DETAILED ACTION
Applicant’s amendment and Arguments/Remarks received on 13 July 2026 have been entered. Claims 35-66, 68, and 70-73 were previously pending in the application. Claims 61, 65, 71, and 72 have been cancelled, and no new claims have been added by Applicant. Claims 35-60, 62-64, 66, 68, 70, and 73 are currently pending in the application. Claims 35, 46, 51, 52, 53, and 54 are independent claims.
The election of Group III, drawn to a method for analyzing the on-target effect of gene editing or the on-target effect of a single-base editing tools, remains in effect in the instant application.
The following election of species remains in effect in the instant application:
Gene editing: CRISPR-mediated gene editing
Enzyme for cutting a nucleic acid target site: Cas9
Detectable marker: Fluorescent signal molecule
Analysis tool: Mutect2.
Claims 35-53 remain withdrawn from consideration as being directed to a nonelected invention, there being no allowable generic or linking claim.
Claims 63-64 and 73 remain withdrawn from consideration as being directed to a nonelected species, there being no allowable generic or linking claim. Amended claim 66 is newly withdrawn as newly being directed to only non-elected species in that each of BE1, BE2, BE3, BE4, ABE7.10, ABE 6.3, ABE 7.8, and ABE 7.9 comprise either a dCas9 or Cas9n as the enzyme for cutting a nucleic acid target site and do not comprise the elected Cas9 as the enzyme for cutting a nucleic acid target site.
Claims 54-60, 62, 68, and 70 are currently pending and under examination in the instant application. An action on the merits follows.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA . The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Priority
The present application is a 35 U.S.C. 371 national stage filing of International Application No. PCT/CN2019/119842, filed 21 November 2019, which claims priority to CN 201910153546.3, filed 28 February 2019, and CN 201910494323.3, filed 09 June 2019. Filing of a certified copy of CN 201910153546.3, filed 28 February 2019, and CN 201910494323.3, filed 09 June 2019, is acknowledged.
Thus, the earliest possible priority for the instant application is 28 February 2019.
37 CFR 1.121(c)
The claim amendments filed 13 July 2026 is objected to under 37 CFR 1.121(c) because Applicant’s claim listing is not in compliance with 37 CFR 1.121(c) which states that the claim listing must provide the status of all claims, that all claims being currently amended in an amendment paper shall be presented in the claim listing, indicate a status of "currently amended," and be submitted with markings to indicate the changes that have been made relative to the immediate prior version of the claims, and that the text of all pending claims not being currently amended shall be presented in the claim listing in clean version, i.e., without any markings in the presentation of text. The presentation of a clean version of any claim having the status of "original," "withdrawn" or "previously presented" will constitute an assertion that it has not been changed relative to the immediate prior version, except to omit markings that may have been present in the immediate prior version of the claims of the status of "withdrawn" or "previously presented."
Specifically, claims 54, 55, 62, and 66 are marked “Currently Amended” and comprise markings indicating insertions and/or deletions from the previous version. However, the amended claims also comprise a vertical line in the left margin such as from “tracked changes” in a document, which is not an allowed amendment format.
In the interests of compact prosecution, the claim listing has been entered. However, future claim listings must include the correct status of all claims, including appropriate mark-ups for changes to the claims, in order for the claim listing to meet the requirements for entry under 37 CFR 1.121(c) or a Notice of Non-Compliant Amendment will be mailed to applicant.
Claim Objections
The objection to amended, newly withdrawn, and cancelled claims 66, 70, and 72 for informalities are withdrawn.
*The following new objection is necessitated by amendments to the claims.
Amended claim 54 is newly objected to because of the following informalities: claim 54 newly recites, “wherein, the” on line 8, which comprises an extra space between “wherein” and “the”.
Claim 54 additionally newly recites, “gRNA-dependent” in line 24, which appears to be a typographical misspelling of “sgRNA-dependent”.
Claim 54 is missing a “,” or “;” following “sequence” on line 28 to separate the subsequence wherein phrase.
Claim 54 recites a “,” following the term “wherein” in lines 29, 32, and 35 which are not grammatically correct.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
The rejection of amended, previously presented, newly withdrawn, and cancelled claims 54-62 and 65-66, 68, and 70-72 under 35 U.S.C. 112(b) as failing to particularly point out and distinctly claim the subject matter which the inventor(s) regards as the invention multiple issues of indefiniteness is withdrawn over cancelled and newly withdrawn claims 61, 65, 66, 71, and 72 and maintained over amended and previously presented claims 54-60, 62, 68, and 70. Applicant's amendments to the claims and arguments have been fully considered but have not been found persuasive in overcoming the rejection for reasons of record as discussed in detail below.
Applicant’s amendments to the claims have addressed most of the identified issues of indefiniteness. However, the amendments to the claims have not resolved the following issues of indefiniteness.
Independent claim 54 still recites, “the labeled cells” in lines 13, 14, 15, 19, 22, and 26. There is still insufficient antecedent basis for this limitation in the claim. Claim 54 has prior recitations of “labeling the 1 to n-1 cells… to produce 1 to n-1 labeled cells” in line 6, “the 1 to n-1 labeled cells” in lines 7 which have had a gene edit induced, “labeled cells derived from the 1 to n-1 labeled cells” in lines 10-11. Therefore, it is still unclear to which labeled cells “the labeled cells” of lines 13, 14, 15, 19, 22, and 26 are referring.
Independent claim 54 was also previously rejected for reciting “the SNV analysis of the labeled cells” in lines 25, “the indel analysis of the labeled cells” in line 26, “the indel analysis of the unlabeled cells” in lines 26-27, “the SNV analysis or indel analysis of the labeled cells” in line 29, and “the SNV analysis or indel analysis of the unlabeled cells” in line 30 for having insufficient antecedent basis in the claim. Claim 54 had prior recitations of “analyzing the cells by sequencing to generate sequencing data from the labeled cells and the unlabeled cells” in lines 17-18, “analyzing the sequencing data … to generate a SNV analysis or an indel analysis”, but had no prior recitation of SNV or indel analyses specifically for each of the labeled and unlabeled cells, respectively. Although claim 54 has been amended to now recite “analyzing the labeled cells and the unlabeled cells by sequencing to generate sequencing data from the labeled cells and the unlabeled cells” in lines 14-15, the amendment of claim 54 to recite “analyzing the sequencing data… to generate a SNV analysis or an indel analysis for both the labeled cells and the unlabeled cells” in lines 17-19 does not fully clarify this issue. Specifically, recitation of “both” indicates that the analysis is including both the labeled cells and the unlabeled cells together rather than separate analyses of each of the labeled cells and the unlabeled cells. Therefore, there is still no prior recitation of SNV or indel analyses specifically for each of the labeled and unlabeled cells, respectively.
Additionally, Applicant’s amendments to independent claim 54 have introduced the following new issues of indefiniteness.
Claim 54 now recites, “step (1) comprises: introducing an enzyme for cutting a nucleic acid target site together with a corresponding guide sequence… and performing gene editing” in lines 29-31 and “wherein, the method analyzes sgRNA-dependent off-target gene editing effects when Cas9 is introduced, or the method analyzes both sgRNA-dependent and sgRNA-independent off-target gene editing effects when nCas9 is introduced” in lines 32-34, which has multiple issues of indefiniteness.
Firstly, it is unclear whether the step of introducing an enzyme for cutting a nucleic acid target site within step (1) is meant to be part of the “labeling” sub-step or the “inducing a gene edit” sub-step.
Secondly, it is unclear whether “performing gene editing” is meant to be the “inducing a gene edit step” recited in line 7 or an additional gene editing step and whether the “performing gene editing” is meant to be performed by the enzyme for cutting a nucleic acid target site or by some other additional means.
Thirdly, it is indefinite whether the enzyme for cutting a nucleic acid target site is meant to be a CRISPR-mediated single-base gene editing enzyme, whether the enzyme for cutting a nucleic acid target site is meant to be limited to Cas9 or nCas9, or whether the Cas9 and nCas9 are merely representative of enzymes for cutting a nucleic acid target site which could be used in the method.
Fourthly, recitation of “corresponding” is indefinite because it is unclear what the guide sequence is meant to correspond to. For example, it is unclear whether “corresponding” is meant to imply that the guide sequence is capable of binding to the enzyme for cutting a nucleic acid target site and/or capable of annealing to the target site or some other site. Further, it is unclear whether the “corresponding guide sequence” is meant to be the sgRNA for which the off-target gene editing is either dependent or independent.
Claim 54 also now recites, “if high sequence similarity” in line 35 and “if no sequence similarity” in lines 36-37. The terms “high sequence similarity” and “no sequence similarity” in claim 54 are relative terms which render the claim indefinite. The terms “high sequence similarity” and “no sequence similarity” are not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. The specification teaches examples of high sequence similarity [pg 24 ln 24] and no sequence similarity [pg 19 ln 5-6, pg 23 ln 5-6, pg 24 lns 4, 23]. The drawings also teach alignments of example sequences for predicted off-target sites and actual off-target sites which show different levels of sequence identity among the various sites, wherein 23 nucleotide regions are displayed [Figure 13C]. However, the disclosure does not teach how the similarity calculation is performed to compare the off-target and target sites nor what constitutes “high” similarity or “no” similarity. For example, neither the claim nor the disclosure teaches the bounds for the range of sequences around off-target and target sites for comparison nor what degrees of identity over such ranges constitute the different levels of similarity which would be classified as “no” or “high”.
As such, the metes and bounds of the claim still cannot be determined.
Applicant argues that the issues of claims 54 and 70have been addressed. However, as discussed above, the issue of claim 70 has been resolved, but not all of the issues of claim 54 have been addressed sufficiently to overcome a finding of indefiniteness. As such, Applicant’s arguments do not overcome a finding of indefiniteness under 35 U.S.C. 112(b).
Claim Rejections - 35 USC § 112(a)
The rejection of cancelled claim 72 under 35 U.S.C. 112(a) for lacking enablement is withdrawn.
Claim Rejections - 35 USC § 103
The rejection of amended, previously presented, newly withdrawn, and cancelled claims 54-62, 65-66, 68, and 70-71 under 35 U.S.C. 103 as being unpatentable over Wang et al. 2017, Cell Research, Vol. 27(6), 815-829, IDS, in view of Katayama et al. 2010, Biology of Reproduction, Vol. 82, 1237-1247; Callari et al. 2017, Genome Medicine, Vol. 9(35), 1-11; and Iyer et al. 2018, PLOS Genetics, 1-17; is withdrawn over cancelled and newly withdrawn claims 61, 65-66, and 71 and maintained over amended and previously presented claims 54-62, 68, and 70. Applicant's amendments to the claims and arguments have been fully considered but have not been found persuasive in overcoming the rejection for reasons of record as discussed in detail below.
Applicant amended the claims to address issues of indefiniteness.
Amended independent claim 54 newly recites, “wherein, the gene editing is CRISPR-mediated single-base gene editing” inline 8. Note that Wang was cited for teaching wherein the on-target gene edit comprises a single nucleotide insertion, thereby teaching that the CRISPR/Cas9 gene editing tool is capable of single-base gene editing [Figure 1F, S2].
Additionally, Applicant amended independent claim 54 to recite “introducing an enzyme for cutting a nucleic acid target site together with a corresponding guide sequence into the 1 to n-1 labeled cells” in lines 29-30, thereby requiring the enzyme for cutting a nucleic acid target site (i.e., the elected Cas9) be introduced simultaneously with the corresponding guide sequence (i.e., the sgRNA), as previously recited in cancelled claim 65, and newly requiring that the introducing together be into the 1 to n-1 labeled cells.
Wang was cited for teaching a method of CRISPR/Cas9-mediated genome editing comprising obtaining an n-cell stage (e.g., 2-cell stage) mouse embryo, subjecting one cell thereof to gene editing by injection of sgRNAs into the cytoplasm of one blastomere of the late two-cell stage embryos having Cas9 expression, and observing or detecting the occurrence of gene editing in the downstream development stages of the embryo (e.g., sequencing of FACS-enriched tail cells from newborn pups, TA cloning and sequencing from multiple organs (brain, testis, thymus, and liver), sequencing of blastocysts derived from the edited mouse’s sperm, and sequencing of F1 and F2 offspring) [title, abstract, column 3 ¶ 2, column 5 ¶ 1, column 5 ¶ 2-column 7 ¶ 1, column 16 ¶ 2, Figure 1, S2], as recited in claims 54-55, 61-62, and 71. Wang discloses that the Cas9 mRNA was injected into the one-cell zygote, and then the Tet1 or Tet3-targeted sgRNA guide sequences were injected into one blastomere of late two-cell stage embryos [column 5 ¶ 2, column 7 ¶ 2], as such, Wang teaches introducing an enzyme for cutting (e.g., Cas9) a nucleic acid target site into the zygote, which is then present within the n-1 labeled cell into which a corresponding guide sequence is subsequently introduced.
Although the method performed by Wang involved introduction of the Cas9 into the zygote, wherein only the sgRNA was introduced specifically and directly into the one cell of the 2-cell embryo, as discussed in the prior actions, Wang provides teachings and motivation for introducing the Cas9 and the sgRNA together with a corresponding guide sequence into the 1 to n-1 labeled cells. Specifically, Wang teaches introducing the Cas9 and sgRNA together as a ribonucleoprotein complex into one cell of a two-cell stage embryo to simplify the protocol and reduce off-target effects [column 15 ¶ 1]. Therefore, an ordinarily skilled artisan would have been motivated to introduce an enzyme for cutting a nucleic acid target site together with a corresponding guide sequence into the 1 to n-1 labeled cells for off-target gene editing analysis to both simplify the protocol of Wang and to reduce the amount of off-target gene editing within the cells. As such, it would have been prima facie obvious to an ordinarily skilled artisan to modify the method of Wang to introduce the Cas9 together with the sgRNA into the one cell of the 2-cell stage mouse embryo with a reasonable expectation of success.
Therefore, Applicant’s amendments do not overcome a finding of obviousness under 35 U.S.C. 103 over Wang, Katayama, Callari, and Iyer.
Applicant argues that:
neither Wang nor Iyer teach CRISPR-mediated single-base editors would generate sgRNA-independent off-target gene editing, and so a person skilled in the art would have had no motivation to develop a method for analyzing sgRNA-independent gene editing effects of base editing tools; and
the method taught by Wang comprises two injections during gene editing by injecting Cas9 mRNA at the zygote stage and injecting sgRNA into one cell at the 2-cell stage and the method of Iyer includes injecting Cas9 protein into the zygote, which introduces unpredictable interference to the cells at the zygote stage and may in turn bring unpredictable effects on subsequence gene editing operations, whereas the present invention does not perform any manipulations on cells at the zygote stage but instead comprises injecting a mixture of a cleavage enzyme or nickase and sgRNA into cells at the 2-cell stage, wherein the cleavage enzyme or nickase is in the form of mRNA.
However, this is not agreed.
In response to Applicant’s arguments against the references individually, it is noted that the test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981). One cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). Further, the Examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In addition, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971).
Specifically, regarding Applicant’s argument 1), as discussed in the prior action, note that Applicant has elected CRISPR-mediated gene editing in which Cas9 cuts a nucleic acid target site, and has specifically not elected Cas9n nor base editors such as BE1, BE2, BE3, BE4, ABE7.10, ABE6.3, ABE7.8, or ABE7.9. Independent claim 1 has been amended to recite, “wherein, the gene editing is CRISPR-mediated single-base gene editing” in line 8 and has not been amended to require non-elected base editor species. Also note that independent claim 54 has been amended to recite “[s]gRNA-dependent or sgRNA-independent off-target gene editing” in lines 1-2 and 24, along with “wherein the method analyzes sgRNA-dependent off-target gene editing effects when Cas9 is introduced” in lines 32-33. Accordingly, as elected, the claims as written do not require that a person skilled in the art would have had motivation to develop a method for specifically analyzing sgRNA-independent gene editing effects of base editing tools.
Regarding Applicant’s argument 2), note that the claims as written do not require introduction of an mRNA encoding the Cas9 cleavage enzyme.
As discussed above, although the method performed by Wang involved introduction of the Cas9 into the zygote, wherein only the sgRNA was introduced specifically and directly into the one cell of the 2-cell embryo, Wang provides teachings and motivation for introducing the Cas9 and the sgRNA together with a corresponding guide sequence into the 1 to n-1 labeled cells. Specifically, Wang teaches introducing the Cas9 and sgRNA together as a ribonucleoprotein complex into one cell of a two-cell stage embryo to simplify the protocol and reduce off-target effects [column 15 ¶ 1]. Therefore, an ordinarily skilled artisan would have been motivated by the teachings of Wang to introduce both the Cas9 and the sgRNA into one cell of a 2-cell stage embryo.
Accordingly, Applicant’s arguments do not overcome a finding of obviousness under 35 U.S.C. 103 over Wang, Katayama, Callari, and Iyer, and the rejection is maintained.
Conclusion
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 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.
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DR. KATIE L. PENNINGTON
Examiner
Art Unit 1634
/KATIE L PENNINGTON/Examiner, Art Unit 1634
Dr. A.M.S. Wehbé
/ANNE MARIE S WEHBE/Primary Examiner, Art Unit 1634
Prosecution Insights