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 .
Detailed Action
Election/Restrictions
Applicant’s election without traverse of Group 1 (claims 1, 2, 4, 8, 12, 14, 18-19, 26, 29, 34-37, 45 and 53) in the reply filed on 07/31/2025 is acknowledged.
Election of the following species on the reply filed on 07/31/2025 is acknowledged:
1) a ligation site at the end of a stem structure (claim 1),
2) a ligating enzyme t4 RNA Ligase 2,
3) a ligation site at least 2 bp from loop and 3 bp from bulge,
4) a combination of gRNA structural features: upper stem, lower stem and bulge.
Priority
The present application is a 35 U.S.C. 371 national stage filing of International Application No. PCT/JP2021/014495, filed 04/05/2021.
Applicant’s claim for the benefit of a prior-filed parent provisional application JP2020-141335, filed on 08/25/2020, JP2020-068266 filed 04/06/2020 under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, or 365(c) is acknowledged.
Certified copies for JP-2020-068266 and JP-2020-141335, both filed 10/05/2022 are acknowledged. However English translations of the foreign priority documents are absent.
Thus, the earliest possible priority for the instant application is 04/05/2021.
Claims Status
Claims 2, 12-19 and 31 are canceled, claims 27-29 have been withdrawn from consideration as being drawn to non-elected subject matter, and claims 1, 3-11, 20-26 and 30 have been considered on the merits. All arguments have been considered.
Withdrawn Objections & Rejections
Applicant's response filed 03/13/2026 has been considered. Rejections and/or objections not reiterated from the previous Office action mailed 11/28/2025 are hereby withdrawn.
The objections and rejections presented herein represent the full set of objections and rejections currently pending in the application.
Claim Rejections - 35 USC § 103 (New)
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.
Claims 1, 3-4 and 26 are rejected under 35 U.S.C. 103 as being unpatentable over Canver et al (JBC (2014) 289:31; 21312-21324) in view of Supharattanasitthi et al (Scientific Reports (2019)9:174;1-7) and as evidenced by evidenced by MedLineCST3 (CST3 gene [online]. NIH MedlinePlus [retrieved on 11/05/2025]. Retrieved from the Internet <URL: chrome-extension://efaidnbmnnnibpcajpcglclefindmkaj/https://medlineplus.gov/download/genetics/chromosome/20.pdf).
The rejection relies on Cowan et al (not of previous record) to address the new claim limitations in amended claim 1.
Regarding claims 1, 3-4 and 26: Claim 1 recites the term “target region”. The instant specification defines “target region” as “a genomic region that is subject to genome engineering” (p24 ¶0017). Therefore any region of the genome is considered a target region as defined by the special definition recited in the instant specification.
Claim 26 recites “single-cell cloning is not performed in a process up to the selection of the cell in which the two or more alleles are engineered”. This is interpreted as single-cell cloning is not performed before selection of the cell with two or more engineered alleles.
Canver teach methods using CRISPR/Cas9 to mediate genomic deletions in mammalian cells with deletions ranging from 1.3 Kb to greater than 1 Mb (abstract, Fig 1B). Canver teach the cells were MEL cells with two copies for each chromosome studied (p21313 col1 ¶4).
Canver teach introducing a genome engineering system comprising a sequence-specific nucleic acid cleaving molecule capable of targeting a target region in two or more alleles in the chromosomal genome (e.g. Cas9); Canver teach CRISPR/Cas9 is targeted to specific genomic sites using sgRNA and that each sgRNA specified sequences exonic, intronic, or intergenic with respect to a RefSeq gene (p21313 col1 ¶2,3).
Canver teach both monoallelic deletion clones and biallelic deletion clones were identified after introduction of the gene editing system into the cells (p21313 col1 ¶3; Fig 1C).
While Canver teach biallelic deletion clones are identified, Canver do not teach introducing two or more donor DNAs for selective markers which comprise DNAs having distinguishably different selective marker genes.
While Canver teach selecting cell based on GFP expression (the top 3% GFP positive cells were sorted and editing status determined by PCR amplification (p21313 col2 ¶2)), Canver do not teach selecting a cell expressing at least two distinguishably different selective marker genes.
Canver do not teach antibiotic selection of the cells, and thus Canver do not teach single-cell cloning is not performed before the antibiotic selection.
Supharattanasitthi teach using CRISPR/Cas9 to edit bi-allelic genes by introducing double antibiotic donor templates (abstract). Supharattanasitthi teach the approach can be applied to similar in vitro experiments using CRISPR/Cas9 editing (p2 ¶3). Supharattansitthi teach the dual antibiotic approach is effective and simple in principle and applicable to many cell types (p5 ¶4). Supharattanasitthi also teach the method has an efficient outcome and is less time consuming compared to standard approaches (p5 ¶4).
Supharattanasitthi teach electroporation of iPSCs with two donor plasmids together and a CRISPR/Cas9 plasmid encoding CRISPR/Cas9 (p2 ¶2).
Supharattanasitthi also teach the two donor DNAs (donor plasmids) comprise nucleotide sequences encoding two different selective marker genes unique to each type of donor DNA (puromycin and blasticidin resistance genes) (p2 ¶2). Supharattanasitthi teach the donor template has a 5’ and 3’ homology arms (upstream and downstream homology arms) (p5 ¶7). The purpose of a 5’ and 3’ homology arm is for homologous recombination upstream (5’) and downstream (3’) of a nucleotide sequence of a target region. Thus, this reads on claim 1(a)(ii).
Supharattanasitthi also teach the nucleotide sequence of the selective marker gene (puromycin/blasticidin) is between the 5’ and 3’ homology arms (Fig1b plasmid donor).
Supharattanasitthi teach dual antibiotic selection of the cells after gene editing (p2 ¶1). This reads on “selecting a cell having the different selective marker genes” and “expressing all the distinguishably different selective marker genes thus introduced” because in order to successfully achieve dual antibiotic selection both antibiotic resistance markers must be expressed.
Supharattanasitthi further teach that single cell cloning is not performed until after antibiotic selection to identify cells with biallelic modifications. Supharattanasitthi teach after electroporation cells are seeded into 10 cm dishes, and 3 days post-transfection, dual antibiotic selection is applied (p7 ¶2). After dual antibiotic selection, individual clones are picked with a loop (p6 ¶2).
It would have been obvious to one of ordinary skill in the art to modify the method of Canver, drawn to a CRISPR/Cas9 system which introduces biallelic deletions of up to 1,025 kb in human cells with the teachings of Supharattanasitthi, to incorporate two donor DNAs (donor plasmids) which comprise nucleotide sequences encoding two different selective marker genes unique to each type of donor DNA (puromycin and blasticidin resistance genes) (p2 ¶2) and selecting a cell based on expression of the different selection markers before performing single cell cloning.
One would have been motivated to modify the method of Canver with the teachings of Suparattanasitthi to introduce the two donor DNAs with different selection markers and perform single-cell cloning after antibiotic selection because Canver teach genomic deletion frequency has an inverse relationship between size and frequency of deletion, and more clones need to be screened as the intended deletion size increases to reliably retrieve biallelic deletion clones (p21313 col2 ¶2). Suparattanasitthi also teach that introducing by two DNA templates with different antibiotic resistance genes, clones surviving the selection process should theoretically contain the desired change in both alleles (p4 ¶4). The method of Canver which does not comprise selection using donor DNAs with distinguishable selection markers reports 28% of the deletions are biallelic (Fig 7A). Thus one would have been further motivated to modify the method of Canver to perform single-cell cloning after antibiotic selection because theoretically all of the clones surviving the dual selection would contain the desired change in bothe alleles. One would also be motivated because Supharattanasitthi teach the method has an efficient outcome and is less time consuming compared to standard approaches (p5 ¶4)
One would have had a reasonable expectation of success in modifying the method of Canver with the teaching of Suparattanasitthi because Suparattanasitthi teach the approach can be applied to similar in vitro experiments using CRISPR/Cas9 editing (p2 ¶3) and that the dual antibiotic approach is effective and simple in principle and applicable to many cell types (p5 ¶4).
Claims 5-7, 8-10 and 20-21 are rejected under 35 U.S.C. 103 as being unpatentable over Canver and Supharattanasitthi as applied to claims 1, 3-4 and 26 above, and further in view of Ikeda et al (WO 2019/018534 A1).
Regarding claims 5-7: The claim recites optional limitations. Optional limitations are interpreted as not required to be present in the present claim analysis.
The claim recites the term “target sequence”. The instant specification defines this term as “a DNA sequence, in the genome, to be cleaved by the sequence-specific nucleic acid cleaving molecule” (p26 ¶0021).
The teachings of Canver and Supharattanasitthi are discussed supra. As discussed supra, Supharattanasitthi teach the donor plasmid comprises antibiotic resistance genes, which reads on a marker gene for positive selection.
Supharattanasitthi further teach a second genome editing step (after step (b)) which uses negative selection to identify cells with the edits (figure 1). Supharattanasitthi teach the second editing step is mediated by Cre.
Supharattanasitthi also teach step (d) of the instant claim; removal of the insertion cassettes results in a loss of GFP expression which is used identify successful gene editing (p3 ¶2; Figure 1). Figure 3c shows an iPS clone that has been identified via negative selection (selected for lack of GFP expression) (p4).
Supharattanasitthi teach the second editing step is mediated by Cre, and does not require a donor DNA. Supharattanasitthi do not teach the second gene editing step comprises a second donor plasmid comprising a marker gene for positive selection and a negative selective marker.
Ikeda teach a method comprising a two step cell editing method using a CRISPR/Cas9 mediated genome editing system to conduct scarless genome editing (abstract). Ikeda teach “scarless” editing does not leave remnants of the editing process in the form of a selection marker or silent mutation, while still retaining the ability to generate HDR-mediated genome editing (p2 ln15-20).
Ikeda teach a second Cas9-mediated homology directed repair genome editing step which comprises a second donor polynucleotide that comprises a left homology arm and a right homology arm flanking a sequence complementary to the target genomic sequence as modified by integration of the first donor polynucletotide into the genomic DNA (claim 1c)i)). Ikeda teach the second donor polynucleotide comprises a deletion of at least one selection marker. Ikeda further teach selecting (isolating) the genetically modified cell comprising the edit based on negative selection for at least one selection marker, wherein the expression cassette encoding the selection marker is deleted (claim 1 d).
Ikeda teach introducing a second genetic modification via homology directed repair (HDR) to a genetically modified cell which comprises bi-allelic modifications from a first genetic modification (abstract, Figure 1A ‘1st step HR’ and ‘2nd step HR’). The 2nd HDR step comprises a donor DNA for recombination comprising upstream and downstream homology arms which can be homologously recombined with upstream and downstream nucleotide sequences of the target sequence (claim 1c(i)).
It would have been obvious to one of ordinary skill in the art to modify the method disclosed by the combination of Canver and Supharattanasitthi drawn to a method of introducing biallelic deletions of up to 1,025 kb in human cells using CRISPR/Cas9 wherein the method comprises incorporation of two donor DNAs (donor plasmids) which comprise nucleotide sequences encoding two different selective marker genes unique to each type of donor DNA (e.g. puromycin and blasticidin resistance genes) (p2 ¶2) and selecting a cell based on expression of the different selection markers before performing single cell cloning with the teachings of Ikeda, to include a second step of gene editing comprising donor sequences comprising a second donor nucleotide which has a selective marker gene for positive selection and an marker gene for negative selection.
One would have been motivated to modify the method of Canver and Supharattanasitthi with the teachings of Ikeda because Ikeda teach a method of “scarless” editing does not leave remnants of the editing process in the form of a selection marker or silent mutation (p2 ln15-20).
One would have had a reasonable expectation of success because the methods are drawn to genome editing using the well-known CRISPR/Cas9 system.
Regarding claims 8-10: The teachings of Canver and Supharattanasitthi are discussed supra. Canver and Supharattanasitthi and Cowan are silent on the length of the region between the upstream homology arm and the downstream homology arm of the donor DNA.
Ikeda teach the teach the region between the upstream homology arm and the downstream homology arm of the donor DNA for the RUNX1 1st integration has a length of 5.8 kbp (Fig 3A). Figure 3b shows the marker integrated product is more than 5kbp larger than the marker-free product.
It would have been obvious for one of ordinary skill in the art at the time of the effective filing date to combine the teaching of Canver and Supharattanasitthi with the teaching of Ikeda because it would have been obvious to combine prior art elements according to known methods to yield predictable results. Combining the donor DNA taught by Canver and Supharattanasitthi with an insert length greater than 5 kbp as taught by Ikeda would have led to predictable results with a reasonable expectation of success because both inventions are drawn to donor DNA and one of ordinary skill in the art would understand that the sequence between the homology arms can have a variable length and is selected depending on the intended purpose of the genome edit.
Regarding claims 20 and 21: The teachings of Canver and Supharattanasitthi are discussed supra. Supharattanasitthi do not teach the donor DNA for recombination has upstream and downstream sequences that are seamlessly linked.
The teachings of Ikeda are discussed supra. Ikeda also teach scarless editing which comprises no nucleotide sequence in the region between the upstream homology arm and the downstream homology arm of the donor DNA (Fig 1B Donor2).
It would have been obvious for one of ordinary skill in the art at the time of the effective filing date to combine the teaching of Canver and Supharattanasitthi with the teaching of Ikeda because it would have been obvious to combine prior art elements according to known methods to yield predictable results.
Combining the donor DNA taught by Canver and Supharattanasitthi with no insert between the upstream and downstream homology arms as taught by Ikeda would have led to predictable results with a reasonable expectation of success because both inventions are drawn to donor DNA and one of ordinary skill in the art would understand that the sequence between the homology arms can have a variable length depending on the intended purpose of the genome edit.
Claims 11 and 30 are rejected under 35 U.S.C. 103 as being unpatentable over Canver and Supharattanasitthi as applied to claims 1, 3-4 and 26 above, and further in view of Rezza et al (Scientific Reports (2019)9:3486;1-8).
Regarding claims 11 and 30: The teachings of Canver and Supharattanasitthi are discussed supra. Canver and Supharattanasitthi are silent on the length of the region between the upstream homology arm and the downstream homology arm of the donor DNA.
Rezza teach insert sizes of greater than 8 kbp are successfully integrated by Cas9n (Figure 2A). Rezza also teach CRISPR/Cas9 technology is efficient for the insertion of large DNA fragments (p6 ¶4).
It would have been obvious for one of ordinary skill in the art at the time of the effective filing date to combine the teaching of Canver and Supharattanasitthi with the teaching of Rezza because it would have been obvious to combine prior art elements according to known methods to yield predictable results. Combining the donor DNA taught by Canver and Supharattanasitthi with an insert greater than 8 kbp as taught by Rezza would have led to predictable results with a reasonable expectation of success because both inventions are drawn to donor DNA and one of ordinary skill in the art would understand that the sequence between the homology arms can have a variable length and is dependent on the intended purpose of the genome edit.
Claims 22-25 are rejected under 35 U.S.C. 103 as being unpatentable over Canver and Supharattanasitthi as applied to claims 1, 3-4 and 26 above, and further in view of Addgene (CRISPR Guide [online]. Addgene [retrieved on 11/13/2025]. Retrieved from the Internet: <URL: https://web.archive.org/web/20201212234421/https://www.addgene.org/guides/crispr/).
Regarding claims 22-25: The teachings of Canver and Supharattanasitthi are discussed supra. Supharattanasitthi further teach the CRISPR/Cas9 cleavage site on the WT sequence is shown with an arrow, and do not show the cleavage site on the plasmid donor sequence (Fig 1(b). While this implies the CRISIPR/Cas9 cleavage site is absent from the donor plasmid, Supharattanasitthi do not explicitly state that the target sequence of the site-specific recombinase is absent in the engineered two or more alleles in the chromosomal genome.
Addgene(2020) teach when designing a repair template (donor DNA) for CRISPR, one can exclude the nuclease specific target sequence (PAM sequence) from the repair template (donor DNA) to prevent the repair template from being a target for Cas9 cleavage (p4 ¶2).
It would have been obvious to one of ordinary skill in the art to adapt the methods of Supharattanasitthi and Cowan drawn to a method of biallelic genetic modification of a cell by doing altering the PAM sequence (target sequence of the site-specific recombinase) in the repair template such that the target sequence of the site-specific recombinase is absent in the engineered chromosomal genome as taught by Addgene(2020).
One of ordinary skill in the art would have been motivated to modify the target sequence of the site-specific recombinase as taught by Addgene to for the purposes of increasing the efficiency of the CRISPR system by preventing cleavage of the repair template.
One would have had a reasonable expectation of success because the method of Supharattanasitthi is directed to the use of CRISPR/Cas9 to modify cells and Addgene(2020) discloses standard methods for the use of CRISPR/Cas9.
Response to Arguments
The responses are directed to the Arguments filed 03/13/2026, all arguments have been considered.
Regarding Arguments directed to 35 USC § 112(b):
Claims 5-7: The claim amendment omitting the term “desired” overcomes the rejection as written and the rejection is withdrawn.
Claims 5-7; 8-11, 10-21 and 30: Applicant submits “two or more donor DNAs for selective markers” and “donor DNA for recombination” refer to functionally distinct elements which are described in the instant specification, and thus one of ordinary skill in the art would understand the distinct elements and thus the term “a donor DNA” is not indefinite.
The argument is unpersuasive because the instant specification defines “donor DNA” as “for use in the repair of double-strand break of DNA and refers to DNA that can be homologously recombined with neighboring DNA of a target region” (p24 [0017]). Therefore donor DNA is by definition used for recombination and the specific designation of a donor DNA as “a donor DNA for recombination” does not distinguish the molecule from another donor DNA “for selective markers” because donor DNA for selective markers also undergoes homologous recombination according to the claimed method.
Amending the claim 1(ii) to recite “a first pair of two or more donor DNAs for selective markers” and claim 5 to recite “where in each of the first pair of two or more donor DNAs” would overcome that part of the rejection as written.
Amending claim 5 (c) to recite “introduce a second donor DNA for recombination” and claim 5(iv) to recite “the second donor DNA for recombination” would overcome that part of the rejection as written.
Amending claims 8-11, 20-21 and 30 similar to claims 1 and 5 would overcome the rejection as written.
Claims 5-7 and 22-25: Applicant submits that claim 1 does not recite "a target sequence," but rather recites "a target region" which is a different element as defined in the instant specification.
This argument is persuasive.
The instant specification defines “target region” as “a genomic region
that is subject to genome engineering” (p24 [0017]).
The instant specification defines “target sequence” as “a DNA sequence, in the genome, to be cleaved by the sequence-specific nucleic acid cleaving molecule” (p26 [0021]).
Thus the target region in interpreted as broader than the target sequence and to encompass the target sequence.
The rejection over “a target sequence” is withdrawn.
Regarding Arguments directed to 35 USC § 102:
Claim 1 has been amended to recite a method comprising “selecting a cell having deletion of the target region in the two or more alleles” which overcomes the rejection as written. The rejection is withdrawn.
Regarding Arguments directed to 35 USC § 103:
Applicant submits the claim amendment to include a deletion overcome the rejection as written. This is persuasive and the rejection is withdrawn.
In response to Arguments relevant to the current rejection:
Applicant argues that the rejection does not address “selecting a cell having deletion of the target region”. This argument is addressed in the instant rejection under 103.
Applicant argues that the rejection does not address “a target region has a length of 5 kbp or more”. This limitation is addressed in the instant rejection under 103.
Conclusion
No claims are 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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ANDREA LYNNE MORRIS SPENCER whose telephone number is (571)272-3328. The examiner can normally be reached Monday-Friday 9:00-5:00.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, James (Doug) Schultz can be reached at 571-272-0763. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. 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.
/ANDREA LYNNE MORRIS SPENCER/Examiner, Art Unit 1631
/TAEYOON KIM/Primary Examiner, Art Unit 1631
Prosecution Insights