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 3/30/2026. Claims 1-7 and 9-13 are pending. Claims 1 and 4-7 have been amended. Claim 8 has been cancelled. All pending claims are currently under examination.
Any rejection or objection not reiterated herein has been overcome by amendment. 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 § 112 – Maintained
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.
Claim 7 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.
Regarding claim 7, claim 7 recites “more specific gRNA binding sites” in relation to either Site 1 or Site 2 integration sites. It is unclear what is meant by “more specific” in the context of this claim. Although claim 7 has been amended to compare two sites (“more specific gRNA binding sites than the other integration site”) it is still unclear as to what the measure of specificity means in claim 7. It is unclear how either Site 1 or Site 2 would be “more specific” because it is unclear to as to what the site’s “specificity” is being measured against.
Response to Arguments
The Applicant’s arguments filed 3/30/2026 have been considered but are not persuasive. The Applicant argues that their amendments are sufficient to obviate the 112(b) rejection. This argument is not persuasive because the amendments still render claim 7 unclear for the reasons set forth above.
112(d) – New Rejection Necessitated by Amendment
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.
Claim 4 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.
Regarding claim 4, claim 4 depends from claim 1. Claim 1 recites that step b of the method requires culturing the cells in the presence of both the first and second antibiotic selection reagents. Claim 4 recites that step b is directed to culturing the cells of claim 1 in presence of one of the antibiotic reagents and then the other. Thus, in claim 4, the cells are not cultured in the “presence of” both of the antibiotic reagents. Claim 4 thus does not include the limitations of claim 1, which requires the presence of both antibiotic reagents during culturing.
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.
Claim Rejections - 35 USC § 103 – New Rejection Necessitated by Amendment
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 1-7, 9, and 11-13 are rejected under 35 U.S.C. 103 as being unpatentable over Sharma (Sharma A et al. Curr Protoc Hum Genet. 2018 Jan 24;96:21.11.1-21.11.20) in view of Kelley (Kelley et al. Cytotherapy Volume 17, Issue 6, Supplement, June 2015, page S53) and Brown (Brown A et al. ACS Synth Biol. 2016 Jul 15;5(7):582-8).
Regarding claim 1, Sharma is a research article that teaches Cas9-mediated tagging of endogenous target proteins in human cells (Title, Abstract, and see document). Sharma teaches a method for producing cells expressing a fusion protein of a marker protein and a cell endogenous target protein from the endogenous locus of the target protein (Abstract). Furthermore, Sharma teaches specific protocols for producing cells expressing a fusion protein of a marker protein and a cell endogenous target protein from the endogenous locus (see sections entitled “Basic Protocol 1-3” from pages 4-16 and “Critical Parameters and Troubleshooting,” pages 17-20). Sharma therefore teaches a general protocol and approach to creating cells with fusion markers attached to endogenous sites and expressed from endogenous loci (pages 4-16 and 17-20). Sharma teaches transfection of a Cas9 plasmid comprising a nucleic acid which confers resistance to a first selection marker (e.g., page 5 first paragraph and first line of “Materials” on page 5 which lists a puromycin resistant Cas9 plasmid and see Figure 1). Sharma teaches the transfection of a circular donor plasmid encoding a marker protein (e.g., see Figure 2). Furthermore, Sharma teaches that the template/donor plasmids comprise a resistance marker (see pages 7-8, sections 9-13). Sharma teaches that the marker protein is flanked 3’ and 5’ by nucleic acids homologous to the integration site in the cell where one of the flanking homologous nucleic acids is homologous to the terminal coding sequence of the target protein (see Figure 2B). Furthermore, Sharma also teaches that tags/markers can be at either the N- or C- terminus of a given protein target, which would include the terminal coding sequence of a target protein (page 3, second paragraph). Sharma teaches transfecting a guide RNA plasmid, which would contain crRNA and tracrRNA (e.g., Figure 2B). Sharma therefore teaches transfecting three plasmids, where the plasmids contain either the Cas enzyme, the donor template, or the guide RNA. Sharma teaches culturing cells in the presence of antibiotic resistance markers in order to select for cells with the correct plasmids (e.g., page 12, steps 2-3). Sharma teaches selecting cells that have survived the selection process which comprise fusion proteins (pages 14-15). Sharma teaches that the cells are mammalian (e.g., human hiPSCs, page 8, fifth paragraph).
Sharma teaches culturing cells in the presence of only one of the selection reagents, but not both, as the donor template is an E. coli resistance gene (for example, puromycin resistance on the Cas plasmid, see Step 2 of page 12).
Sharma, while teaching transfecting gRNA plasmids (i.e., single guide RNA comprising crRNA and tracrRNA in a plasmid) does not teach transfecting individual crRNA and tracrRNA molecules i.e., dual-guide RNAs (Sharma, Figure 2).
Kelley is a research abstract and poster presentation from the 21st International Society for Cell and Gene Therapy (ISCT) meeting (Kelley, pages 1-2). Kelley teaches that Cas9 plasmids can be co-transfected with crRNA and tracrRNA, and that such co-transfection of synthetic crRNA/tracrRNA with a Cas9 nuclease allows for fast, high efficiency gene editing without the need for cloning (see sections entitled “Co-transfection of Cas9 plasmid and synthetic crRNA:tracrRNA” and “Conclusions”). Kelley therefore teaches a direct motivation to transfect individual crRNA and tracrRNA molecules. Furthermore, Kelley reduced this method to practice in combination with a Cas expressed from a plasmid; the results are therefore predictable (see section entitled “Co-transfection of Cas9 plasmid and synthetic crRNA:tracrRNA,” page 2 Poster of Kelley). Furthermore, Kelley teaches the use of Cas9 plasmids with antibiotic resistance markers and targeted gene editing and therefore directly overlaps in the field of endeavor and subject matter of Sharma (Title, Abstract, and Conclusions of Kelley).
Furthermore, Brown is a research article that focuses on genome editing using nucleases such as Cas9 (Title, Abstract, and throughout). Brown and Sharma therefore overlap in subject matter and field of endeavor because they are directed to the same reagents and general methods (i.e., editing genomes using Cas9 vectors/nucleases, and plasmids). Furthermore, Brown also teaches that Cas9 nuclease vectors and HDR donor template vectors can be used for endogenous gene tagging ((page 582, right column, first paragraph); the methods and teachings of Brown are therefore directly applicable to Sharma who teaches endogenous tagging (throughout). Brown teaches that donor vectors can be introduced with antibiotic resistant markers which streamline screening (page 582, left column, third paragraph)
Brown teaches:
“By simply cotransfecting Cas9, a sgRNA targeting the CTTN locus and a universal sgRNA targeting two separate transfer vectors that encode puromycin or hygromycin resistance expression cassettes we successfully integrated one vector into each allele of the CTTN gene (Figure 3A). Simultaneous selection with hygromycin and puromycin ensured that most clonal populations generated contained biallelic modifications,” (page 58, right column, first paragraph).
Brown therefore teaches that, when applying Cas nuclease applications using multiple vectors, it is advantageous to use methods such as dual selection, where the plasmids/vectors contain separate antibiotic resistance genes, so that the selection process ensures that the desired components are present in the cell (page 58, right column, first paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of generating marker fusions taught by Sharma to include separate crRNA/tracrRNA molecules as taught by Kelley because such a combination is the simple substitution of one known element for another to arrive at predictable results. In the present case, the invention is the simple substitution of the sgRNA plasmid delivery of crRNA/tracrRNA to the cell, as taught by Sharma, for the direct co-transfection of a dual-guide crRNA/tracrRNA as taught by Kelley. Furthermore, the combination is not simply a combination of known elements: a practitioner would be motivated to adopt the teachings of Kelley to Sharma because Kelley teaches that co-transfection of crRNA/tracrRNA allows for fast and highly efficient gene editing without the need for cloning (Kelley, Conclusions). Furthermore, the results are predictable because both Sharma and Kelley are using similar reagents for the same goal of gene editing and Kelley has already reduced to practice and demonstrated that crRNA and tracrRNA can be co-transfected with a plasmid encoding a Cas enzyme.
Regarding the limitation that the cells are cultured in the presence of both selection markers, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the antibiotic selection process taught by Sharma to include the dual selection process taught by Brown, as such as combination is the simple application of a known technique to a similar product or method in the same way. In the present case, the use of dual selection for multiple plasmids in Cas9/donor methods is already known as taught by Brown. Furthermore, it is not simply a combination of elements: the practitioner would be motivated to combine the teachings or Brown because Brown teaches that dual selection using two antibiotic selection reagents is simple, and offers the advantage that it “ensures” that the desired components of the plasmids are present. Furthermore, the combination is predictable because both Sharma and Brown are drawn to the same reagent types, where furthermore both Sharma and Brown teach donor templates and endogenous tagging. Thus, the combination can further be viewed as the simple substitution of one known prior art element for another with predictable success.
Regarding claim 2, Sharma does not teach that the elements are transfected at the same time.
Kelley teaches that Cas9 plasmids and crRNA/tracrRNA are transfected simultaneously (“cotransfected”) and further that such a method allows for fast gene editing (sections entitled “Co-transfection of Cas9 plasmid and synthetic crRNA:tracrRNA” and “Conclusions”). Brown also teaches cotransfection of reagents, where such a method is simple (page 584, right column, first paragraph).
It would have been obvious to a person of ordinary skill in the art before the time of the effective filing date of the claimed invention to modify the nucleic acid delivery method of Sharma to deliver the components simultaneously, as taught by Kelley, because such a combination is the simple combination of known prior art elements to yield predictable results. In the present case, Kelley has already taught the simultaneous delivery of Cas9 plasmids with crRNA and tracrRNA nucleic acids (“Co-transfection of Cas9 plasmid and synthetic crRNA:tracrRNA” and “Conclusions”); furthermore, a practitioner would be motivated to deliver the nucleic acids simultaneously because Kelley teaches that this is a fast and highly efficient method (“Conclusions”). The practitioner is further motivated by Brown, who similarly teaches cotrransfection of reagent elements, where such methods are simple.
Regarding claim 3, Sharma teaches selecting cells that have been selected based on resistance marker selection, and further that cells are selected based on the presence of fusion proteins (Sharma, protocol from page 12, “Perform puromycin selection and monoclonal expansion of HDR-edited hiPSCs” to page 16, step “e”).
Regarding claim 4, Sharma teaches culturing and selecting cells that perform cell division in the presence of only a first selection marker (page 12, “Perform puromycin selection and monoclonal expansion of HDR-edited hiPSCs”). Sharma teaches selecting cells that perform cell division where the fusion protein has been detected, and providing cells that express the fusion protein or a marker protein and a cell endogenous target protein from an endogenous gene locus of the target protein (Sharma, protocol from page 12, “Perform puromycin selection and monoclonal expansion of HDR-edited hiPSCs” to page 16, step “e”).
Sharma, does not explicitly teach that the cells are cultured sequentially in the presence of the first and then second antibiotic resistance genes.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of generating marker fusions taught by Sharma to include the use of dual selection performed in succession. A practitioner could immediately envision using dual selection of antibiotic resistant plasmids in succession, simply to ensure that the each of the plasmids are present within a desired cell before the introduction of an additional plasmid, where such methods could easily be achieved and envisioned in a two-plasmid selection system such as those taught by Brown.
Regarding claim 5, Brown teaches that the first and second selection markers are hygromycin B and puromycin (page 584, right column, first paragraph).
Regarding claim 6, Brown teaches that the concentration of puromycin was 0.5 ug/ml and the concentration of hygromycin was 100 ug/ml (page 586, left column, second paragraph). Sharma teaches that the concentration of puromycin can be a variable range from 0.5-1 ug/mL.
Sharma/Brown do not teach that the puromycin concentration is 2 ug/mL, or that the hygromycin B concentration is 50 ug/mL.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the concentration of selection marker present during culturing conditions, as such concentrations could be arrived at through routine laboratory optimization procedures (see MPEP 2144.05, section II). MPEP 2144.05 section II states that, in general, differences in concentration will not support the patentability of subject matter encompassed by prior art, particularly in light of the fact that the recited concentrations are within the same general ranges and orders of magnitude as those known in the art, as taught by Sharma and Brown.
Regarding claim 7, the broadest reasonable interpretation of claim 7 is that the marker protein is integrated into a site in genomic DNA, where the site can be at or near the start codon of a first intron-exon junction. Sharma teaches that the insertion of the marker gene can occur at the N-terminus, which reasonably includes tagging of the protein at or near the start codon ATG at the first intron-exon junction (page 3 second paragraph).
Regarding claim 9, Sharma teaches that the marker protein is inserted N-terminally to the target protein (Figure 2, which depicts marker eGFP inserted N-terminally to target gene sequence). Sharma teaches that the marker protein is inserted in the endogenous gene locus so that it is directly before the first codon of the target protein in the mRNA of the fusion protein (Figure 2B and 2C). Note that although Figures 2B and 2C depict a linker, the caption underneath Figure 2 states that “we recommend a glycine-serine linker.” The linker is therefore inherently optional, and a practitioner could envision an embodiment without the linker, as the linker taught by Sharma is taught as a recommendation but not a requirement (Figure 2 with caption beneath Figure 2). Thus, Sharma teaches that the marker can be directly before the first codon of the target protein in the mRNA fusion protein (Figures 2B-2C and Figure 2 caption). Sharma teaches that the 3’ flanking region, which would include the “target gene coding sequence (N-terminus)” as depicted in Figures 2B and 2C, includes the start codon of the nucleic acid encoding the target protein (see “M” marked in Figure 2B).
Regarding claims 11-12, Sharma teaches that the marker protein is GFP (Figures 2B-2C).
Regarding claim 13, Sharma teaches that the homologous flanking regions should be at least 500 bp in length, and can also be lengthened further (page 18, second paragraph). Furthermore, Sharma teaches that they and others have observed that longer homology arms (greater than 500 bp) tend to increase the efficiency of HDR, and can thus enhance the integration rate of fluorescent cassettes into cellular genomes (page 3, final paragraph). Sharma therefore teaches that their homology arms are at least 500 bp, are adjustable in length, and furthermore teaches a direct motivational teaching to increase the length of homology arms because of increased efficiency.
Sharma does not directly teach that the lengths of the homology arms are “about 1000 bp.”
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the lengths of the homology arms taught by Sharma, which are 500 bp or higher, to be “about 1000” base pairs in length because Sharma also teaches that increasing the length of homology arms is known to improve the efficiency of cassette integration. Furthermore, it could also be argued that Sharma does teach homology arms which are “about 1000” base pairs in length since Sharma teaches homology arms that are “greater than 500 bp” (page 3, final paragraph). The specification does not offer a clear definition of “about” in the context of the claim, and a length of “greater than 500 bp” as taught by Sharma could reasonably be interpreted to be “about 1000 bp,” particularly in light of Sharma’s teaching that increasing the length improves HDR efficiency; thus, a practitioner could easily envision homology arms greater than 500 bp, such as 800 bp (i.e., “about” 1000 bp).
Claims 10 and 13 are rejected under 35 U.S.C. 103 as being unpatentable over Sharma (Sharma A et al. Curr Protoc Hum Genet. 2018 Jan 24;96:21.11.1-21.11.20) in view of Kelley (Kelley et al. Cytotherapy Volume 17, Issue 6, Supplement, June 2015, page S53) and Brown (Brown A et al. ACS Synth Biol. 2016 Jul 15;5(7):582-8), as applied to claims 1-7, 9, and 11-13, above, and further in view of Roberts (Roberts B et al. Mol Biol Cell. 2017 Oct 15;28(21):2854-2874).
A discussion of the combination of Sharma and Kelley and Brown is given above and incorporated here. Sharma teaches the integration of marker proteins to tag human iPSCs target genes using Cas9/gRNA systems (e.g., Figure 2, Title, Abstract, and see document).
Sharma does not teach that the marker protein does not contain a start codon.
Roberts is a research article that teaches gene tagging of endogenous targets using Cas9 in human iPSCs (Title, Abstract, and see document). Thus, Roberts and Sharma are in the exact same field of endeavor, use similar methods, and are even targeting the same cell types. Furthermore, Roberts also teaches donor plasmid constructs comprising 3’ and 5’ homology arms, a GFP marker protein, a portion of the target sequence, and crRNA plasmids (see Figure 1). Furthermore, Roberts teaches that the GFP marker protein is “inserted after the endogenous start codon (ATG) in frame with the first exon,” (caption beneath Figure 1). Roberts therefore teaches that the marker protein does is not required to comprise a start codon and can inserted after the endogenous start codon (Figure 1).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the marker protein taught by Sharma to not include a start codon, as taught by Roberts, because such a combination is the simple substitution of a known prior art element for another to obtain predictable results. In the present case, a practitioner would substitute the marker protein design taught by Sharma for that of Roberts and not include a start codon as taught by Roberts, as such a construct is already known to exist as a design strategy depending on where the practitioner would want to insert the marker. Furthermore, Sharma and Roberts teach very similar designs in the exact same field of endeavor, with the same goal of tagging target genes with fluorescent markers using Cas/gRNA systems. Thus, the results of the combination of the prior art documents are predictable. Given the teachings of Sharma and Roberts, the field appears to be replete with knowledge of inserting fluorescent markers into target proteins to generate fusions, and the design of such constructs is obvious and predictable given the overlapping subject matter of the prior art.
Regarding claim 13, Roberts teaches that the homology arms are 1000 bp (see Figure 1A, where homology arms “HA” are taught to be “1 kb”).
Response to Arguments
The Applicant’s arguments filed 3/30/2026 have been considered but are not persuasive to place the claims in condition for allowance. The Applicant argues that Sharma, while teaching the Cas plasmid with puromycin resistance, failed to teach the instantly recited claim limitations because the second antibiotic resistance marker taught by Sharma is the E. coli kanamycin resistance gene, which is inoperable in the presently recited mammalian cell, dual antibiotic selection method. This argument is found to be persuasive. However, the Applicant’s amendments to the independent claims, which are now drawn to mammalian cells which are cultured in the presence of two antibiotic resistant agents, has prompted a new search. Presently, the claims are not patentable over Sharma, Kelley, and Brown for the reasons discussed above. Briefly, the general endogenous tagging method recited in claim 1 is known as taught by Sharma, where the inclusion of dual selection for Cas-associated plasmids is a known technique as taught by Brown, where Brown teaches advantages to using dual selection to ensure the presence of the required components of the system. As reiterated in the above 103 rejection, Kelly teaches the cotransfection of crRNA and tracrRNA, as well as advantages to such a methodological approach. As such, the present claims are not patentable over Sharma, Kelley, and Brown.
The Applicant argues that they have demonstrated surprising results by showing that using dual selection prompted a 10-fold increase in integration efficiency. This argument is not persuasive. As an initial matter, Brown teaches dual selection using Cas and Cas-associated plasmids, where the use of dual editing efficiencies is simple and ensures that the desired components of a system are present (page 584, right column, first paragraph). Thus, even if it were argued that the results are unexpected, which the office does not concede, the discovery would be the result of following suggestions already present in the prior art. The fact that the inventor alleges that they recognized another advantage which would flow naturally from following the suggestion of the prior art cannot be the basis for patentability when the differences would otherwise be obvious. See Ex parte Obiaya, 227 USPQ 58, 60 (Bd. Pat. App. & Inter. 1985).
Furthermore, the Applicant’s assertions are not supported by the specification. The Applicant points to Tables 1 and 4 to show that using dual selection improved integration efficiency by 10-fold. A closer examination of the data shows that this assertion does not accurately represent the data or the implications of the data in the specification. For instance, Tables 1 and 4 are reproduced, below:
Table 1 (Singe selection)
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233
699
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Greyscale
Table 4 (dual selection)
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241
815
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Greyscale
The Applicant argues that the dual selection system has a 10-fold increase in efficiency, based on the 24.3% efficiency in Table 4 compared with the 2.3% efficiency in Table 1. However, this is an improper conclusion drawn from the two tables.
As an initial matter, Table 1 comprises data from Gate P3, which are weaker FITC positive cells (see row 1, “Plates from gate P3” in Table 1 and Figure 8). The “gate P4” or high FITC cells are eGFP positive cells, which are the cells which would be expected to have the integration of eGFP into the genomic locus (see specification, page 14, first paragraph). Table 4 shows data that is only focused on the eGFP positive cells, and does not include “Gate P3” cells. Hence, the data in Table 4 are conditioned to the specific population of cells which would be expected to have integration of eGFP.
Furthermore, Table 4 includes crRNA3, which appears to be highly efficient crRNA (i.e., has the highest overall efficiency). crRNA3 was not tested or reported in Table 1. The lack of inclusion of the highest efficiency crRNA3 from Table 1 could therefore lower the efficiency reported in Table 1. The contributions which crRNA3 makes to the efficiency calculation in Table 4 therefore must be removed from the calculation when comparing Tables 1 and 4, as crRNA3, the crRNA with highest efficiency, was not tested in Table 1.
The relevant data to compare from Tables 1 and 4 are therefore the following: crRNA1 and crRNA 2, taken from gate P4 (and excluding P3, as Table 4 does not report P3), where furthermore the parameters used in Table 1 are circular donor plasmids (see specification, bottom of page 13). With these considerations in mind, with respect crRNA1, gate P4, Table 1 shows 11/68, or 16% efficiency, where Table 4 shows 40/75 = 53% efficiency. However, with respect to crRNA2, gate P4, Table 1 shows 9/64 = 14% efficiency, while Table 4 shows 1/98 = less than 1% efficiency. The overall relevant efficiency then which should be compared for the two conditions (single selection versus dual selection) would be (14+16)/2 = 15% for single selection and (53+1)/2 = 27% efficiency, or about 1.8 times the efficiency for dual selection. However, even this data is not a surprising result, as it is derived from only two crRNAs that were tested. Furthermore, with respect to crRNA2, the single selection method appears to be at least 14-fold higher for integration efficiency than its dual selection counterpart (compare gate P4, crRNA2 9/64 = 14% in Table 1 compared to circular donor DNA/crRNA2, 1/98 = ~1% in Table 4). Thus, contrary to the Applicant’s assertions regarding the data in Tables 1 and 4, the dual selection is not “surprisingly” 10-fold higher than the single selection method. Instead, the only data which can be properly compared between the two tables show two crRNAs, 1 and 2, which function in widely varying efficiency percentages based upon which selection method is used (single or dual), where for instance crNRA2 is significantly more efficient in the single selection method compared with negligible efficiency using dual selection. The data therefore do not support the Applicant’s assertions regarding the surprising enhancement in integration efficiency when using dual selection, as such a trend can not be derived from the relevant data to compare between Tables 1 and 4.
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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