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 .
Application Status
This action is written in response to applicant’s correspondence received on 12/11/2025. Claims 31, 40, 49-50, 64-68, 70-72, and 77-79 are pending. Claims 31, 49, 64-66, and 70 have been amended. Claims 1-30, 32-39, 41-48, 51-63, 69, and 73-76 have been cancelled. Claims 49-50 have been withdrawn from consideration. Claims 78-79 are newly added. Claims 31, 40, 64-68, 70-72, and 77-79 are currently under examination.
Any rejection or objection not reiterated has been overcome. Applicant’s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow. This Office Action is Final.
Claim Rejections - 35 USC § 103 – Maintained/Updated in Response to Amendments
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.
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.
Claims 31, 40, 64-68, 70-72, and 77-79 are rejected under 35 U.S.C. 103 as being unpatentable over May et al. (WO 2014/150624 A1, of record) in view of Yin (WO 2015/191693 A2, published 12/17/2015, earliest effective filing date 06/10/2014, of record), Potter et al. (WO 2016/065364 A1, earliest effective filing date October 24, 2014, provided in an IDS), and Maeder (WO 2015/153791 A1, of record). Claim 31 is further evidenced by Dubitzky (Dubitzky et al. Encyclopedia of Systems Biology, DOI 10.1007/978-1-4419-9863-7, published 2013, of record). The rejection is further evidenced by GenBank L29345 (NCBI GenBank Database, accession number L29345.1, GFP genetic sequence, published 12/30/1994, accessed 12/13/2024).
Regarding claim 31, May describe a gene editing system, comprising a gRNA fusion molecule and at least one Cas9 molecule (complex comprising a site-directed polypeptide, a nucleic acid-targeting nucleic acid, and a donor polynucleotide can be delivered to a target nucleic acid; paragraph [0851], nuclease, e.g., Cas9; paragraph [0850]). May et al. describe a gRNA complex molecule, comprising a gRNA molecule and a template nucleic acid (complex comprising a gRNA and a donor polynucleotide; paragraph [0851], figure 30). Furthermore, the final sentence of paragraph 284 of May teaches that the 3’ tracrRNA sequence can comprise one or more hairpins (i.e., at least two hairpins). The 3’ tracrRNA sequence is identified as 130 in Figure 1A (paragraph 284), which clearly depicts this sequence/structure at the 3’ end of the molecule. Furthermore, paragraph 333 teaches that the 3’ tracrRNA sequence comprises a stem loop structure. Thus, May teaches a stem loop structure at the 3’ end of their gRNA molecules. May therefore teaches that the 3’ end of their gRNA molecule comprises one or more hairpin loops (paragraph [0284]). May further teaches that their template nucleic acids comprise single-stranded DNA, or double-stranded DNA (in some embodiments, the donor polynucleotide can be single stranded. In some embodiments, the donor polynucleotide can be double stranded; paragraph [0860], furthermore, May defines a “donor polynucleotide” as a nucleic acid [00159], and states that a nucleic acid can be DNA [00174]; thus, May teaches that their donor/template polynucleotides can be single- or double-stranded DNA).
May also teaches Figure 30, wherein the description of Figure 30 on page 49 states that Figure 30 “depicts exemplary methods of the disclosure of bringing a donor polynucleotide to a modification site in a target nucleic acid”, paragraph 121. Furthermore, paragraph 849-860 of May teaches that their method includes bringing the donor polynucleotide into close proximity of the site directed target by associating, in various ways (Figure 30A-30F) the donor with the gRNA, using hybridization strategies, and thereby enhancing the insertion of the donor polynucleotide into the site by increasing its proximity to the target site by directly linking/attaching it with the gRNA (Figure 30A-30F and paragraphs 849-860). Thus, May teaches enhanced delivery of donor nucleic acids to target sites by directly linking the donor nucleic acid with gRNA.
May teaches that the donor polynucleotide/template is hybridized/annealed to the 3’ end of the gRNA through complimentary base pairing/annealing (Figure 30A-30E) or through RNA/DNA binding protein interactions (Figure 30F). May therefore teaches non-covalent linkage of the gRNA to the template/donor nucleic acid (Figure 30). May teaches that the 5’ end of donor can bind the 3’ end of the gRNA (Figure 30B).
Furthermore, May teaches that site-directed mutations may be made to regions and domains of the Cas9 such as the HNH domain, where such modifications can alter the nuclease activity of the domain (e.g., paragraphs 499-500). May also teaches that mutations may be made in Cas proteins to form nickases, where the nuclease activity of a domain is targeted to form a nickase (e.g., paragraph 602). Furthermore, May teaches that such adoption of nickase strategies can be used for targeting genomic loci, where the target cleavage sites can be within the general region of a target site (e.g., paragraph 604). Furthermore, May teaches that target DNA targeted using nickases can be used, where the gRNA targets 5’ of a target sequence (Figure 34). Per the instant specification, the drawing presently recited in claim 31 broadly includes a nickase Cas9 which nicks between 1-10,000 bases 5’ of a target site (bottom of page 136 into top of page 137 of the present specification). Thus, the teaching of May, who teaches that nickases can cut at a target site or 5’ of a target site for insertions or deletions reasonably reads on the presently recited drawing of target position and cleavage event recited in the drawing of claim 31 (paragraphs 602-607).
May teaches that Cas9 can comprise two or more nuclease domains including RuvC and an HNH domain (paragraph 246). May further teaches that the HNH domain can be modified to contain mutations (section entitled “Modifications to the HNH domain”, paragraphs 547-557). May further teaches that “The nuclease used in the methods of the disclosure (e.g., Cas9) can comprise nickase activity in which the nuclease can introduce single-stranded breaks in a target nucleic acid” paragraph 859. Therefore, May teaches that the Cas9 they used in the study can comprise a RuvC and HNH domain, and also teaches mutations in the HNH domain, and further teaches that the Cas9 they teach can be in the form of a nickase.
May does not teach that the donor polynucleotide is covalently linked with the gRNA in their molecule complexes, or that this covalent linkage is a phosphodiester bond between the 3’ end of the gRNA molecule and the 5’ end of the template nucleic acid. May, while teaching Cas9 nickases which comprise a RuvC domain and an HNH domain that can be inactivated to form the nickase, and teaches a motivation to make such a Cas9 to target DNA, does not specifically reduce the Cas9 nickases to practice which has an inactive HNH domain.
Yin is a patent document focused on enhancing gene editing tools (paragraph 3). Specifically, Yin teaches methods related to modifying gene sequences using CRISPR/Cas9 editing systems, and delivery systems for achieving gene modification using gRNAs and donor nucleic acids (Abstract, and e.g., paragraphs 6 and 83). The teachings of Yin and May therefore directly overlap in subject matter and stated goals because both relate to the use of gRNA/CRISPR/donor nucleic acid template delivery (see Abstracts of both, and see documents) Yin teaches that the gRNA and repair templates used in their methodology can be covalently linked to each other and non-covalently linked to each other (paragraphs 6, 15, 23, 83, and claims 18, 22, 27, 54 114, and 116). Paragraph 15 of Yin teaches that, while the gRNA and the repair template/donor polynucleotide can be covalently linked, the repair template can also be partially annealed to the gRNA, as was also taught by May (May, Figure 30). Thus, Yin teaches both partial annealing (non-covalent) and covalently linking gRNA with a repair template, and also that these two methods of associating a gRNA and a template/donor/repair template can be used interchangeably as they accomplish the same task, as evidenced by the fact that they are recited in the alternative as methods of linking gRNA with template a nucleic acid (e.g., paragraphs 6 and 83). Additionally, Yin teaches in paragraph 15 that the repair template that is covalently linked with the gRNA can be DNA.
Furthermore, Yin teaches that as a result of linking the gRNA to the repair template DNA, which can be bound either covalently or non-covalently as functional alternatives, the efficiency of nucleotide sequence modification by which the repair template is efficiently directed to the nucleus of the cell is “greatly improved,” (paragraph 83). Thus, Yin teaches that there are two ways to bind gRNA to donor templates: either non-covalently or covalently, that such binding can be used interchangeably, and that a result of linking gRNAs to donor templates is significant improvement of sequence modification (paragraph 83).
Furthermore, Potter et al. describe a gRNA fusion molecule, comprising a gRNA molecule and a template nucleic acid (donor nucleic acid is covalently linked to guide RNA; paragraph [0036]), wherein the template nucleic acid comprises single-stranded DNA, or double-stranded DNA (donor nucleic acid will typically be DNA and may be single-stranded or double-stranded; paragraph [0050]).
Furthermore, Potter and May directly overlap in subject matter because Potter also teaches gRNAs with 3’ hairpin structures that are attached to donor/template nucleic acids (e.g., Figures 1,7, and 9). Potter also teaches the goal of improving the efficiency of homologous recombination in CRISPR systems for gene editing by increasing the concentration and proximity of donor/template nucleic acids to target sites (Abstract, and paragraphs 2-4). Thus, Potter, May, and Yin are all in the same field of endeavor, use similar reagents and methods, and overlap in objective, aim, and subject matter.
Potter teaches Figures 1, 7, and 9, along with a description of Figure 1 in paragraph [0006] stating that “the hairpin nucleic acid molecule is guide RNA.” Figures 1, 7, and 9 of Potter depict nucleic acid molecules with two regions of secondary structure, wherein one region of secondary structure is in the middle of the molecule and one is at the 3’ end. The structures at the 3’ end of the gRNAs in Figures 1, 7, and 9, contain a region which has folded back (i.e., complimented) with itself, forming a stem structure and, because these regions at the 3’ end contain stem structures, these regions must also inherently contain loop structures, as evidenced by Dubitzky. Dubitzky is an encyclopedia of terms and definitions used in systems biology. Dubitzky teaches that “hairpin structure is a pattern that can occur in single-stranded DNA or, more commonly, in RNA. The structure is also known as a stem-loop structure”, “Definition”, first paragraph. Dubitzky therefore teaches that the term “hairpin” (which is the term Potter uses to describe the structure in Figure 1 in paragraph 6) is synonymous with stem-loop structure, and corroborates that the hairpin structure recited by Potter inherently contains a stem loop. Furthermore, Dubitzky teaches that “the formation of a hairpin structure is dependent on the stability of the resulting helix and loop regions. The first prerequisite is the presence of a sequence that can fold back on itself to form a paired double helix”, Formation and Stability, first paragraph, and that “the stability of the loop also influences the formation of the hairpin structure. “Loops” that are less than three bases long are sterically impossible and do not form”, “Formation and Stability”, second paragraph. Figures 1, 7, and 9 of Potter clearly depict a gRNA nucleic acid molecule that has a secondary structure at the 3’ end, wherein said secondary structure contains a region that has “folded back” on itself, i.e., has formed a stem structure. Furthermore, as taught by Dubitzky, it is physically impossible for such a “folding back” stem structure to occur with a loop less than three nucleotides in length. Given that the 3’ end of the gRNA molecules depicted by Potter in Figures 1, 7, and 9 clearly contain a region that has complimented with itself to form a stem, and that Dubitzky teaches that it is impossible to have a stem without at least a four base loop, the secondary structure at the 3’ end of Figures 1, 7, and 9 are inherently hairpin loop structures.
Thus, the teachings of May and Potter directly overlap in scope and subject matter because each teach gRNAs linked with template nucleic acids, where the 3’ end of the gRNA molecule comprises a hairpin loop, and where the template nucleic acid comprises single-stranded DNA or double-stranded DNA.
Furthermore, Potter teaches that the 3’ end of their gRNA molecule is linked to the 5’ end of the template nucleic acid by a phosphodiester bond (gRNA-azido-dATP, figures 7 and 9).
Potter teaches that their invention “involves enhancing homologous recombination by increasing the concentration of donor nucleic acid at or in close proximity to the junction of a break in a nucleic acid molecule resident in a cell,” (paragraph 4). Potter therefore teaches that their methods, which use gRNAs comprising 3’ hairpin loops covalently bound to template/donor DNA enhances recombination/gene editing events (paragraph 4). Thus, Potter directly teaches a motive to incorporate their teachings into similar systems, such as those taught by May and Yin (paragraph 4).
Furthermore, Potter, like May, also teaches that either the HNH or RuvC domain of Cas9 can be targeted to inactivate one of these nuclease sites for the beneficial outcome of making a nickase, and that this is a known, useful strategy to generate a nickase version of the protein (paragraph 6). Furthermore, Potter also teaches that, when using such nicakses comprising for instance an inactive HNH domain, a target locus can be cut within the surrounding region of the target (paragraph 31). Thus, Potter also teaches that cleavage sites can be in the surrounding region of a target site, and therefore teaches the targeting strategy depicted in the drawing in claim 31 (paragraph 31, where the specification at pages 136-137 recite that the gRNA can cut within 1-10,000 bps of the target, where Potter’s teaching of cutting in the region of a target reasonably reads on 1-10,000 bases of the target locus, paragraph 31 or Potter).
Regarding a method of generating a Cas9 with functional RuvC domain and inactive HNH domain, such a strategy is already known and reduced to practice as taught by Maeder. For instance, Maeder is a patent document related to methods of CRISPR gene editing and targeting (Description). Maeder teaches that “the gRNAs are used with a Cas9 nickase molecule having RuvC activity, e.g., a Cas9 molecule having the HNH activity inactivated, e.g., a Cas9 molecule having a mutation at N863, e.g., the N863A mutation”, (page 806, lines 17-19). Therefore, Maeder teaches that Cas9 has a RuvC domain and an HNH domain, and further teaches that a mutation at N863 renders the Cas9 molecule a nickase enzyme with RuvC activity remaining intact while inactivating the HNH domain. Thus, Maeder teaches a known method of rendering a Cas9 protein into a nickase by making a mutation at position N863, which leaves the RuvC domain intact but inactivates the HNH domain. Thus, Maeder teaches that mutational strategies to render Cas9 nickases, where the mutation leaves an active RuvC domain and inactive HNH domain (as taught by both May and Potter) are already known and reduced to practice. Thus, the teachings of May regarding inactivating the HNH domain are predictable and known in the art.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the gRNA/donor templates of May with Yin and Potter, to arrive at a gRNA fusion molecule comprising a gRNA covalently linked to a template nucleic acid, wherein the 3’ end of the gRNA molecule comprises two hairpin loops, wherein the 3’ end of the gRNA molecule is linked to the 5’ end of the template nucleic acid by a phosphodiester bond, and wherein the template nucleic acid comprises DNA. The combination of May, Yin, and Potter is the simple substitution of one method of linking gRNA and template DNA (non-covalent annealing, May) for another known method of linking gRNA and template DNA (covalent phosphodiester 3’/5’ linkage, as taught by Potter/Yin) to obtain predictable results. In addition, the combination is also obvious to try, where a practitioner would be choosing from only two options (covalent or non-covalent bonds), where the solution is predictable because both are taught to be viable ways of binding nucleic acid molecules, and further where the expectation of success is reasonable because phosphodiester/covalent bonds are known to work to link nucleic acids (Potter). Furthermore, a practitioner would be motivated to combine the teachings of May, Yin, and Potter because Yin and Potter teach that their methods can greatly improve the efficiency of gene editing (Yin, paragraph 83, Potter paragraph 4).
Furthermore, May already teaches atCas9 comprising a RuvC and inactive HNH domain are known, and are useful for targeting DNA regions as a nickase. The product recited in claim 31 is further obvious in view of Maeder, who teaches specific strategies to make the HNH-inactive Cas protein already taught by both May and Potter. Thus, an inactive HNH domain is not a novel feature, nor is it unpredictable as it is well-known in the art as taught by May, Potter, and Maeder, where further a practitioner would be motivated to make such an HNH-inactivation based on the teachings of May so that they could target specific DNA targets with nicakse Cas9 enzymes using the strategy of Maeder.
Furthermore, regarding the drawing in claim 31 depicting a targeting/cleavage strategy, Potter teaches that nickases cleave in the general surrounding area of a target as a known strategy of DNA targeting (paragraph 31). Thus, the targeting/cleavage strategy using an HNH-inactive nickase is simply the known targeting strategy when using such enzymes, as taught by Potter (paragraph31). It would therefore be obvious to a person of ordinary skill in the art to adopt the nickases taught by May to use the targeting strategy as taught by Potter, to arrive at the targeting/cleavage strategy depicted in claim 31.
Regarding claim 40, May teaches a cell comprising the gRNA fusion molecule or gene editing system (tagged cell can be generated by contacting the cell with a donor polynucleotide, and a complex comprising a site-directed polypeptide and a nucleic acid-targeting nucleic acid; paragraph [0826]).
Regarding claim 64, May teaches that the 3' tracrRNA sequence can comprise one or more hairpins; paragraph [0284].
Regarding claim 65, May teaches that their nucleic acids can comprise an MS2 binding site (paragraph 741).
Regarding claim 66, May teaches ligation of nucleotides using T4 DNA ligase, paragraph 866. Claim 66 is therefore obvious as it would be obvious to a practitioner of ordinary skill in the art that such nucleic acids could be ligated together, particularly in light of the fact that the gene editing system, comprising covalent linkage and phosphodiester bond is rendered obvious in view of May, Yin, and Potter. Ligation is therefore a know method taught by May of joining nucleic acids together.
Furthermore, claim 66 is not drawn to a method of ligation, but to a product. Thus, recitation of the limitation that the gRNA is ligated to the template nucleic acid does not change the structure of the product recited, as the claim depends from claim 31 and requires covalent linkage with a phosphodiester bond. Thus, even though it would be obvious to use a ligase to join the nucleic acids, the limitations of claim 66 regarding the how the gRNA and template are joined do not add structural limitations to the claim limitations recited in claim 31.
Regarding claims 67, 68, and 71, May teaches that the site-directed polypeptides used in their methods can be enzymatically active (paragraph 611), and further states that “in some embodiments, the polypeptide is Cas9”, paragraph 17.
Regarding claim 70, Maeder teaches that “the gRNAs are used with a Cas9 nickase molecule having RuvC activity, e.g., a Cas9 molecule having the HNH activity inactivated, e.g., a Cas9 molecule having a mutation at N863, e.g., the N863A mutation”, (page 806, lines 17-19). May teaches SpCas9 (paragraph 888).
Regarding Claim 72, both May and Potter teach the formation of ribonucleoprotein complexes for Cas/gRNA systems (May paragraph 214, Potter paragraph 101).
Regarding claim 77, May teaches that donor polynucleotides can comprise a reporter element such as GFP (paragraph 769). As evidenced by GenBank L29345, the nucleic acid sequence of GFP is 922 basepairs (page 2). Thus, by teaching that donor polynucleotides can comprise a nucleic acid sequence encoding GFP, which is 922 bp, May teaches that template nucleic acids can be at least 150 nucleotides in length (May paragraph 769, GenBank L29345, page 2).
Regarding claim 78, claim 78 recites that the gRNA was ligated using splint ligation. However, claim 78 is not drawn to a method claim, but a product. Thus, recitation of the method by which the gRNA molecule is covalently linked to the template nucleic acid (i.e., by “Splint Ligation”) does not add any structural limitation to what is actually being claimed: a gRNA fusion molecule. Thus, the newly recited limitations are not relevant to the overall structure of what is being claimed, as the claim is directed to a structure and not the method of the creation of the structure, or what was “used” to generate the structure/gRNA fusion.
This claim interpretation is supported by the specification. For instance, page 210 of the specification, second paragraph, describes Figures 5A, 6A, and 7A, and the ligation of DNA donor templates with gRNA using splint ligation. Note that the second paragraph concludes by stating that “The ligation product [i.e., the gRNA/donor fusion molecule] was purified under denaturing conditions, which removed the splint, leaving a single stranded hybrid species” (page 210, second paragraph, bracketed note added for clarity). Thus, the recitation in claim 78 of “Splint Ligation” in order to generate the gRNA fusion does not ultimately change the structure of the gRNA fusion molecule, as the specification indicates that the splint is removed to generate the final product. Claim 78 is therefore broadly drawn to the same structural limitations of claim 31, and is therefore rejected for the same reasons given in the rejection of claim 31.
Recitation of the method by which the gRNA fusion molecule is made (i.e., “Splint Ligation”) is irrelevant because the splint can be ultimately removed after the fusion molecule is created, per the specification at page 210, second paragraph.
Regarding claim 79, Potter also teaches that, when using nicakses comprising for instance an inactive HNH domain, a target locus can be cut within the surrounding region of the target (paragraph 31). Thus, Potter teaches that cleavage sites can be in the surrounding region of a target site, and therefore teaches the targeting strategy depicted in the drawing in claim 31, where Potter’s teaching of cutting in the general region of a target reasonably reads on 1-10,000 bases of the target locus, paragraph 31 or Potter). Potter teaches that when using dual nickase targeting systems, the nicks are made in the surrounding region of the target (i.e., within 1-10,000 basepairs)
It would have been obvious to a person of ordinary skill in the art before the effective filing date to target to design the Cas9 to cleave a single strand (i.e., nick) within 1-10,000 base pairs of the target site because Potter teaches that, when using nickases, this is the known strategy of targeting DNA. Thus, a practitioner would be motivated to nick a site within 1-10,000 basepairs of the target, so that they could carry out the known strategy taught by Potter.
Additional Rejection of claim 78 – This rejection is given with the interpretation that Splint Ligation is required for the claim
Claim 78 is rejected under 35 U.S.C. 103 as being unpatentable over May et al. (WO 2014/150624 A1), Yin (WO 2015/191693 A2, published 12/17/2015, earliest effective filing date 06/10/2014), and Potter et al. (WO 2016/065364 A1, earliest effective filing date October 24, 2014, provided in an IDS), as applied to claims 31, 40, 64-68, 70-72, and 77-79, above, and further in view of Lohman (Lohman GJ et al. Nucleic Acids Res. 2014 Feb;42(3):1831-44, of record), and Kershaw (Kershaw et. al. R.T. (2013). Splint Ligation of RNA with T4 DNA Ligase. In: Conn, G. (eds) Recombinant and In Vitro RNA Synthesis. Methods in Molecular Biology, vol 941. Humana Press, Totowa, NJ, or record). The rejection is further evidenced by Dubitzky (Dubitzky et al. Encyclopedia of Systems Biology, DOI 10.1007/978-1-4419-9863-7, published 2013) and GenBank L29345 (NCBI GenBank Database, accession number L29345.1, GFP genetic sequence, published 12/30/1994, accessed 12/13/2024).
The teachings of May, Potter, and Yin are given above. May, Potter, and Yin do not explicitly teach splint ligation.
With regards to ligation methods known in the art, Lohman is a research article that teaches efficient methods of nucleic acid ligation (Title, Abstract, and see document). Lohman teaches the ligase PBCV-1 DNA ligase, and further teaches that PBCV-1 efficiently ligates ssRNA and ssDNA donors using complementary DNA splints (page 1832, left column, second paragraph). Lohman further teaches that PBCV-1 ligation is a particularly efficient method with high sensitivity (page 1832, left column, second paragraph). Thus, Lohman teaches that the ligation of RNA and DNA molecules using a splint is a known strategy that is highly efficient and sensitive, and therefore teaches a motivation to use such splint ligation strategies to ligate oligos together with a reasonable prediction of success (page 1832, left column, second paragraph).
Furthermore, Kershaw is a molecular biology textbook which teaches splint ligation of oligonucleotides such as RNA, where a splint that is complementary to two molecules is used to ligate two oligos together using a ligase (see pages 257-269). Kershaw therefore teaches that splint ligation is a textbook technique known in molecular biology to join two oligos together using a complementary splint. Kershaw further teaches that splint ligation methods are useful tools in molecular biology, specifically for the assembly of synthetic RNAs into longer RNAs such as those taught by May, Potter, and Yin. Given that Kershaw teaches that splint ligation is a known, textbook technique to generate fusion oligos, and also that such techniques are taught by Kershaw to be useful for generating synthetic oligos, a practitioner would be sufficiently motivated to use this widely known with a reasonable expectation of success given that it has been reduced to practice with detailed methodological steps available as taught by Kershaw (Kershaw, pages 257-269 and Abstract). Thus, given the teachings of Lohman and Kershaw, the art appears to be replete with textbook knowledge of using splint ligation to ligate oligonucleotides. Kershaw further teaches that the splint is complementary to the 3’ end of on donor molecule and the 5’ end of a second molecule to be ligated (e.g., Figure 2 on page 260).
Furthermore, given that it is obvious to generate the recited gRNA fusion molecules in light of May, Yin, and Potter (see rejection of claim 31, above), it is further obvious to use a known method to generate such a molecule using splint ligation, as taught by both Kershaw and Lohman. Lohman already teaches splint ligation of oligos, and furthermore teaches a motivation to use splint ligation techniques because using splint ligation oligos is efficient and sensitive (see above). Furthermore, splint ligation is a known technique in molecular biology, where it is known to be useful for generating synthetic oligos such as those taught by May, Yin, and Potter, as taught by Kershaw (Abstract on page 257, and pages 257-269). The art is replete with knowledge with regards to the use of splint ligation to generate oligos, as taught and evidenced by Kershaw who teaches textbook protocols for performing splint ligation. The results are therefore both predictable and obvious.
Response to Arguments
The Applicant’s arguments filed on 12/11/2025 have been considered but are not persuasive.
The Applicant argues that the prior art fails to teach each of the limitations, and also fails to identify a reason to combine the May, Yin, and Potter references with a reasonable expectation of success. This argument is not found to be persuasive. As will be discussed further below, the prior art references cited do teach all of the recited limitations an furthermore provide sufficient motivation to combine the references. The general thrust of the Applicant’s arguments appear to be piecemeal analysis, where each individual reference is attacked individually for what they teach and/or do not teach. In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
In response to applicant's argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007).
In this case, as discussed above, May teaches each of the claim elements of claim 31, with the exception that they do not teach covalently linking the gRNAs with the donor template and does not reduce the Cas9 nickase to practice (although they do teach HNH inactivation where the RuvC domain remains active, see above). However, Yin teaches that linking gRNAs to donor templates by partial annealing/non-covalent linkages is interchangeable with using covalent bonds (Yin, paragraph 83) to accomplish the same task as May (i.e., to bring donor template DNA in proximity to its target). Furthermore, Yin teaches that linking gRNA and donor molecules greatly improves CRISPR/Cas gRNA systems because the repair/donor template can be efficiently directed to its intended location (paragraph 83). The Applicant has therefore not originated the concept of linking gRNAs with donor templates because Yin already teaches this design and its advantages. Yin therefore teaches a strong motivation to link gRNAs to template/donor nucleic acids using either covalent bonds and/or non-covalent bonds as ready, interchangeable alternatives. The Applicant has therefore not invented the concept of covalently linking gRNAs with template donors.
Potter also teaches the same principle, where increasing the proximity of donor/template DNA by binding it directly to gRNA improves editing efficiency (Summary, Figures 1, 7, and 9). Thus, a practitioner is taught a known motivation to bind gRNA to donor/template DNA, because this is a known strategy to bring the donor template in close proximity to its target (Yin, Potter, May). Furthermore, Yin teaches that non-covalent and covalent linkages are readily interchangeable while Potter teaches that binding gRNA to donor templates using phosphodiester covalent bonds is a known method (e.g., Figures 1, 7, and 9). In addition, not only does Potter teach covalent binding of gRNA to donor templates using a covalent phosphodiester bond, the gRNA of Potter also contains a 3’ hairpin structure similar to May (Figures 1,7,9 Potter, Figure 30 of May). Thus, there is a reasonable expectation of success in combining May, Yin, and Potter, because these systems are all gRNA donor/template DNA systems which use the same class of molecules to accomplish the same task, where the gRNA of Potter is structurally very similar to that of May (May, Figure 30, Potter, Figures 1, 7, and 9).
Furthermore, the KSR rationale of a simple substitution of one known art element for another to obtain predictable results is applied with regards to the use of either a non-covalent or covalent bonds (see the rejection of claim 31, above). A conclusion of obviousness based on this rationale is sufficient to show obviousness per MPEP 2141. In the present case, it was already known that covalent bonds can be used to attach gRNAs with donor templates, as taught by Yin. Thus, the KSR rationale is satisfied, as the two bonds (non-covalent and covalent) are functional alternatives of one another in the context of the present invention, a taught by Yin (see 103 rejection, above). Thus, the rejection can be viewed as the simple the substitution of one known element for another with predictable results.
Furthermore, May teaches HNH inactivation as a useful strategy to make a nickase. A practitioner is therefore motivated to make such a nicakse because May teaches that the nuclease domains can be inactivated to make nickases (see above). Thus, combining Maeder with May is certainly obvious because Maeder teaches specific mutation strategies to make the nickases already taught by May. Thus, the practitioner is motivated to combine the teachings of Maeder because May has already taught a motivation to make a nickase, where Maeder simply teaches known strategies to make what is suggested by May (i.e., mutation in a nuclease domain such as HNH).
Furthermore, Potter teaches that their invention “involves enhancing homologous recombination by increasing the concentration of donor nucleic acid at or in close proximity to the junction of a break in a nucleic acid molecule resident in a cell,” (paragraph 4). Potter therefore teaches that their methods, which use gRNAs comprising 3’ hairpin loops covalently bound to template/donor DNA enhances recombination/gene editing events (paragraph 4). Thus, Potter directly teaches a motive to incorporate their teachings into similar systems, such as those taught by May and Yin, including the use of covalent linkages (paragraph 4).
Furthermore, the fact that Potter teaches positive results (“enhancing homologous recombination,” see above paragraph) would motivate a practitioner to combine the teachings of Potter with May.
Thus, all of the elements of claim 31 are known elements in analogous art, and furthermore Yin and Potter teach that linking gRNAs and donor templates increases reaction efficiency and therefore teaches motivation to combine these elements with a reasonable expectation of success (Yin paragraph 83, Potter paragraph 4). Thus, bringing gRNA and donor templates within close proximity is not inventive because Yin, May, and Potter also teach the localization of donor templates to target site by binding donor templates to gRNAs (see 103 rejection). May also teaches gRNAs with two hairpin loops, to be used to non-covalently bind to donor template DNA (Figure 30). Thus, the cited prior art, taken together, provides the limitations of the claims.
In response to applicant's arguments against the references individually, one cannot show non-obviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). In the present case, all of the recited prior art elements are known, and furthermore, as discussed in the 103 rejection and above, a practitioner would be motivated to combine the references with a reasonable expectation of success because May, Yin, and Potter all overlap in the same field of endeavor, use the same reagents in the same ways to produce the same effect of targeted gene editing, and in the case of May and Yin, Yin directly teaches that non-covalent partially bonded donor templates can be substituted by covalent bonded donor templates with the same effect (paragraph 83, and see Abstracts and Backgrounds of May, Yin, and Potter). Furthermore, both May and Potter teach gRNAs comprising 3’ hairpin loop structures bonded directed to donor templates (Figure 30, May, Figures 1, 7, and 9 of Potter). Thus, the results and teachings of May, Yin, and Potter can be combined with a reasonable expectation of success.
Furthermore, Dubitzky and GenBank are merely evidentiary references, where any argument made against what they lack in teaching is moot as they are only used to provide background information (i.e., they are evidentiary references).
The Applicant argues that Maeder does not teach the specific design and use of the claimed gRNA fusion molecules. However, Yin, Potter, and May render obvious such gRNA fusions to be used with known Cas9 molecules such as those disclosed by Maeder. The Applicant’s argument is therefore not persuasive.
Furthermore, regarding the design of cleavage/targeting depicted in the drawing of claim 31, such targeting strategies are known to be standard targeting strategies when using Cas enzymes such as nickases, where a nickase can cleave in the general region of a target, especially if paired with an additional nickase (see for example Potter, paragraph 31). Thus, such a cleavage/target design is a known strategy of using Cas nickases.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/D.C.R./Examiner, Art Unit 1635
/RAM R SHUKLA/Supervisory Patent Examiner, Art Unit 1635
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