K929A/H930A/Y1013A mutations, wherein the specific mutations improved the specificity of the SpCas9 ([0163]; see Table 5). Liu teaches that the mutant was designed, synthetized, and both its activity and specificity tested ([0163]). Liu does not specifically teach the use of a Cas9 having an amino acid substitution at position K929, wherein the substitution at position K929 is not K929A (Claim 1). Liu does not specifically teach the use of a Cas9 having a K929T amino acid substitution (Claims 2 and 41). However, Liu further teaches that the disclosed substitutions are not limited to alanine depending on the design needs ([0011]). Liu also teaches that alanine to serine is a conservative amino acid substitution ([0092]). Liu teaches that serine to threonine is a conservative amino acid substitution ([0092]). Miyata is drawn towards a study concerned with how tolerated certain amino acid substitutions are in proteins (Abstract). Miyata teaches the calculation of a value, termed pair distance “d” that quantitatively classifies the physico-chemical properties of amino acids including polarity, volume, and composition (pg. 220). Miyata teaches that substitutions between amino acids that have similar physico-chemical properties, and have been calculated to have a pair distance of less than 1.0, may not result in any significant change of tertiary structure over almost all variable sites in the protein (i.e., Miyata teaches that amino acids with a pair distance of less than 1.0 could be substituted for one another, and that substitution is tolerated by the protein such that it would not result in the change of the protein’s tertiary structure or resulting function derived from the tertiary structure) (pg. 232). Miyata teaches that alanine and serine have a pair distance of 0.51 (pg. 222; see Table 1). Miyata teaches that serine and threonine have a pair distance of 0.89 (pg. 222; see Table 1). Therefore, regarding claims 1-2 and 41, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have arrived at the claimed K929T mutation because it would have merely amounted to a simple substitution of one known amino acid for another that results in a similar protein function. Because Miyata teaches that alanine-to-serine, and serine-to-threonine, substitutions are tolerated and are not predicted to alter a protein’s tertiary structure, it would have been predictable to have substituted the alanine present at position K929 for a threonine (i.e., resulting in a K929T substitution), and the resulting conservative substitutions not altering the improved specificity of the Cas9 as seen with the K929A mutation of Liu. The conservative amino acid substitutions would not be expected not alter the Cas9 protein’s improved specificity because the substitutions would not be expected to alter the tertiary structure of the Cas9. Regarding claims 5, 42, and 44 Liu teaches the use of an SpCas9 mutant comprising N588A/K929A/H930A/Y1013A mutations that improved the specificity of the SpCas9 ([0163]; see Table 5). Regarding claims 8 and 28, it is noted that the claimed SEQ ID NO: 22 is 100% identical to the SEQ ID NO: 1 of Liu, except for the presence of a K929T and H930A double mutation ([0012]; see SEQ ID NO: 1 in previously attached sequence alignment). Because Liu teaches that SEQ ID NO: 1 may comprise two simultaneous mutations at positions K929 and H930 ([0012]) and recites a specific disclosure of N588A/K929A/H930A/Y1013A mutations within SEQ ID NO: 1 that resulted in an SpCas9 variant with improved specificity ([0163]; see Table 5), for the same reasons as discussed above as applied to claim 1, it would have been obvious to one of ordinary skill in the art to modify the Cas9 of Liu such that it comprised a K929T mutation in addition to the disclosed H930A mutation. Further, a person of ordinary skill in the art would have recognized that Liu previously identified positions K929 and H930 as mutational sites of interest that could generate a Cas9 with improved specificity. Regarding claims 9, 29-31, and 43, Liu teaches that the unmutated Cas9 molecule has 100% identity to the claimed SEQ ID NO: 1 ([0012]; see SEQ ID NO: 1 in previously attached sequence alignment). Accordingly, a Cas9 mutant that comprises 1 or two mutations, as rendered obvious above, would result in a Cas9 molecule that has at least 80% identity to the claimed SEQ ID NO: 1. Regarding claims 10 and 32-34, Liu teaches that the Cas9 variant may comprise a nuclear localization signal ([0042]). Regarding claims 11 and 35-37, it is noted that the functional limitations in the claim do not impart any structural limitations onto the claimed SpCas9 variant. Further, Liu teaches that the N588A/K929A/H930A/Y1013A mutations within SEQ ID NO: 1 resulted in an SpCas9 that had higher specificity (i.e., reduced off-target editing activity) when compared to a wild type protein ([0163]; see Table 5). Therefore, as discussed above, the conservative K929T substitution rendered obvious by Liu and Miyata would not alter the improved specificity of the Cas9 as seen with the K929A mutation of Liu. Regarding claims 12 and 38-40, Liu teaches that the SpCas9 may be complexed with a sgRNA that targets a DNA target site in order to edit the target DNA ([0043]). It is noted that the functional limitations in the claim do not impart any structural limitations onto the claimed SpCas9 variant. Further, Liu teaches that the N588A/K929A/H930A/Y1013A mutations within SEQ ID NO: 1 resulted in an SpCas9 that had higher specificity (i.e., reduced off-target editing activity) when compared to a wild type protein ([0163]; see Table 5). Therefore, as discussed above, the conservative K929T substitution rendered obvious by Liu and Miyata would not alter the improved specificity of the Cas9 as seen with the K929A mutation of Liu. Response to Arguments Applicant's arguments filed 20 August 2026 have been fully considered but they are not persuasive. Applicant alleges that the instant specification provides evidence that a K929T mutation demonstrates substantial improvements in the off-target effects relative to a K929A mutant (Remarks; pg. 8). Applicant alleges that FIG. 3B demonstrates that several K929 mutants that are not K292A demonstrate very low off-target effects relative to a K929A mutant (Remarks; pg. 8). Applicant alleges that accordingly, that a skilled artisan would recognize that K929 mutants, such as K929T mutants, as not being simple or obvious variants of K929A (Remarks; pg. 8). These arguments are not found persuasive because the unexpected results alleged by Applicant are not commensurate in scope with the claimed invention. Although Applicant provides evidence that specific SpCas9 variants that had better off target effects than an SpCas9 with a K929A mutation and a H930A mutation (i.e., a variant termed “Variant 9” in Table 5 and “Mutant 9” in FIGs. 2C, 3B, and 4B (pg. 36; see Table 5; see FIGs. 2C, 3B, and 4B)), the instant specification further discloses SpCas9 variants that fall within the claimed genus but do not demonstrate the alleged unexpected results. The instant specification provides evidence that SpCas9 variants with better off-target effects relative to the disclosed Mutant 9 in the specification were the following SpCas9 variants: K929T/H930H (i.e., Variant 1 in Table 5, “V10” in FIGs. 2C, 3B, and 4B), K929T/H930D (i.e., Variant 5 in Table 5, “Mutant 5” in FIGs. 2C, 3B, and 4B), K929T/H930T (i.e., Variant 6 in Table 5, “Mutant 6” in FIGs. 2C, 3B, and 4B), K929Y/H930A (i.e., Variant 7 in Table 5, “Mutant 7” in FIGs. 2C, 3B, and 4B), K929D/H930A (i.e., Variant 8 in Table 5, “Mutant 8” in FIGs. 2C, 3B, and 4B) ([0018]-[0020]; pg. 36; see Table 5; see FIGs. 2C, 3B, and 4B). However, as evidenced by FIGs. 2A, 3A, and 4A, “Mutant 3” (i.e., Variant 3 in Table 5 comprising K929T/H930H mutations: an SpCas9 comprising a species of mutations inclusive of a K929 mutation that is not K929A) had worse off-target effects than the disclosed Mutant 9 ([0018]-[0020]; pg. 36; see Table 5; see FIGs. 2A, 3A, and 4A). Additionally, as evidenced by FIG. 3B, “Mutant 4” (i.e., Variant 4 in Table 5 comprising K929T/H930Y mutations: an SpCas9 comprising a species of mutations inclusive of a K929 mutation that is not K929A) had worse off-target effects than the disclosed Mutant 9 ([0018]-[0020]; pg. 36; see Table 5; see FIG. 3B). Accordingly, the instant specification provides evidence that the alleged unexpected result does not occur over the entirety of the claimed genus of SpCas9 proteins comprising a substitution at K929, wherein the substitution at K929 is not K929A (see Claim 1) because the instant specification provides working examples of two species of SpCas9 falling within the claimed genus that do not have better off-target effects relative to an SpCas9 comprising a K929A mutation. Conclusion Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a). A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action. Any inquiry concerning this communication or earlier communications from the examiner should be directed to KYLE T REGA whose telephone number is (571)272-2073. 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Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000. /KYLE T REGA/Examiner, Art Unit 1636 /NEIL P HAMMELL/Supervisory Patent Examiner, Art Unit 1636