DETAILED ACTION
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. Applicant’s Request for Continued Examination, Amendment and Arguments/Remarks received on 09 October 2025 have been entered. Claims 35-66, 68, and 70-71 were previously pending in the application. New claims 72-73 have been added by Applicant. Claims 35-66, 68, and 70-73 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 a 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Claims 35-53 remain withdrawn from consideration as being directed to a nonelected invention, there being no allowable generic or linking claim. Claims 63-64 remain withdrawn from consideration as being directed to a nonelected species, there being no allowable generic or linking claim. Claim 73 is newly withdrawn as being directed to a nonelected species, there being no allowable generic or linking claim.
Claims 54-62 and 65-66, 68, and 70-72 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.
Specification
The objection to the specification of the disclosure for the brief description of the drawings not describing each panel of Figure 16 is withdrawn in view of the amendment to the specification individually describing each panel A-C.
Claim Objections
Previously presented claim 66 is objected to because of the following informalities: claim 66 recites an “enzyme for cutting a nucleic acid target site is selected from the group consisting of:” followed by a list of enzymes, wherein the list does not recite “and” before the final option. Appropriate correction is required.
Previously presented claim 70 is objected to because of the following informalities: claim 70 recites, “The method according to claim 69”. However, claim 69 is cancelled, and therefore, the actual method steps which Applicant intends to claim as part of the invention of claim 70 cannot be determined. Appropriate correction is required. Further examination of this claim under 35 U.S.C. 101, 102, 103, 112(a), and double patenting is precluded.
New claim 72 is objected to because of the following informalities: claim 72 recites, “p.V2897MM” on line 3, which appears to be a typographical error duplicating the M.
Additionally, new claim 72 recites a list of SNV, wherein the SNV is selected from the group consisting of the recited list. However, the list does not recite “and” before the final option.
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.
Amended, previously presented, and new claims 54-62 and 65-66, 68, and 70-72 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. Claims 55-62, 65-66, 68, and 71 are included in this rejection due to their dependence on independent claim 54.
Amended independent claim 54 has multiple issues of indefiniteness.
Applicant elected the species of CRISPR-mediated gene editing wherein the enzyme for cutting a nucleic acid target site is Cas9, which requires an sgRNA to direct the target-specific cutting. Amended claim 54 now recites, “analyzing an sgRNA-independent gene editing effect”, and so it is unclear to what extent the “sgRNA-independent gene editing” is meant to encompass the target-specific (e.g., on-target) and non-target (e.g., off-target) editing, such that encompassing on-target editing within the sgRNA-independent gene editing conflicts with the elected species. Additionally, independent claim 54 was edited to recite that the analyzing an sgRNA-independent gene editing effect is analyzing an effect of “a single-base gene editing tool”, which is unclear whether it is meant to encompass any tool capable of editing a single base (e.g., introducing a single base edit) or whether it is meant to encompass only tools which specifically edit only one base at a time. If the latter, then “single-base gene editing tool” conflicts with the elected species of CRISPR/Cas9 in that Cas9, while being capable of single-base gene editing, is capable of a variety of multi-base editing applications as well. Further, claim 54 as written merely recites, “inducing a gene edit”, and has no recitations which address whether or not the edit is single-base nor whether or not the edit is sgRNA-dependent.
Further, recitation of “analyzing an sgRNA-independent gene editing effect of a single-base gene editing tool” in lines 1-2, is further indefinite because the claim later recites “labeling the 1 to n-1 cells with a detectable marker” in line 6 and “inducing a gene edit in at least one target sequence in the n-1 labeled cells”, and as such, claim 54 requires at least the introduction of a detectable marker along with at least one induced gene edit. Additionally, “inducing a gene edit in at least one target sequence” encompasses introducing multiple edits into the same cell, which necessarily introduces more than a single base gene edit into the cell.
Claim 54 recites “the labeled cells” in lines 14, 17, 25, 26, and 29. There is 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 n-1 labeled cells” in lines 8-9 which have had a gene edit induced, “labeled cells derived from the 1 to n-1 labeled cells” in lines 11-12. Therefore, it is unclear to which labeled cells “the labeled cells” of lines 14, 17, 25, 26, and 29 are referring.
Claim 54 recites “the cells” in line 17. There is insufficient antecedent basis for this limitation in the claim. Claim 54 has multiple prior recitations of various cells. Therefore, it is unclear to which cells “the cells” of line 17 is referring.
Claim 54 recites “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.. There is insufficient antecedent basis for this limitation in the claim. Claim 54 has 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 has no prior recitation of SNV or indel analyses specifically for each of the labeled and unlabeled cells, respectively.
Claim 54 recites, “the sgRNA-independent off-target gene editing” in line 29. There is insufficient antecedent basis for this limitation in the claim. Claim 54 has not prior recitation of any sgRNA-independent off-target gene editing.
Recitation in claim 54 of “the sgRNA-independent off-target gene editing” in line 29 is further indefinite because it is unclear to what extent “sgRNA-independent off-target gene editing” is meant to differ from any other “off-target gene editing” observed by the claimed methods in that the method of claim 54 has no recitation of the use of any sgRNA. Although Applicant has elected CRISPR-mediated gene editing wherein Cas9 cuts a nucleic acid target site, claim 54 does not recite any particular gene editing methods and merely recites “introducing a gene edit” in line 8.
Claim 54 also recites the limitation "the SNV" in line 25. There is insufficient antecedent basis for this limitation in the claim. Claim 54 has prior recitations of “SNV analysis” but not of SNVs themselves.
As such, the metes and bounds of the claim cannot be determined.
Previously presented claim 70 recites, “The method according to claim 69”. However, claim 69 is cancelled, and, therefore, claim 70 is indefinite because the actual method steps which Applicant intends to claim as part of the invention cannot be determined. Further examination of this claim under 35 U.S.C. 101, 102, 103, 112(a), and double patenting is precluded.
New claim 72 recites the limitation "the SNV" in line 1. There is insufficient antecedent basis for this limitation in the claim. Claim 72 depends on claim 54, which recites “a single nucleotide variation (SNV) analysis tool” in lines 20-21, “a SNV analysis” in line 21, “the SNV analysis” in lines 24, 29, and 30, and “the SNV analysis” in line 25, “the SNV” in line 25. As discussed above for independent claim 54, “the SNV” recited in claim 54 line 25 itself lacks antecedent basis.
Additionally, to the extent to which “the SNV” is meant to refer to the sequence variant detected in the SNV analysis, new claim 72is further indefinite in that it is unclear whether Applicant intends to encompass only wherein the recited variants are actually detected as off-target edits in the labeled cells, or whether Applicant intends to encompass wherein the method comprises analyzing the locus for potentially exhibiting the recited variant(s).
As such, the metes and bounds of the claim cannot be determined.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
New claim 72 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
New claim 72 is drawn to the method of claim 54, wherein the SNV is sgRNA-independent and is selected from the group consisting of 26 specifically recited variants. The claims as written read broadly on any gene editing tool capable of a single-base gene edit, and Applicant has specifically elected a CRISPR/Cas9 gene editing tool. The claims also read broadly on any n-stage embryo from any organism.
The specification teaches that the recited variants are all mouse single nucleotide variants observed as off-target edits when using a BE3 base editing tool [page 24 ¶ 5, Table 7]. The specification further teaches that no sequence similarity was observed between off-target and target sites, and off-target sites predicted by computer showed high sequence similarity with the targeted sites of BE3; and, as such, the specification teaches that the BE3 off-target SNVs are sgRNA-independent and may be caused by overexpression of APOBEC1 [page 24 ¶ 4]. The specification also teaches that the single-base editing tool BE3 will cause a large number of off-target SNVs, which the inventors expect are off-target variations caused by the overexpression of APOBEC1 and its binding to single-stranded DNA [page 27 ¶ 3].
Example 1 of the working examples discloses off-target analysis using whole genome sequencing for CRISPR-Cas9, BE3, and ABE7.10 in mouse embryos, wherein the Cas9-sgRNA average SNVs were 0, 16, and 19 for Pde6b, Tyr-A, and Tyr-B sgRNAs, respectively, which the specification teaches may be caused by spontaneous mutations during gene replications in that the amount of mutations is within the range of spontaneous mutations [page 18 ¶ 3-page 19 ¶ 1, Table 2]. The specification also teaches that the number of SNVs in the CRISPR/Cas9-edited cells was similar to the number of SNVs in the unedited cells, indicating that CRISPR/Cas9 editing did not produce off-target effects and that the SNVs observed by the inventors came from spontaneous mutations [page 19 ¶ 2]. The specification also states that the SNVs detected in the samples treated with Cre or Cas9 may be caused by spontaneous mutations during genome replication during development because the number of SNVs detected is within the range of simulated spontaneous mutations and the adjacent sequence showed no sequence similarity with the target site [page 23 ¶ 2]. In contrast, the specification teaches that the inventors surprisingly found an average of 283 SNVs per embryo in embryos edited by BE3, which is at least 20 times higher than the levels observed in embryos treated with Cre or Cas9 [page 23 ¶ 3].
Examples 2-3 of the working examples in the instant specification specifically address the off-target effects of APOBEC1-comprising enzymes, such as BE3, and do not address off-target mutations arising from CRISPR/Cas9 editing.
Therefore, Applicant’s disclosure teaches that the recited variants were specifically found only with the BE3 gene editor, which has a 20x higher SNV mutation rate compared to the Cas9 gene editor, and were not found with Cas9. The specification does not provide an enabling disclosure for detecting the variants recited in new claim 72 in a method comprising inducing a gene edit with a CRISPR-mediated gene editing tool wherein the enzyme for cutting a nucleic acid target site is Cas9.
The art at the time of filing also teaches that CRISPR/Cas9 does not produce significant SNV variants; for example, Iyer found that if CRISPR/Cas9-mediated mutagenesis performed under the conditions they described were causing SNV or indel off-target mutations in treated embryos, then the number of such mutations is not statistically distinguishable from the background rate of de novo mutations occurring due to other processes [Iyer et al. 2018, PLOS Genetics, 1-17, cited in a prior action, page 5 ¶ 4].
Given the lack of evidence of any Cas9-induced off-target SNVs, the ordinarily skilled artisan at the time of filing would not have considered the detection of any specific off-target SNVs resulting from CRISPR/Cas9-mediated gene editing to be predictable.
Applicant’s disclosure does not overcome this art recognized unpredictability as the disclosure does not provide sufficient guidance for detecting the recited variants in cells edited with CRISPR/Cas9.
Therefore, in view of the state of the art at the time of filing for off-target analysis of CRISPR-edited genomes, the art recognized unpredictability for observing any specific off-target SNVs resulting from Cas9-mediated gene editing, the limitation of the working examples to teaching specific mouse variants only for BE3-mediated gene editing (including the specific mouse variants recited in new claim 72) in mouse embryos, and the breadth of the claims, it would have required undue experimentation to practice the instant method for analyzing an sgRNA-independent gene editing effect of a CRISPR/Cas9 gene editing tool in any n-cell stage embryo, wherein the specific mouse SNVs recited in claim 72 are detected by the method without undue experimentation.
Claim Rejections - 35 USC § 103
The rejection of amended and previously presented 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 maintained. 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.
Regarding Applicant’s amendments to independent claim 54, as discussed above for the 35 U.S.C. 122(b) rejection of amended independent claim 54, note that the metes and bounds of the claim are unclear. For purposes of compact prosecution, Examiner has applied a broadest reasonable interpretation for amended independent claim 54 consistent with the elected species which encompasses an on-target gene editing which is sgRNA-dependent, wherein the gene editing tool is capable of single-base gene editing, such that the method is analyzing an sgRNA-independent effect of the gene editing wherein the sgRNA-independent effect being analyzed is an off-target gene editing effect.
Wang teaches 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].
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]. Wang also 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].
Additionally, Wang teaches the labeling of 1 cell of a 2 cell stage embryo with the detectable marker eGFP to produce 1 to n-1 labeled cells along with the CRISPR-Cas9/sgRNA targeting of a gene of interest (e.g., Tet1 or Tet3), thereby inducing gene editing in a target sequence in the 1 to n-1 eGFP-labeled cells [column 5 ¶ 2- column 6 ¶ 1].
Wang teaches observing the occurrence of on-target and off-target gene editing in the downstream development stages of the embryo, wherein the embryo comprises eGFP+ labeled cells derived from the 1 to n-1 labeled cells and unlabeled cells that were not subjected to gene editing, wherein the observing comprises
(a) FACS sorting the developmentally downstream cells from the embryo (e.g., ESC lines from Tet-1-targeted or Tet-3-targeted blastocysts) to separate the eGFP+ labeled cells derived from the 1 to n-1 eGFP labeled cell (e.g., mG+) and the unlabeled cells (e.g., cells not labeled with eGFP, mT+) that were not subjected to gene editing [column 5 ¶ 2-colulmn 7 ¶ 2, column 10 ¶ 1, column 16 ¶ 2-3 and 4, supplemental methods Off-target Analysis, Figures 1-2, S2-S4, and Table S4];
(b) analyzing the cells by sequencing (e.g., Sanger sequencing or Illumina deep sequencing) to generate sequencing data from the labeled and unlabeled cells and detecting a frequency of on-target editing in the unlabeled cells (e.g., 0% on-target edits in unlabeled cells) [column 5 ¶ 2-column 6 ¶ 1, Figure 1];
(c) analyzing the cell by sequencing data for indel mutations and mismatches, and including only variants with an allele frequency higher than the frequency of on-target editing in the unlabeled cells in the analysis (e.g., which necessarily have an allele frequency higher than 0%)[column 5 ¶ 2-column 6 ¶ 1, Figure 1]; and
(d) comparing the analysis of the labeled cells with the analysis of the unlabeled cells to detect on-target and off-target gene editing effects, including detecting mismatches and indel mutations and determining frameshift status [column 5 ¶ 2-colulmn 7 ¶ 2, column 10 ¶ 1, column 16 ¶ 2-3 and 4, supplemental methods Off-target Analysis, Figures 1-2, S2-S4, and Table S4], wherein the sequence variant is not found in the at least one target sequence [Table S4, supplemental methods Off-target Analysis].
Wang does not explicitly teach that the detecting off-target gene editing i) is detecting the sgRNA-independent off-target gene editing, nor ii) is detecting a sequence variant in the analysis of the labeled cells that is not found in the analysis of the unlabeled cells.
However, Iyer was cited for teaching a method of analyzing the effect of gene editing comprising obtaining a 1 cell stage embryo, subjecting the 1 cell therefore to gene editing by microinjection of Cas9 ribonucleoproteins (RNPs) comprising Cas9 and the gRNAs to introduce a mutation into the Tyr gene which results in the detectable trait of a complete loss of coat color in the biallelic mutants [page 5 ¶ 6-page 6 ¶ 1, page 7 ¶ 1-3]. Iyer further teaches observing or detecting the occurrence of off-target gene editing in the downstream development stages of the embryo comprising extracting genomic DNA from macerated whole 12.5 d.p.c. embryos (e.g., edited cells) and from the unedited kidney of parent animals followed by analyzing the DNA by whole genome sequencing to generate sequencing data from the cells that have undergone gene editing and the cells that have not undergone gene editing [page 2 ¶ , page 7 ¶ 4, page 8 ¶ 1-pge 9 ¶ 1, Figure 1]. Iyer also teaches analyzing the sequencing data using at least an SNV analysis tool or an indel analysis tool to generate a SNV analysis and/or an indel analysis, wherein only variants with an allele frequency greater than or equal to 10% are included in the analysis (which is greater than the on-target editing in the unedited cells, which Iyer teaches to be 0%), and comparing the SNV analysis and/or the indel analysis of the edited cells to that of the unedited cells to identify de novo mutations in the edited cells which were not present in the unedited cells, thereby identifying potential off-target mutations [page 8 ¶ 6, page 9 ¶ 2-page 10 ¶ 1, page 10 ¶ 4, Table S3, S8, Figure 1]. Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to compare sequencing data for edited and unedited cells to detect a sequence variant in the analysis of the edited/labeled cells that is not found in the analysis of the unedited/unlabeled cells for the detection of off-target edits.
Additionally, Iyer teaches analyzing the sgRNA-independent gene editing off-target effect of the CRISPR/Cas9 gene editing tool to detect sgRNA-independent off-target gene editing, in that Iyer teaches using whole genome sequencing data to look for any/all SNVs or indels which were not present in the unedited parents and/or the unedited no injection, sham, and Cas9-only controls, and as such was not limited to detecting only sgRNA-dependent off-target editing [page 5 ¶ 1-2, page 9 ¶ 3- page 10 ¶ 1, page 14 ¶ 4, Table 1, S7].
Therefore, given the teachings and motivation of Iyer to compare whole genome sequencing data analyses for edited and unedited cells to detect any/all sequence variant(s) in the analysis of the edited/labeled cells that is/are not found in the analysis of the unedited/unlabeled cells for the detection of potential off-target edits, it would have been prima facie obvious to an ordinarily skilled artisan at the time of filing the instant application to modify the method of Wang to include whole genome sequencing to look for all SNV or indel variants within the edited cells which are not present in the unedited cells to analyze both the sgRNA-dependent and the sgRNA-independent off-target gene editing effects with a reasonable expectation of success.
As such, Applicant’s amendments do not overcome a finding of obviousness over Wang, Katayama, Callari, and Iyer under 35 U.S.C. 103.
Applicant argues that:
none of the cited references teach or suggest the claimed features because the claimed method is directed to determining off-target gene editing in sgRNA-independent single-base editing;
the cited references do not teach detecting a sequence variant in the SNV analysis or indel analysis of the labeled cells that is not found in the SNV analysis or indel analysis of the unlabeled cells, wherein the sequence is not found in the at least one target sequence;
the sgRNA-independent off-target edits observed by the instant inventors share no sequence similarity with the targeted sites and may be caused by overexpression of APOBEC1 in the BE3 construct.
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), note that the claims as written merely recite analyzing an sgRNA-independent gene editing effect of a single-base gene editing tool in the preamble, “inducing gene editing” in line 8 the claim body, and “to detect the sgRNA-independent off-target gene editing” in line 27 of the claim body, which indicates merely that the sgRNA-independent effect being analyzed is the off-target effect of the gene editing, and not that the gene editing tool itself is sgRNA-independent. Further, limiting the claims to only on-target gene editing which is sgRNA-independent would conflict with the elected species in 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, or BE4. Therefore, the on-target gene editing encompassed by the claims as written and as elected necessarily includes sgRNA-dependent gene editing.
Regarding Applicant’s argument 2), as discussed above, Wang was cited for teaching off-target analysis in which SNVs and indel sequence variants were detected in sites which were not the at least one target sequence. Additionally, Iyer teaches analyzing the sequencing data using at least an SNV analysis tool or an indel analysis tool to generate a SNV analysis and/or an indel analysis and comparing the SNV analysis and/or the indel analysis of the edited cells (e.g., downstream developmental stage of the edited embryo) to that of the unedited cells (e.g., parents) to identify de novo mutations in the edited cells which were not present in the unedited cells, thereby identifying potential off-target mutations [page 9 ¶ 2-page 10 ¶ 1, page 10 ¶ 4, Table S8].
Regarding Applicant’s argument 3), note that Applicant has elected CRISPR-mediated gene editing in which Cas9 cuts a nucleic acid target site. Therefore, functions attributable specifically to a non-elected species (e.g., BE3-based gene editing) cannot overcome a finding of obviousness for the elected invention.
Therefore, Applicant’s amendments and arguments do not overcome a finding of obviousness over Wang, Katayama, Callari, and Iyer under 35 U.S.C. 103, and the rejection is maintained.
Conclusion
No claim is allowed.
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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