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
1. 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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 October 17, 2025 has been entered. Claim 2 have been cancelled. Claims 1, 7 and 8 have been amended.
2. Claims 1 and 3-8 are currently being examined.
Priority
3. Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, or 365(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, or 365(c) as follows:
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of the first paragraph of 35 U.S.C. 112. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
The disclosure of the prior-filed application, KR 10-2021-0052619 fails to provide adequate support or enablement in the manner provided by the first paragraph of 35 U.S.C. 112 for one or more claims of this application. Examiner has established a priority date of April 22, 2022 for claims 1 and 3-8 because the claims as currently constituted recite “a CRISPR/Cpf1 system comprising crRNA (CRISPR RNA) and a donor nucleic acid molecule that complementarily binds to a target DNA,”, “wherein the donor nucleic acid molecule is in single-stranded or double-stranded form”, and wherein a point mutation is induced by the donor nucleic acid molecule into a target DNA. The donor nucleic acid as claimed can be a part of the crRNA or a separate nucleic acid in single-stranded or double-stranded form and a review of the KR 10-2021-0052619 does not reveal a CRISPR/Cpf1 system comprising said donor nucleic as broadly claimed. Applicant is invited to submit evidence pointing to the serial number, page and line where support can be found establishing an earlier priority date.
Regarding the foreign priority document of KR10-2022-0049739, it is not in English and no translation has been received and thus the examiner cannot determine if KR10-2022-0049739 describes the now-claimed invention. Therefore for the purposes of applying prior art, the effective filing date of claims 1 and 3-8 is April 22, 2022, the date the present application was filed.
Response to Arguments
4. Applicant argues that support for a 3’end truncated crRNA consisting of 15 to 20 consecutive nucleotides complementary to a target DNA can be found at Figs. 2(A)(1), 2(A)(3), 3 and 4 and ¶ 0038 of KR ‘619.
Applicant’s arguments have been considered, but have not been fully persuasive. Although KR ‘619 teaches a 21 nucleotide CRISPR repeat sequence TRS complementary to a target DNA in Fig. 2A and making 1-6 nt truncations to the 3’end of the crRNA in ¶¶ 0037-0038 of KR ‘619, KR ‘619 does not teach a donor nucleic acid that binds a target DNA that is separate from the crRNA as encompassed by the claims and can induce a point mutation in the target DNA.
Additionally KR ‘619 teaches that crRNA with a 6 nt truncation or a 5 nt truncation and a single mismatch disrupted the CRISPR/Cpf1 gene editing function and inhibited its ability to induce double strand DNA breaks measured by the reduction in CFUs. See ¶¶ 0035-0038. Thus, KR ‘619 does not provide support for a donor nucleic acid molecule that complementarily binds to a target DNA,, and wherein a point mutation is induced by the donor nucleic acid molecule into a target DNA when the that crRNA has a 5 or 6 nt truncation (16 or 15 consecutive nucleotides) and a mismatch with the target DNA. Thus, the effective filing date of claims 1 and 3-8 is April 22, 2022 for the reasons previously set forth and above.
Reinstated Rejections
Claim Rejections - 35 USC § 103
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.
5. Claims 1 and 3-7 are rejected under 35 U.S.C. 103 as being unpatentable over Kim (Microbiol. Biotechnol. August 2020, 30, 1583– 1591, of record) in view of Kleinstiver (Nat Biotechnol, 2016, Vol. 34, No. 8, pages 869-74 and Supplementary Fig. 3, of record) essentially for the reason of record set forth below.
Regarding claim 1:
Kim teaches: A method for single-base genome editing based on CRISPR/Cpf1 (see title and abstract), comprising crRNA and a donor nucleic acid molecule that complementarily binds to a target DNA, wherein the method comprises generating one mismatched nucleotide between the target DNA and the crRNA sequence by the donor nucleic acid molecule (see p. 1584 par. 7- “To construct perfect-matched and mismatched crRNA expression plasmids targeting crtEb, pHK473 was used as a template to amplify a ~1.9-kb fragment and a ~2.5-kb fragment. These two fragments were digested with DpnI and purified for isothermal assembly to generate pHK493. Other crRNA expression vectors (pHK494–pHK499) were generated using the same method as that used for pHK493 and confirmed through Sanger sequencing, using P35”; bottom of p. 1586 to the top of p. 1587- “In the case of pHK497 (single-mismatched)… all T-to-G single-base edits were successful, as intended. These results indicated that the use of target-mismatched crRNA is not only an efficient but also an accurate negative selection method for single-base genome editing using the CRISPR/Cpf1 system”; bottom of p. 1589 to the top of p. 1590- “With single-base-mutagenic oligonucleotides, different target-mismatched crRNA plasmids were transformed for single-base editing of T150G (i.e., introduction of TAG stop codon) in crtEb.”; Fig. 4-5). However, Kim does not expressly teach truncating the crRNA by 5 nucleotides at the 3’-end.
Kleinstiver teaches truncation of the 3’-end of a crRNA by 4 to 6 nucleotides, and that doing so reduces mismatch tolerance while maintaining robust mutagenesis efficiency (see Fig. 2C, spacer length of 18 nt in particular; see Fig. 2B; p. 869 col. 2 to p.870 col. 1- “to assess the specificities of AsCpf1 and LbCpf1, we examined the tolerance of these nucleases to mismatches at the crRNA-protospacer DNA interface. We mismatched adjacent pairs of bases within the complementarity regions of crRNAs targeted to three different endogenous target sites in the human DNMT1 gene (Fig. 2a)… Further testing of AsCpf1 and LbCpf1 editing at two of the three DNMT1 target sites using crRNAs with single mismatches along the length of the protospacer complementarity region … Taken together, these experiments suggest that both AsCpf1 and LbCpf1 are highly sensitive to mismatched crRNA nucleotides at most positions between 1 and 18, whereas recognition of bases at the 3′ end of the crRNA may be dispensable for efficient mutagenic activity… We further evaluated the importance of bases at the 3′ end of the crRNA for editing by testing AsCpf1 and LbCpf1 with a series of crRNAs bearing variable-length 3′ end deletions and extensions. When Cpf1 was paired with truncated crRNAs against three DNMT1 sites, T7E1 analysis revealed that mutagenesis efficiencies remained robust even when four to six bases were removed from the 3′ end of the crRNA”)
Kleinstiver also teaches that 3’ truncations of 5 nucleotides in the crRNA are efficient at inducing mutations. See Supplementary Figure 3a.
In light of these teachings, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Kim to incorporate the teachings of Kleinstiver, by truncating the Cpf1 crRNA by 4-6 nucleotides at the 3’-end, and producing a 3’-end truncate crRNA comprising a region consisting of 15 to 20 consecutive nucleotides complementary to the target DNA, because 4-6 nucleotides falls within the range set forth by Kleinstiver (i.e. truncation by 4-6 nucleotides) (See MPEP § 2144.05), Kleinstiver also teaches that 3’ truncations of 5 nucleotides are efficient at inducing mutations, and Kleinstiver suggests truncating the crRNA by 4-6 nucleotides at the 3’-end reduces mismatch tolerance while maintaining robust mutagenesis efficiency. Furthermore, it would have been obvious to try truncating the crRNA by 5 nucleotides in particular (as shown in Fig. 2C of Kleinstiver), as Kleinstiver teaches a finite number of identified, predictable solutions (i.e., truncation by 4-6 nucleotides), with a reasonable expectation of success in maintaining editing efficiency (See MPEP § 2143).
Regarding claim 3, Kim teaches the method wherein the target DNA comprises a nucleotide of a sequence complementary to the crRNA and a protospacer-adjacent motif (PAM) (see p. 1584 par. 7).
Regarding claim 4, Kim teaches the method wherein the donor nucleic acid molecule is ssODN (i.e., in single-stranded or double-stranded form), and wherein the donor nucleic acid molecule induces a genetic modification on the target DNA (see bottom of p. 1588 to top of p. 1589- “After transformation of IPTG-induced HK1220 cells harboring the RecT plasmid (pHK489) with single-stranded mutagenic oligonucleotides and crRNA plasmids, the surviving cells putatively harboring the desired mutations were obtained through negative selection (Fig. 3A)”; also see Fig. 3).
Regarding claim 6, Kim teaches the method wherein the modifications include a substitution of one nucleotide (see p. 1584 par. 7; bottom of p. 1586 to the top of p. 1587; bottom of p. 1589 to the top of p. 1590; Fig. 4-5).
Regarding claim 7, and as set forth above and in the previous office action, Kleinstiver teaches crRNAs with truncation by 5 nt (spacer lengths 18 nt) resulted in higher editing efficiencies compared to the non-truncated crRNAs with default spacer length, 23 nt, at DNMTl site 7 (see Fig. 2C).
Response to Arguments
5. In the Remarks of January 28, 2025, Applicant argues that Kim is not prior art to the captioned application under § 103 because Applicant asserts Kim is an exception under § 102(b)(1)(A). According to § 102(b)(1)(A), “[a] disclosure made 1 year or less before the effective filing date of a claimed invention shall not be prior art to the claimed invention [...] the disclosure was made by the inventor or joint inventor or by another who obtained the subject matter disclosed directly or indirectly from the inventor or a joint inventor...”.
Applicant argues that for the instant situation, the claimed invention has an effective filing date of April 22, 2021, when the priority application (1.e., KR 10-2021-0052619) was filed.
Applicant argues that as explained above, an English translation of the priority document and a verification document are submitted herewith to establish the effective filing date of April 22, 2021. In comparison, the pending Office Action notes that Kim was published in August 2020. Accordingly, the disclosure of Kim was less than 1 year before the effective filing date of the present application.
Applicant argues that furthermore, Applicant asserts that the co-author of Kim, Se Young Oh, obtained the disclosed subject matter of Kim directly or indirectly from inventors Hyun Ju Kim and Sang Jun Lee. Accordingly, a declaration of attribution under 37 C.F.R. § 1.130 is submitted herewith. In view of the declaration, Kim is not prior art to the captioned application. Therefore, in summary, Kim is an exception under § 102(b)(1)(A) and does not qualify as prior art to the captioned application. For this reason alone, the rejection under 35 U.S.C. § 103 over Kim in view of Kleinstiver is moot. Withdrawal of the rejection of claims 1 and 3-7 under 35 U.S.C. § 103 over Kim in view of Kleinstiver is respectfully requested.
Applicant’s arguments have been considered, but have not been found persuasive. The Declaration of Sang Jun Lee under 37 CFR 1.130(a) filed January 28, 2025 is insufficient to overcome the rejection of claims 1 and 3-7 under 35 U.S.C. 103 as being unpatentable over based upon Kim (Microbiol. Biotechnol. August 2020, 30, 1583– 1591) in view of Kleinstiver (Nat Biotechnol, 2016, Vol. 34, No. 8, pages 869-74) because KR 10-2021-0052619 does not provide adequate support or enablement in the manner provided by the first paragraph of 35 U.S.C. 112 for claims 1 and 3-7 as currently constructed as set forth above. In particular, KR 10-2021-0052619 does not teach “a CRISPR/Cpf1 system comprising crRNA (CRISPR RNA) and a donor nucleic acid molecule that complementarily binds to a target DNA,”, “wherein the donor nucleic acid molecule is in single-stranded or double-stranded form”, and wherein a point mutation is induced by the donor nucleic acid molecule into a target DNA. The donor nucleic acid as claimed can be a part of the crRNA or a separate nucleic acid in single-stranded or double-stranded form and a review of the KR 10-2021-0052619 does not reveal a CRISPR/Cpf1 system comprising said donor nucleic as broadly claimed.
Thus, the priority date is April 22, 2022 for claims 1 and 3-7. Given that Kim was published greater than 1 year prior to April 22, 2022 the exceptions under § 102(b)(1)(A) do not apply. Therefore the rejection is made and maintained for the reasons set forth above.
6. Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Kim (supra) in view of Kleinstiver (supra), as applied to claims 1 and 3-7 above, and further in view of Hur (Nat Biotechnol, 2016, 34, 807–808, of record) for the reasons of record set forth below.
The teachings of Kim, Kleinstiver, and Hur are discussed above and/or the previous office action, and are incorporated herein.
Regarding claim 8:
Kim teaches constructing a donor nucleic acid molecule that complimentary binds to the target DNA and induces one mismatched nucleotide between the target and the crRNA sequence (see p. 1584 par. 7).
As set forth above and in the previous office action, Kleinstiver teaches constructing a 3'-truncated crRNA in which four to six bases are truncated from the 3'-end of the crRNA comprising a nucleotide sequence complementary to the target DNA (see page 870, col. 1, par. 2; and section titled “Plasmids and oligonucleotides” under section titled “Online Methods” on the page after p. 874). The term “constructing” is not defined by the applicant, so the term is interpreted as the possession, teaching, and use thereof. Kleinstiver et al possesses and teaches the use of a 3’-truncated crRNA as well as a donor nucleic acid molecule, and as a result, meets the limitation of “constructing” in claim 8. Kleinstiver further suggests the further improvement, evaluation, and broader use of a CRISPR/Cpf1 system for the development of “highly specific therapeutics,” (see p. 873, col. 2, last par.).
While Kim teaches contacting the donor nucleic acid molecule into bacterial cells to be edited, Kim does not teach doing so into a “subject”, such as in animals or humans, and Kleinstiver fails to remedy this deficiency.
As set forth above and in the previous office action, Hur teaches a method using CRISPR/Cpf1 system to genetically modify the genes of mice embryos, functioning as the “subjects,” and obtaining genetically modified mice by growing said mice embryos in surrogate mothers (see p. 807, col. 3, lines 7 to 9- “electroporated AsCpf1 RNPs, targeting two sites in the Foxn1 exon 7, into mouse embryos,”; p. 808, col. 1 lines 10 to 14- “transplanted mouse embryos after Cpf1 microinjection or electroporation into surrogate mothers and obtained mice with targeted mutations in Foxn1 or Tyr”).
In light of these teachings, it would have been prima facie obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to have modified the methods of Kim and Kleinstiver, to incorporate the teachings of Hur, by implementing these methods into a subject, as Kleinstiver suggests the broader use of a CRISPR/Cpf1 system for the development of “highly specific therapeutics” (i.e., in vivo). Furthermore, there would have been a reasonable expectation of success, given the knowledge that a method using CRISPR/Cpf1 system to genetically modify the genes of a subject was previously known in the art, as taught by Hur.
Response to Arguments
7. In the Remarks of January 28, 2025, Applicant argues that in the present response, claim 8 has been amended. As amended, claim 8 is not obvious over Kim in view of Kleinstiver and further in view of Hur.
Applicant argues that As stated in Section II. above, Applicant asserts that Kim is not prior art to the captioned application under § 103 because Applicant asserts Kim is an exception under § 102(b)(1)(A). For this reason alone, the rejection under 35 U.S.C. § 103 over Kim in view of Kleinstiver, and further in view of Hur is moot. Withdrawal of the rejection of claim 8 under 35 U.S.C. § 103 over Kim in view of Kleinstiver and further in view of Hur is respectfully requested
Applicant’s arguments have been considered, but have not been found persuasive. The Declaration of Sang Jun Lee under 37 CFR 1.130(a) filed January 28, 2025 is insufficient to overcome the rejection of claims 8 under 35 U.S.C. 103 as being unpatentable over based upon Kim (Microbiol. Biotechnol. August 2020, 30, 1583– 1591) in view of Kleinstiver (Nat Biotechnol, 2016, Vol. 34, No. 8, pages 869-74) and further in view of Hur (Nat Biotechnol, 2016, 34, 807–808) as set forth in the last Office action because KR 10-2021-0052619 does not provide adequate support or enablement in the manner provided by the first paragraph of 35 U.S.C. 112 for claim 8 as currently constructed as set forth above. In particular, KR 10-2021-0052619 does not teach ) (a) constructing a donor nucleic acid molecule that complementarily binds to the target DNA and induces one mismatched nucleotide between the target DNA and crRNA sequence; (b) constructing a 3'-truncated crRNA in which 5 nucleotides are truncated from the 3'-end of the crRNA comprising a nucleotide sequence complementary to the target DNA; and (c) contacting the donor nucleic acid molecule of step (a) and the 3'-truncated crRNA of step (b) into the subject to be edited, thereby editing the target DNA of the subject.
Thus, the priority date is April 22, 2022 for claim 8. Given that Kim was published greater than 1 year prior to April 22, 2022 the exceptions under § 102(b)(1)(A) do not apply. Therefore the rejection is maintained for the reasons of record.
8. Claims 1 and 3-7 are rejected under 35 U.S.C. 103 as being unpatentable over Yan (Appl Environ Microbiol 83:e00947-17, of record) in view of Kleinstiver (supra) for the reasons of record set forth below.
Regarding claim 1:
Yan teaches a CRISPR-Cpf1-based gene editing method in bacteria comprising crRNA and a donor ssDNA or dsDNA that complimentarily binds to a target DNA, method comprising generating one mismatched nucleotide between the target DNA and the cRNA sequence, i.e., a point mutation, by the donor nucleic acid molecule (see abstract; Fig. 1; p. 2 par. 4- "our results show that CRISPRCas12a-assisted recombineering can rapidly and efficiently generate point mutations, deletions, and insertions in Escherichia coli, Yersinia pestis, and Mycobacterium smegmatis"; p. 2 par. 5- “we tested this system by attempting to introduce point mutations into the lacZ gene using ssDNA oligonucleotide recombination (Fig. 1A). We designed recombinogenic lacZ disruption oligonucleotides targeting the leading and lagging strands of DNA replication, whereby successful recombination introduced a stop codon within the lacZ open reading frame (ORF) (Fig. 1B)”; p.4 par. 4- “Point mutations were successfully introduced into two sites (located in hmsT and y4098) of the chromosome in Y. pestis KIM6with recombination efficiencies of 83% and 81%, respectively, using ssDNA oligonucleotides. This result suggests CRISPR-Cas12a-assisted recombineering can be used for genetic manipulation of chromosomal DNA in Y. pestis”; bottom of p. 5 to p. 7- “Consequently, point mutations can be introduced into the PAM- and crRNA-targeting regions using CRISPR-Cas12a-assisted ssDNA oligonucleotide recombineering. We sought to determine the sites that were susceptible to mutation by testing the recombination efficiency of a series of lagging-strand oligonucleotides designed to introduce a stop codon in the gfp gene by mutation of one to three nucleotides (Fig. 4A)… We found that a 1-bp insertion or deletion was efficiently generated at positions 1 to 18 of the protospacer sequence but not at positions 19 and 22 (Fig. 4D). Taken together, these experiments suggest that our system can efficiently introduce point mutations into PAM- and crRNA-targeting regions in M. smegmatis”; also see Fig. 4A in particular). However Yan does not expressly teach truncation of the 3’-end of a crRNA by 5 nucleotides.
As set forth above, Kleinstiver teaches truncation of the 3’-end of a crRNA by 4-6 nucleotides, and that doing so reduces mismatch tolerance while maintaining robust mutagenesis efficiency (see Fig. 2C, spacer length of 18 nt in particular, as spacer length 18 nt corresponds to 5 nt truncation; see Fig. 2B; p. 869 col. 2 to p.870 col. 1).
Kleinstiver also teaches that 3’ truncations of 5 nucleotides in the crRNA are efficient at inducing mutations. See Supplementary Figure 3a.
In light of these teachings, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have developed a CRISPR-Cpf1-based gene editing method in bacteria, method comprising generating one mismatched nucleotide between the target DNA and the crRNA sequence by the donor nucleic acid molecule, as taught by Yan, specifically wherein the Cpf1 crRNA is truncated by 5 nucleotides at the 3’-end, as taught by Kleinstiver. One would have been motivated to do so because the prior art of Kleinstiver suggests truncating the crRNA by 4-6 nucleotides at the 3’-end reduces mismatch tolerance while maintaining robust mutagenesis efficiency, and Kleinstiver also teaches that 3’ truncations of 5 nucleotides are efficient at inducing mutations, and the claimed method of truncating the crRNA and producing a 3’-end truncate crRNA comprising a region consisting of 15 to 20 consecutive nucleotides complementary to the target DNA falls within the range set forth by Kleinstiver (i.e., truncating the crRNA by 4-6 nucleotides) (See MPEP § 2144.05). Furthermore, it would have been obvious to try truncating the crRNA by 5 nucleotides in particular (as shown in Fig. 2C of Kleinstiver or Supplementary Figure 3a), as Kleinstiver teaches a finite number of identified, predictable solutions (i.e., truncation by 4-6 nucleotides), with a reasonable expectation of success in maintaining editing efficiency (See MPEP § 2143). Moreover, it would have been obvious to implement a single base change with donor nucleic acids, such as the ssDNA or dsDNA, as donor nucleic acids are known in the art to introduce both small and large changes in a target sequence during CRISPR/Cpf1 mediated gene editing, per the teachings of Yan and Kleinstiver.
Regarding claim 3, Yan teaches the method wherein the target DNA comprises a nucleotide of a sequence complementary to the crRNA and a protospacer-adjacent motif (PAM) (see Fig. 1B; p. 10 par. 6 to p. 11 par. 1- “Two complementary oligonucleotides containing the target sequence adjacent to 5=-YTN-3= were synthesized, annealed to yield a protospacer cassette with BpmI or BsaI overhangs at the 5= and 3= ends, respectively, and then cloned into the pAC-crRNA plasmid (Fig. S1)… To mutate a particular gene in mycobacteria, two complementary oligonucleotides containing the target sequence adjacent to =-YTN-3= were synthesized, annealed to yield a protospacer cassette with BpmI and HindIII overhangs at the 5= and 3= ends, respectively, and then cloned into pCR-Zeo or pCR-Hyg. All plasmids constructed in this study are listed in Table S1. The oligonucleotides used in this study are listed in Table S2”).
Regarding claim 4, Yan teaches the method wherein the donor nucleic acid molecule is ssDNA or dsDNA (i.e., in single-stranded or double-stranded form), and wherein the donor nucleic acid molecule induces a genetic modification on the target DNA (see in particular: p. 2 par. 5; p.4 par. 4; bottom of p. 5 to p. 7; and Fig. 1).
Regarding claim 6, Yan teaches the method wherein the modifications include a substitution of one nucleotide (see bottom of p. 5 to p. 7; Fig. 4A)
Regarding claim 7, and as set forth above and in the previous office action, Kleinstiver teaches crRNAs with truncation by 5 nt (spacer lengths 18 nt) resulted in higher editing efficiencies compared to the non-truncated crRNAs with default spacer length, 23 nt, at DNMTl site 7 (see Fig. 2C).
Response to Arguments
9. In the Remarks of January 28, 2025, Applicant argues that to establish a prima facie case of obviousness, the following requirements must be met: (1) there must be some suggestion or motivation, within the references themselves or in the knowledge generally available to one of ordinary skill in the art, to modify the reference or to combine reference teachings, (2) there must be a reasonable expectation of success, and (3) the prior art reference combination must teach or suggest all the claim limitations. In re Royka, 490 F.2d 981, 180 USPQ 580 (CCPA 1974). In re Vaeck, 947 F.2d, 20 USPQ2d 1438 (Fed. Cir. 1991)
Applicant argues that the pending claims in the present application are a “method comprising (a) generating one mismatched nucleotide between the target DNA and the crRNA sequence by the donor nucleic acid molecule; and (b) truncating the 3’-end of crRNA in which 5 nucleotides are truncated from the 3’-end of the crRNA comprising a nucleotide sequence complementary to the target DNA.” Therefore, the objective of the present application is to induce a single point mutation at a desired site using genome editing of microorganisms.
Applicant argues that genome editing of microorganisms using Cpf1 results in the death of cells where no mutation occurs at the target site. These cells are then recognized as targets by CRISPR/Cpf1, leading to a double strand break in the cell's genomic DNA. Cells with a mutation in the target DNA sequence induced via oligonucleotide-directed mutagenesis are not recognized as targets and survive. This process results in obtaining the mutated strain through negative selection.
Applicant argues that however, due to the mismatch tolerance of the CRISPR/Cpf1 system, targets where a single-base point mutation has occurred are recognized and destroyed in the same manner as targets without any mutation. As a result, a method of inducing a single-base mutation at the desired site was unknown. The present application, in contrast, discloses a method for obtaining a single-base mutated strain through negative selection. Using a donor DNA that induces one mismatched nucleotide at the target DNA, along with a crRNA where 5 nucleotides (nt) are truncated from the 3'-end, the inventors of the application enabled the efficient acquisition of a single-base mutated strain.
Applicant argues that as shown in Example 2 of the present application, both the cleavage of 1 to 6 nt at the 3'-end of crRNA and the introduction of a single base mismatch were simultaneously performed. As demonstrated, DNA with cleavage of 4 or fewer nucleotides at the 3'-end, despite the mutation, is recognized as having no mutation and is destroyed. On the other hand, when a 3' 5-nt truncation exists in the crRNA, a double-strand break occurs in the cell, leading to cell death (i.e., mismatch tolerance occurs). However, when both a single mismatch and a 3' 5-nt truncation are present simultaneously, mismatch tolerance does not occur, and a mutated strain can be obtained (FIG. 3A and 3B).
Applicant argues that additionally, as further illustrated in Example 3, the presence of a 5-nt truncation at the 3'-end of the crRNA of CRISPR/Cpf1 induces a double-strand break in targets where no mutation has occurred. However, it was further demonstrated that targets with a single-base mismatch due to mutation are not recognized as targets, making it possible to obtain a strain with a single-base edit at the precisely targeted site. In other words, it was confirmed that when 5 nucleotides are truncated from the 3'-end of the crRNA of CRISPR/Cpf1, the tolerance to single- base mismatches is overcome, resulting in an increase in single-base editing efficiency.
Applicant argues that more specifically, when attempting to induce a single-base mutation in galK and xvyB, using a crRNA with a 5-nt truncation at the 3'-end, a significant increase in the ratio of white colonies generated by single-base editing at both the ga/K 504th base and the xvyB 643rd base was observed (as shown using McConkey’s selective medium containing galactose, where mutated cells are white, and unmutated cells form red colonies). Subsequent sequence analysis confirmed that only base 504 of ga/K and base 643 of xylB were precisely altered (FIG. 4 and 5).
Applicant argues that in contrast, the combined teachings of Yan and Kleinstiver do not teach or suggest the limitations of claims 1 and 3-7. Yan teaches CRISPR-Cas12a-assisted recombineering to introduce mutations, deletions, and insertions in bacterial genomes, focusing on marker less and scarless genetic modifications. Regarding mismatch tolerance, Yan shows that the Cas12a nuclease is not uniformly sensitive to mismatches between the crRNA and the target DNA, and the degree of mismatch tolerance varies depending on the position within the complementary region between the crRNA and the target DNA. Kleinstiver establishes baseline specificity and efficiency of Cpfs in human cells. However, Kleinstiver does not address single- base editing or the use of 3'-truncated crRNAs. Therefore, the pending claims are not obvious over the combined teachings of Yan and Kleinstiver because the claimed method provides a method to overcome mismatch tolerance for sing-base precision. The combination of Yan with Kleinstiver does not teach or suggest the pending claims, and a person of ordinary skill in the art would not combine the teachings of Yan with the teachings of Kleinstiver to arrive at the pending claims.
Applicant argues that furthermore, a person of ordinary skill in the art would be motivated to arrive at the pending claims up on reading the teachings of Yan and Kleinstiver. Yan teaches for a “single” mismatch consistent tolerance without differences in mismatches at positions 1, 8, 9, and 19-23 (FIG. 2b), and Yan states that base recognition at the 3' end of the crRNA is “dispensable” for inducing effective mutation activity. This is contrary to the pending claims, in which the effect is achieved by truncating the 3' end of the crRNA to effectively induce a single- base mutation.
Applicant argues that according to the teachings of Yan, if base recognition at the 3' end of the crRNA is “dispensable” for inducing effective mutation activity, a person of ordinary skill in the art would not be motivated to truncate the 3’ end of the crRNA to efficiently induce a single-base mutation, as in the present invention. In particular, as taught in Yan, there is no difference in mismatches at positions 1, 8, 9, and 19-23. Thus, it would be difficult for a person of ordinary skill in the art to envision a significant effect could be achieved by truncating the '5nt' at the 3' end of the crRNA, as in the pending claims.
Applicant argues that similarly, Kleinstiver teaches that even when 4 to 6 nucleotides are removed from the 3' end of the crRNA, the efficiency is still maintained. Kleinstiver further discloses that that 4 to 6 nucleotides at the 3' end of the crRNA are dispensable for target site cleavage. It follows that, even if 4 to 6 nucleotides are cleaved from the 3' end of the crRNA, target site cleavage still occurs. In contrast, the pending claims truncate the 5 nt at the 3' end of the crRNA to prevent target site cleavage, with the aim of obtaining a viable strain with a single mutation. As a result, as taught by Kleinstiver, a person of ordinary skill in the art would find it difficult to conceive of a configuration that truncates the '5nt' at the 3' end of the crRNA as claimed, and further, it would also be difficult to anticipate the excellent effect of obtaining a strain with a single nucleotide mutation without killing it when truncating the 5nt.
Applicant argues that in other words, a person of ordinary skill in the art, upon reading the teachings of Yan combined with the teachings of Kleinstiver, would not appreciate that truncating the 3' end of the crRNA would have any effect on the single nucleotide mutation efficiency. Therefore, it would be unlikely for a person of ordinary skill in the art to expect that truncating the 3' end of the crRNA, as in the pending claims, would achieve the effect of obtaining single nucleotide mutations as provided by the pending claims. As taught by Kleinstiver, “[w]hen Cpfl was paired with truncated crRNAs against three DNMT1 sites, T7E1 analysis revealed that mutagenesis efficiencies remained robust even when four to six bases were removed from the 3’ end of the crRNA (Fig. 2c; Supplementary Fig. 3a). ... These findings suggest that just 17 to 19 bases of crRNA protospacer complementarity are required for robust Cpf1 activity and that four to six bases at the 3’ end of a 23 nt spacer may not be necessary for target site cleavage. Previous studies with SpCas9 showed that gRNAs bearing truncated target complementarity regions are usually more sensitive to single mismatches at the gRNA/target DNA interface than their full-length counterparts. However, we observed virtually no difference in AsCpf1 and LbCpf1 activities when bearing 20 instead of 23 nucleotide spacer sequences (Fig. 2d; using singly mismatched crRNAs Supplementary Fig. 3b).”
Applicant argues that in fact, the combined teachings of Kleinstiver and Yan teach away from the pending claims. A prior art reference must be considered in its entirety, i.e., as a whole, including portions that would lead away from the claimed invention. W.L. Gore & Assoc., Inc. v. Garlock, Inc., 721 F.2d 1540, 220 USPQ 303 (Fed. Cir. 1983), cert. denied, 469 U.S. 851 (1984) (emphasis original). As Kleinstiver teaches four to six bases at the 3’ end of the short CRISPR RNA (crRNA) used to program Cpf1 nucleases are insensitive to single base mismatches. In contrast, the pending claims of the present application are a method involving designing a 3'-truncated crRNA by deleting 1 to 5 nucleotides from the 3'-end of a crRNA sequence complementary to a target DNA. This modification overcomes the system's inherent mismatch tolerance, enabling accurate editing and improving the efficiency of single-base substitutions. Therefore, a person of ordinary skill in the art would be led away from the pending claims based on the combined teachings of Yan/Kleinstiver.
Applicant argues that as such, the combination of Yan in view of Kleinstiver: i) does not teach or suggest all the claim limitations, ii) would not motivate a person of ordinary skill in the art to arrive at the pending claims; and iii) teaches away from the pending claims. Accordingly, claims 1 and 3-7 are not obvious
Applicant’s arguments have been considered, but have not been found persuasive. Although the Applicant may have found both when a single mismatch and a 3' 5-nt truncation are present simultaneously in a crRNA, mismatch tolerance does not occur, and a mutated strain can be obtained when targeting the galK gene and the xylB with CRISPR/Cpf1 using a 3′-end truncated crRNA, the claims are not so limited. In particular, the specification teaches the term “crRNA” refers to an RNA specific for a target DNA, capable of forming a complex with a Cpf1 protein, and bringing the Cpf1 protein to the target DNA. See p. 7-[0034]. Thus, the claims encompass any potential crRNA. Additionally, the initial length of the crRNA is not defined so truncation from the 3’-end of the crRNA still encompasses crRNAs of any length comprising a region consisting of 15 to 20 consecutive nucleotides complementary to the target DNA . Thus, the evidence of unexpected results is not commensurate in scope with the invention claimed and is not sufficient to show the non-obviousness of the claimed invention. See MPEP 716.02 (d)
With regard to Yan, Yan does not teach that base recognition at the 3' end of the crRNA is “dispensable” for inducing effective mutation activity in Fig. 2B. Rather Yan teaches using the CRISPR)-Cas12a (Cpf1) system point mutations can be easily generated on the chromosome or native plasmids in Escherichia coli, Yersinia pestis, and Mycobacterium smegmatis. See abstract.
With regard to Kleinstiver, Kleinstiver teaches that 3’ truncations of 5 nucleotides in the crRNA are efficient at inducing mutations. See Supplementary Figure 3a. Thus, Kleinstiver does not teach away from using 3’ truncations of 5 nucleotides in the crRNAs.
Thus one would have been motivated to use the 3’ truncations of 4-6 nucleotides of the crRNA because the Kleinstiver teaches truncating the crRNA by 4-6 nucleotides at the 3’-end reduces mismatch tolerance, while maintaining robust mutagenesis efficiency, and Kleinstiver, teaches that 3’ truncations of 5 nucleotides in the crRNA are efficient at inducing mutations and the claimed method of truncating the crRNA by 5 nucleotides falls within the range set forth by Kleinstiver (i.e., truncating the crRNA by 4-6 nucleotides). Furthermore, it would have been obvious to try truncating the crRNA by 5 nucleotides in particular (as shown in Fig. 2C of Kleinstiver or Supplementary Figure 3a), as Kleinstiver teaches a finite number of identified, predictable solutions (i.e., truncation by 4-6 nucleotides), with a reasonable expectation of success in maintaining editing efficiency. Thus the rejections is maintained for the reasons previously set forth and above.
10. Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Yan (supra) in view of Kleinstiver (supra), as applied to claims 1 and 3-7 above, and further in view of Hur (supra) for the reasons of record.
The teachings of Yan and Kleinstiver are discussed above, and are incorporated herein.
Regarding claim 8:
Yan teaches constructing a donor nucleic acid molecule that complimentary binds to the target DNA and induces one mismatched nucleotide between the target and the crRNA sequence (see section titled “Preparation of recombinogenic DNA.” on p. 11; bottom of p. 5 to p. 7 and Fig. 4A; reads on instant claim 8 element (a)).
As set forth above and in the previous office action, Kleinstiver teaches constructing a 3'-truncated crRNA in which four to six bases are truncated from the 3'-end of the crRNA comprising a nucleotide sequence complementary to the target DNA (see page 870, col. 1, par. 2; and section titled “Plasmids and oligonucleotides” under section titled “Online Methods” on the page after p. 874). The term “constructing” is not defined by the applicant, so the term is interpreted as the possession, teaching, and use thereof. Kleinstiver et al possesses and teaches the use of a 3’-truncated crRNA as well as a donor nucleic acid molecule, and as a result, meets the limitation of “constructing” in claim 8. Kleinstiver further suggests the further improvement, evaluation, and broader use of a CRISPR/Cpf1 system for the development of “highly specific therapeutics,” (see p. 873, col. 2, last par.).
While Yan teaches contacting the donor nucleic acid molecule into bacterial cells to be edited, Yan does not teach doing so into a “subject”, such as in animals or humans, and Kleinstiver fails to remedy this deficiency.
As set forth above and in the previous office action, Hur teaches a method using CRISPR/Cpf1 system to genetically modify the genes of mice embryos, functioning as the “subjects,” and obtaining genetically modified mice by growing said mice embryos in surrogate mothers (see p. 807, col. 3, lines 7 to 9- “electroporated AsCpf1 RNPs, targeting two sites in the Foxn1 exon 7, into mouse embryos,”; p. 808, col. 1 lines 10 to 14- “transplanted mouse embryos after Cpf1 microinjection or electroporation into surrogate mothers and obtained mice with targeted mutations in Foxn1 or Tyr”).
In light of these teachings, it would have been prima facie obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to have modified the methods of Yan and Kleinstiver, to incorporate the teachings of Hur, by implementing these methods into a subject, as Kleinstiver suggests the broader use of a CRISPR/Cpf1 system for the development of “highly specific therapeutics” (i.e., in vivo). Furthermore, there would have been a reasonable expectation of success, given the knowledge that a method using CRISPR/Cpf1 system to genetically modify the genes of a subject was previously known in the art, as taught by Hur.
Response to Arguments
11 In the Remarks of January 28, 2025, Applicant argues that the deficiencies of the combination of Yan and Kleinstiver are detailed in Section IV. above. The teachings of Hur in combination with the teaching of Yan and Kleinstiver fail to address the deficiencies of Yan and Kleinstiver regarding the claimed invention. Accordingly, for the reasons articulated in Section IV, above, claim 8 is not obvious. Withdrawal of the rejection of claim 8 under 35 U.S.C. § 103 over Yan in view of Kleinstiver and further in view of Hur is respectfully requested.
Applicant’s arguments have been considered, but have not been found persuasive. Applicant is reiterating the arguments set forth above, thus for the reasons previously set forth and above the arguments are not found persuasive and the rejection is maintained.
New Grounds of Rejections
Claim Rejections - 35 USC § 112
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.
12. Claim 6 is 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.
Claim 6 recites the limitation "the modifications" in line 1. There is insufficient antecedent basis for this limitation in the claim because claim 5 does not recite modifications.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
13. Claim 5 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 5 is drawn to the method of claim 1, wherein the donor nucleic acid molecule induces a genetic modification on the target DNA. Claim 1 recites that a point mutation is induced by the donor nucleic acid molecule. Genetic modification of the target DNA is a broader genus of mutations than a point mutation. Thus, claim 5 fails to further limit the subject matter of claim 1.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Conclusion
14. No claims allowed.
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/PETER J REDDIG/Primary Examiner, Art Unit 1646