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 .
Continued Examination Under 37 CFR 1.114
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. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 18 December 2025 has been entered.
Application Status
This action is written in response to applicant’s correspondence received 18 December 2025. Claims 2-6, 8-9, and 11-28 are currently pending. Accordingly, claims 2-6, 8-9, and 11-28 are examined herein.
Any rejection not reiterated herein has been overcome by amendment. Applicant' s amendments 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) 2-4, 8-9, 11-14, 18-20, and 23 is/are rejected under 35 U.S.C. 103 as being unpatentable over Suzuki ("In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration." Nature 540.7631 (2016): 144-149), as evidenced by Liu ("C2c1-sgRNA complex structure reveals RNA-guided DNA cleavage mechanism." Molecular cell 65.2 (2017): 310-322), in view of Korona (Journal of developmental biology 5.4 (2017): 16).
Regarding claim 18, Suzuki is directed towards a study concerned with in vivo genome editing via CRISPR/Cas9 mediated homology-independent integration (Abstract). Suzuki teaches the use of homology-independent targeted integration (i.e., HITI) vectors that can be utilized in a method of homology-independent integration of a target gene of interest into a target cell’s chromosome (see Extended Data Figure 1). Suzuki teaches that the HITI vectors function via the use of Cas9 gRNA target sites present on a chromosomal region of interest and Cas9 gRNA target sites flanking a gene of interest present on a donor DNA such that NHEJ repair and homology-independent targeted integration of the gene of interest can occur at the chromosomal region of interest (see Extended Data Figure 1). Suzuki teaches a method of integrating an exogenous gene of interest into a first target intronic region of a chromosome in a first cell comprising delivering an HITI-AAV encoding a Cas9 protein, an AAV-rMertk-HITI donor plasmid comprising an exon 2 of a Mertk gene flanked by two gRNA target sites, and an intron 1 comprising two Cas9 gRNA target sites (pg. 146-148; see Figure 3). Suzuki teaches that vectors encoding gRNAs can be utilized to deliver Cas9 gRNAs to cells of interest (pg. 150; see Extended Data Figure 6). Suzuki teaches that the exon 2 was able to be integrated at intron 1 in order to restore MERTK function in the eye compared to a control vector that utilized homology arms flanking the exon 2 (pg. 148). Suzuki teaches that HITI vectors can be utilized to knock-in Luc or GFP reporter protein into a genomic region of interest to generate knock-in reporters for tracing cells in live animals. (see Extended Data Figure 6).
As evidenced by Liu (Molecular cell 65.2 (2017): 310-322), the Cas9 gRNAs of Suzuki are sgRNAs. Liu is directed towards a review study concerned with how different CRISPR enzymes cleave target nucleic acids of interest (Abstract). Liu teaches that Cas9 associates with a dual-guide RNA structure consisting of a crRNA and a trans-activating CRISPR RNA (tracrRNA) that combine to generate an sgRNA that can directed the Cas9 to a target nucleic acid of interest (pg. 310, 320; see Figure 7).
Suzuki does not teach or suggest that the reporter protein is flanked, 5’ to 3, by a splice acceptor sequence and a splice donor sequence (Claim 18).
However, one of ordinary skill in the art would have considered the teachings of Korona as both references are common fields of endeavor pertaining to the insertion of exogenous DNA sequences into an intronic genomic region of a first target gene of a cell.
Korona is directed towards a study concerned with introducing tags into genes at their endogenous genomic loci (Abstract). Korona teaches that the insertion of tags facilitate imaging approaches at the cellular or subcellular levels (Abstract). Korona teaches the use of a protein trap, termed MiMIC, comprising a donor DNA molecule that comprises, 5’ to 3’, a splice acceptor site, a nucleic acid sequence encoding a GFP protein, and a splice donor site (pg. 5). Korona teaches that the splice donor and slice acceptor sites can be utilized to allow for the incorporation of an artificial exon into a primary transcript and the splicing of the fluorescent reporter into a protein of interest (pg. 4; see Figure 2). Korona teaches that insertion of a gene of interest, via MiMIC, in the correct orientation into an intron of a coding gene will generate a truncated protein due to the splice acceptor and stop codons, thus acting as a gene trap (pg. 4). However, Korona further teaches that MiMIC is a transposon-based approach to genome engineering and that a comprehensive screen requires three different vectors, each with a reporter in a different open reading frame (pg. 4).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the HITI donor vector of Suzuki such that the exon 2 was substituted with an artificial exon comprising a splice acceptor site, a sequence encoding a reporter protein, and a splice donor site, as described by Korona. A person of ordinary skill in the art would have been motivated to do so in order to allow for the targeted integration of an artificial exon comprising a sequence encoding a reporter protein at intronic regions of interest via splice sites without the need for a large amount of vectors in order to tag proteins of interest. A person of ordinary skill in the art would have had a reasonable expectation of success because Suzuki teaches that exons can be targeted and integrated at intronic regions of interest in a target chromosome while Korona teaches the use of an artificial exon that can be integrated at an intronic region of interest in order to tag a protein of interest with a reporter protein.
Regarding claim 2, Suzuki teaches that the first and second binding sites may be the same “Scramble” guide RNA binding site (see Methods and Extended Data Figure 1).
Regarding claim 3, Korona teaches that the reporter protein is a fluorescent GFP protein (pg. 5).
Regarding claim 4, Suzuki teaches the gRNA target sites on the donor molecule (i.e., the first and second sgRNA binding sites) are the same gRNA target sites, termed “Scramble” (pg. 150; see Extended Data Figure 1)
Regarding claims 8-9, Suzuki teaches that the endonuclease is a Cas9 endonuclease (pg. 146-148; see Figure 3).
Regarding claims 11-14, as evidenced by Liu above, the gRNA of Suzuki comprises a fusion of a crRNA and a tracrRNA (pg. 310, 320; see Figure 7).
Regarding claim 19, Suzuki teaches that the exon flanked by gRNA binding sites was able to be integrated at an intronic genomic sequence of a first target gene (pg. 146-148; see Figure 3).
Regarding claim 20, Korona teaches that the reporter protein is expressed when a protein that it was spliced to is expressed (pg. 4; see Figure 2).
Regarding claim 23, Suzuki teaches that reporter proteins can be detected via the use of immunofluorescence images (see Extended Data Figure 1).
Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over Suzuki ("In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration." Nature 540.7631 (2016): 144-149), as evidenced by Liu ("C2c1-sgRNA complex structure reveals RNA-guided DNA cleavage mechanism." Molecular cell 65.2 (2017): 310-322), in view of Korona (Journal of developmental biology 5.4 (2017): 16) as applied to claims 2-4, 8-9, 11-14, 18-20, and 23 above, and further in view of Kamiyama (Nature communications 7.1 (2016): 11046).
Regarding claim 4, Suzuki, as evidenced by Liu, in view of Korona renders obvious claims 2-3, 8-9, 11, 17-20, and 23-24 as described above.
Suzuki, as evidenced by Liu, in view of Korona does not teach or suggest that the reporter is a split fluorescent protein fragment (Claim 4).
However, one of ordinary skill in the art would have considered the teachings of Kamiyama as both references are analogous prior art pertaining to the use of reporter proteins.
Kamiyama is drawn to a study concerned with the use of split fluorescent proteins (Abstract). Kamiyama teaches the use of a functional split fluorescent mCherry reporter protein (Abstract). Kamiyama teaches that small size of the split fluorescent tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated repair (Abstract).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the reporter rendered obvious by Suzuki, as evidenced by Liu, in view of Korona for a split fluorescent mCherry reporter protein, as described by Kamiyama. A person of ordinary skill in the art would have had a reasonable expectation of success because both Kamiyama and Suzuki, as evidenced by Liu, in view of Korona teaches the successful integration of a donor sequence encoding a reporter protein into a genomic target of interest. Therefore, substituting the reporter rendered obvious by Suzuki, as evidenced by Liu, in view of Korona for a split fluorescent mCherry reporter protein, as described by Kamiyama, would have resulted in the predictable outcome of success.
Claims 5-6 and 21-22 are rejected under 35 U.S.C. 103 as being unpatentable over Suzuki ("In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration." Nature 540.7631 (2016): 144-149), as evidenced by Liu ("C2c1-sgRNA complex structure reveals RNA-guided DNA cleavage mechanism." Molecular cell 65.2 (2017): 310-322), in view of Korona (Journal of developmental biology 5.4 (2017): 16) as applied to claims 2-4, 8-9, 11-14, 18-20, and 23 above, and further in view of Suarez (Veterinary parasitology 167.2-4 (2010): 205-215).
Regarding claims 5-6 and 21-22, Suzuki, as evidenced by Liu, in view of Korona renders obvious claims 2-3, 8-9, 11, 17-20, and 23-24 as described above.
Suzuki, as evidenced by Liu, in view of Korona does not teach or suggest the use of a blasticidin resistance gene positioned between the splice donor site and the second sgRNA site or the splice acceptor site and the first sgRNA site (Claims 5-6). Suzuki, as evidenced by Liu, in view of Korona does not teach or suggest that the antibiotic resistance gene is integrated into the first target gene and its presence detected (Claims 21-22).
However, one of ordinary skill in the art would have considered the teachings of Suarez as both references are analogous prior art pertaining to the introduction of nucleic acid sequences of interest into genomic targets.
Suarez is drawn to a review concerned with methods for the transient and stable expression of exogenous genes in target cells (Abstract). Suarez teaches the use of DNA encoding a blasticidin-GFP expression cassette that, when integrated into a target parasite genome, allows for the parasites to grow under the selective pressure of blasticidin (pg. 211-212; see Fig. 4). Suarez teaches that by utilizing blasticidin as a selectable marker, target genes and organisms can be studied successfully in the presence of blasticidin (Abstract).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the donor DNA sequences rendered obvious by Suzuki, as evidenced by Liu, in view of Korona such that it included a blasticidin resistance gene located outside of the splice donor and acceptor sites, but within the first and second sgRNA binding sites, in order to utilize a selection marker that can be utilized to study organisms in the presence of blasticidin, as described by Suarez. A person of ordinary skill in the art would have been motivated to do so in order to utilize a well-known selectable marker in order to study a target protein and organism in the presence of blasticidin. A person of ordinary skill in the art would have had a reasonable expectation of success because both Suarez and Suzuki, as evidenced by Liu, in view of Korona renders teach the successful integration of exogenous DNA into a target nucleic acid of interest.
Claims 15-16 are rejected under 35 U.S.C. 103 as being unpatentable over Suzuki ("In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration." Nature 540.7631 (2016): 144-149), as evidenced by Liu ("C2c1-sgRNA complex structure reveals RNA-guided DNA cleavage mechanism." Molecular cell 65.2 (2017): 310-322), in view of Korona (Journal of developmental biology 5.4 (2017): 16) as applied to claims 2-4, 8-9, 11-14, 18-20, and 23 above, and further in view of Liu (Journal of controlled release 266 (2017): 17-26).
Regarding claims 25-28, Suzuki, as evidenced by Liu, in view of Korona renders obvious claims 2-3, 8-9, 11, 17-20, and 23-24 as described above.
Suzuki, as evidenced by Liu, in view of Korona does not teach or suggest that the endonuclease and donor-specific guide RNAs are encoded on a single nucleic acid molecule (Claim 15). Suzuki, as evidenced by Liu, in view of Korona does not teach or suggest that the endonuclease, the guide RNAs, and the donor plasmids are present on a single nucleic acid molecule (Claim 16).
However, one of ordinary skill in the art would have considered the teachings of Liu as both references are analogous prior art pertaining to the use of Cas9.
Liu is drawn to a review concerned with CRISPR-Cas9 genome editing systems (Abstract). Liu teaches the use of a plasmid-based CRISPR-Cas9 system that encodes a Cas9 protein and sgRNA on the same vector (pg. 19). Liu teaches that a donor DNA molecule can be utilized alongside the CRISPR-Cas9 system in order to insert the donor sequence in a target genomic sequence of choice (pg. 19; see Fig. 3). Liu teaches that a single plasmid approach is advantageous as it avoids multiple transfections of different components (pg. 19).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method rendered obvious by Suzuki, as evidenced by Liu, in view of Korona such that the endonuclease, donor-specific guide RNAs, and donor DNA are encoded on a single nucleic acid molecule, as described by Liu. A person of ordinary skill in the art would have been motivated to do so in order to avoid multiple transfections of different components. A person of ordinary skill in the art would have had a reasonable expectation of success because both Suzuki, as evidenced by Liu, in view of Korona and Liu are drawn towards the use of CRISPR-Cas9 systems that can edit target genes and be encoded on vectors.
Claim(s) 17 is/are rejected under 35 U.S.C. 103 as being unpatentable Suzuki ("In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration." Nature 540.7631 (2016): 144-149), as evidenced by Liu ("C2c1-sgRNA complex structure reveals RNA-guided DNA cleavage mechanism." Molecular cell 65.2 (2017): 310-322), in view of Korona (Journal of developmental biology 5.4 (2017): 16) as applied to claims 2-4, 8-9, 11-14, 18-20, and 23 above, and further in view of Shin (Biochemical and biophysical research communications 502.1 (7 July 2018): 116-122).
Regarding claim 17, Suzuki, as evidenced by Liu, in view of Korona renders obvious claims 2-3, 8-9, 11, 17-20, and 23-24 as described above.
Suzuki, as evidenced by Liu, in view of Korona does not teach or suggest that the endonuclease-encoding nucleic acid sequence, the donor plasmid, the first site-specific intron targeting guide RNA, and the donor plasmid-specific gRNA encoding sequence are present on separate nucleic acid molecules (Claim 17).
Regarding claim 18, Shin is drawn to a study concerned with targeted sequence substitution through the use of microhomology-mediated end joining (i.e., “MMEJ”) (Abstract). Shin teaches the use of a Cas9 (i.e., an endonuclease) encoding nucleic acid sequence, a vector comprising a donor sequence flanked by two sgRNA binding sites, and two different guide RNAs that target a genomic region of interest such that the donor sequence is integrated into the genome of the target cell (pg. 117; see FIG. 1). Shin teaches that vectors encoding the guide RNAs were co-transfected with a vector encoding a Cas9 endonuclease (pg. 118). Shin teaches that the cells were transfected with mixtures of plasmids encoding a Cas9-red fluorescent protein (RFP)-puromycin expression cassettes, gRNA-expressing plasmids, and donor constructs at a 1:1:10 M ratio using Lipofectamine (i.e., Shin teaches that the endonuclease-encoding sequence, the donor plasmid, and the sequences encoding the sgRNAs are present on separate nucleic acid molecules (pg. 117).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the vectors rendered obvious by Suzuki, as evidenced by Liu, in view of Korona for vectors wherein the endonuclease-encoding nucleic acid sequence, the donor plasmid, the first site-specific intron targeting guide RNA, and the donor plasmid-specific gRNA encoding sequence are present on separate nucleic acid molecules, as described by Shin. A person of ordinary skill in the art would have been motivated to do so in order to utilize a known delivery method of delivering vectors to cells of interest via the use of Lipofectamine. A person of ordinary skill in the art would have had a reasonable expectation of success because both Suzuki, as evidenced by Liu, in view of Korona and Shin are drawn towards the use of CRISPR-Cas9 systems that can insert exogenous DNA into a genomic nucleic acid of interest via the delivery of the components of the system via vectors.
Claims 24-28 are rejected under 35 U.S.C. 103 as being unpatentable over Suzuki ("In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration." Nature 540.7631 (2016): 144-149), as evidenced by Liu ("C2c1-sgRNA complex structure reveals RNA-guided DNA cleavage mechanism." Molecular cell 65.2 (2017): 310-322), in view of Korona (Journal of developmental biology 5.4 (2017): 16) as applied to claims 2-4, 8-9, 11-14, 18-20, and 23 above, and further in view of Li (Nature plants 2.10 (2016): 1-6).
Regarding claims 24-28, Suzuki, as evidenced by Liu, in view of Korona renders obvious claims 2-3, 8-9, 11, 17-20, and 23-24 as described above.
Suzuki, as evidenced by Liu, in view of Korona does not teach or suggest integrating the exogenous DNA sequence into an intronic genomic sequence of a second target gene in a second cell by the delivery of the claimed composition and a second site-specific intron-targeting guide RNA that targets a second intronic site in a second target gene (Claim 24). Suzuki, as evidenced by Liu, in view of Korona does not teach or suggest that the exogenous DNA is integrated in two or more cells, wherein the intronic genomic sequence is unique for each of the two or more cells (Claim 25). Suzuki, as evidenced by Liu, in view of Korona does not teach or suggest that the first and second site-specific intron- targeting guide RNA-encoding nucleic acid sequences each target a different intronic site in the first target gene (Claim 26), each target a different intron in the first target gene (Claim 27), or each target a different site in the same intron of the first target gene (Claim 28).
However, one of ordinary skill in the art would have considered the teachings of Li as both references are analogous prior art pertaining to the design and use of guide RNAs that target intronic sequences.
Li is drawn to a study concerned with insertions in different introns in rice via the use of CRISPR- Cas9 (Abstract). Li teaches that guide RNAs can be designed to target different introns in a target EPSPS rice gene (Abstract). Li teaches that the guide RNAs allowed for editing of both the C3 and C5 introns in the target EPSPS rice gene (i.e., Li teaches the use of guide RNAs that are designed such that they different intronic sites and introns in a target gene) (pg. 2; see Figure 1). Li also teaches that multiple base pairs (i.e., different target sites) can be targeted on a singular intron through the use of guide RNAs (pg. 2; see Figure 1). Li teaches that by targeting specific and different intronic sites in different cells, exogenous DNA can be successfully inserted with a high degree of accuracy into a target introns (Abstract).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute at least one set of the genome-specific gRNA molecules described in Suzuki, as evidenced by Liu, in view of Korona for gRNA molecules targeting a different intronic site in first and second target the first target gene, a different intron in the first target gene, and a different site in the same intron of the first target gene, as described by Li, such that the target genomic sequence is unique for each of the two or more cells. A person of ordinary skill in the art would have had a reasonable expectation of success because both Li and Suzuki, as evidenced by Liu, in view of Korona teach and render obvious the use of gRNAs that can target intronic sequences of interest.
Response to Arguments
Applicant’s arguments with respect to claim(s) 2-4, 8-9, 11-14, 18-20, and 23 above have been considered but are moot because the new ground of rejections of the claims do not rely on any reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument.
Insofar as Applicant’s arguments pertain to the previously utilized Kamiyama, Suarez, Li, and Liu references, Applicant’s arguments pertain to how the previously utilized Shin, Sigal, and Sanz references do not teach homology independent integration. Accordingly, as the newly recited references do not utilize the previously utilized Shin, Sigal, and Sanz references, the Kamiyama, Suarez, Li, and Liu references are interpreted as being directed towards valid prior art.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KYLE T REGA whose telephone number is (571)272-2073. The examiner can normally be reached M-R 8:30-4:30, every other F 8:30-4:30 (EDT/EST).
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/KYLE T REGA/Examiner, Art Unit 1636
/NEIL P HAMMELL/Supervisory Patent Examiner, Art Unit 1636