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 .
Election/Restrictions
Applicant’s election of Group I (claims 1-16, which are directed to an engineered composition comprising a site-specific nuclease polypeptide, a DNA polymerase, and a VirD2 polypeptide) in the reply filed on 10 Dec 2025 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Claims 17-22 withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention Group II.
Accordingly, claims 1-16 are examined herein.
Priority
Acknowledgment is made of Applicant’s claim for priority based on a US Provisional Application No. 63/088,420 filed on 06 Oct 2020.
Claim Interpretation
Claims 1 and 5 recite the term “connected” between two polypeptides. The specification does not explicitly define this term. Thus, Examiner is interpreting this term to broadly to encompass any association between the two polypeptides such as, but are not limited to, covalent linkage (e.g., via a peptide or chemical crosslinker), noncovalent linkage (e.g., protein-protein interactions and affinity tags), and two polypeptides are known to associate or function together in a biological context.
Specification
The disclosure is objected to because it contains an embedded hyperlink ([0323], bottom of pg. 99) and/or other form of browser-executable code. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
The disclosure is objected to because of the following informalities:
The specification recites “…a transposon or domain thereof (e.g., an IscB or TpnA domain)...” ([0244]), should be “TnpA domain”.
Appropriate correction is required.
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.
Claims 10 and 11 are 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 10, there is insufficient antecedent basis for the recitation of “the homology sequence”. It is noted that the first recitation of this term is in claim 6. Thus, it is unclear which homology sequence is being further defined in the instant claim. Further, there is insufficient antecedent basis for the recitation of “the guide polynucleotide”. It is noted that the first recitation of this term is in claim 9. Thus, it is unclear which guide polynucleotide is being further defined in the instant claim.
Regarding claim 11, there is insufficient antecedent basis for the recitation of “the Cas polypeptide”. It is noted that the first recitation of this term is in claim 9. Thus, it is unclear which Cas polypeptide is being further defined in the instant claim.
Claim Rejections - 35 USC § 112 – Written Description
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 8 and 10 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
MPEP 2163.II.A.3(a).i) states, "Whether the specification shows that applicant was in possession of the claimed invention is not a single, simple determination, but rather is a factual determination reached by considering a number of factors. Factors to be considered in determining whether there is sufficient evidence of possession include the level of skill and knowledge in the art, partial structure, physical and/or chemical properties, functional characteristics alone or couple with a known or disclosed
correlation between structure and function, and the method of making the claimed invention".
Claim 8 is drawn to possession of an engineered composition comprising a IscB or TnpA nuclease, a DNA polymerase, and a VirD2 polypeptide. The claim required IscB or TnpA to function as nucleases and capable of forming a complex with the aforementioned two proteins to direct them to a specific target site. The specification does not provide description of the structure, sequence of functional characterization of IscB or TnpA. The only disclosure is a citation to Kapitonov et al. J. Bacterial. 2016. 198(5):797-807) ([0133]). However, a mere citation to prior art teaching the sequence similarity of IscB and TnpB proteins to Cas9, is insufficient to demonstrate possession of the claimed nucleases.
The specification lacks description on fusion protein orientations, linker sequences, structural compatibility, retention of nuclease activity, and positioning with DNA polymerase and VirD2 polypeptide. The specification does not describe the mechanism in which IscB or TnpA directs DNA polymerase and VirD2 polypeptide to a target site. The specification does not provide an actual reduction to practice any engineered composition comprising IscB or TnpA nuclease. The specification does not provide the complete structure, partial structure, physical and/or chemical properties, functional characteristics when coupled with DNA polymerase and VirD2 polypeptide.
Regarding the state of the art, Kapitonov teaches IscB and TnpA are Cas9 homologs and they share conserved catalytic and hydrophobic residues (Fig. 1). Kapitonov further teaches the subfamily of IscB proteins contain the HNH endonuclease domain inserted within the RuvC-like domain (pg. 799). However, Kapitonov does not teach any experimental examples in which IscB and TnpA function as nucleases. As of the filling date, there is no prior art disclosing any successful nuclease activity of IscB or TnpA, let alone complexing with additional proteins and retaining its function.
The dependent claims 10 and 11 lack antecedent basis; thus, do not recite additional limitations to claim 8. Based on the preponderance of the evidence, including relevant teachings of the specification the absence of working examples, and the state of the art including knowledge of IscB and TnpA’s nuclease activity, Applicant was not in possession of an engineered composition of claim 8.
Claim 10 is drawn to possession of an engineered composition of claim 8, wherein the homology sequence is complementary to at least a portion of the guide polynucleotide. However, due to lack of antecedent basis for the term “homology sequence” in claim 8, and considering the first recitation of this term appears in claim 6, claim 10 is interpreted as referring to the homology sequence in the donor construct of claim 6. The specification teaches the donor construct can have homology with a guide molecule, wherein “the homology region is located at the 3’end…or to the first region of the sgRNA guide sequence” ([0944]). Figures 3-5 are illustrative drawings of the composition comprising the claimed proteins, without description of exemplary sequences. The specification does not provide an actual reduction to practice any donor construct comprising a homology sequence complementary to at least a portion of the guide polynucleotide. The specification does not provide the complete structure, partial structure such sequence length, sequence identity, or possible secondary structures, physical and/or chemical properties, and functional characteristics when coupled with Cas9 nuclease.
Regarding the state of the art, Pickar-Oliver et al (Nat Rev Mol Cell Biol, 2019, 20, 490-507) teaches donor constructs used in CRISPR-Cas9 genome editing strategies comprised of homology sequences complementary to a portion of the target sequence, not guide polynucleotide (Fig. 2). As of the filling date, there is no prior art disclosing donor constructs used in genome editing strategies involving NHEJ or HDR-mediated repair pathways that comprised of homology sequences complementary to the guide polynucleotide.
Based on the preponderance of the evidence, including relevant teachings of the specification the absence of working examples, and the state of the art including knowledge of donor constructs employed with site-specific nuclease, Applicant was not in possession of an engineered composition of claim 10.
Claim Rejections - 35 USC § 112 – Enablement
Claims 8 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
The specification does not reasonably provide enablement for the use of a composition comprising a) a site-specific nuclease, specifically IscB or TnpA; b) a DNA polymerase; and c) a VirD2 polypeptide. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with the claims.
The test of enablement is whether one skilled in the art could make and use the claimed
invention from the disclosures in the specification coupled with information known in the art without
undue experimentation {United States v. Telectronics., 8 USPQ2d 1217 (Fed. Cir. 1988)). Whether undue
experimentation is needed is not based upon a single factor but rather is a conclusion reached by
weighing many factors. These factors were outlined in Ex pa rte Forman, 230 USPQ 546 (Bd. Pat. App. &
Inter. 1986) and again in In re Wands, 8 USPQ2d 1400 (Fed. Cir. 1988), and the most relevant factors are
indicated below:
Nature of the Invention and Breadth of Claims
With respect to claim breadth, the standard under 35 U.S.C. §112(a) entails determining what the claims recite and what claims mean as a whole. The nature of the invention is directed to a composition comprising a) a site-specific nuclease; b) a DNA polymerase; and c) a VirD2 polypeptide; wherein the site-specific nuclease is connected to the DNA polymerase and VirD2 polypeptide, and directs these two polypeptides to a target nucleic acid. Claim 8 narrows the scope of the invention by limiting the site-specific nuclease to be an IscB or TnpA polypeptide, and claim 10 narrows the scope of the invention by limiting the homology sequence is complementary to at least a portion of the guide polynucleotide.
Guidance of the Specification
The specification does not teach how to make and use any composition comprising IscB or TnpA polypeptides. Further, the specification does not enable any person skilled in the art to which it pertains (i.e. connecting these two site-specific nucleases to a DNA polymerase and a VirD2 polypeptide, wherein the site-specific nucleases direct these two polypeptides to a target polynucleotide) to make and/or use the invention commensurate in scope with the claims. There is a lack of adequate guidance from the specification or prior art with regard to the actual composition comprising a), b), and c) components.
Regarding IscB and TnpA polypeptides, the specification teaches “ the site specific nuclease may be an IscB or TnpB protein or functional domain thereof. See e.g., Kapitonov et al. J. Bacterial. 2016. 198(5):797-807. ([0133]). The specification further teaches the engineered nucleic acid modification system…comprising a transposon or domain thereof (e.g., an IscB or TpnA domain) fused with one or more VirD2 polypeptides, DNA polymerase, and donor polynucleotide, and optionally a guide RNA…and optionally a donor construct” ([0244]). The supplemental information (e.g., drawings) does not provide additional teachings of IscB and TnpA polypeptides.
These general statements do not provide any specific guidance regarding how to make and use the engineered composition.
The specification provides no experimental examples, structural descriptions, or functional characterizations of IscB and TnpA as nucleases with the required two key functions: i) capable of forming a complex with DNA polymerase and VirD2, and ii) capable of directing the DNA polymerase and VirD2 to a target sequence in a target polynucleotide.
State of the Art
At the time of filling, the state of the art relating to IscB and TnpA demonstrated that they share high sequence similarity with Cas9, but no successful example of IscB and TnpA functioning as nucleases and connected or capable of forming a complex with any DNA polymerase and VirD2 polypeptides have been reported.
Malcuit et al (GB 2467167 A; Published Date: 28 Jul 2010) teaches a fusion polypeptide comprising of a TnpA fused to a recombinase A and a Nuclear Localization Signal (NLS) of VirD2 (“tnpA-recA-virD2NLS domain fusion”, pg. 40-41). Malcuit teaches TnpA, as a transposon, binds to the left and right ends of the donor construct and transfers it into the nucleus for integration into chromosomal DNA, and TnpA directs the fused recombinase A to the chromosomal DNA for recombination induction (Abstract). However, Malcuit‘s engineered composition is materially different from the claimed invention, wherein TnpA is claimed as a site-specific nuclease that directs a DNA polymerase, rather than recombinase A, and VirD2 polypeptide, rather than its NLS to a target DNA. Most importantly, in the claimed invention, VirD2 polypeptide associates with the donor construct rather than TnpA. Thus, neither teaches the claimed functional role of TnpA nor suggests engineering TnpA capable of forming a complex with a DNA polymerase or VirD2.
Kapitonov et al (J. Bacterial. 2016. 198(5): 797-807) teaches IscB and TnpA are Cas9 homologs and they share conserved catalytic and hydrophobic residues (Fig. 1). Kapitonov further teaches the subfamily of IscB proteins contain the HNH endonuclease domain inserted within the RuvC-like domain (pg. 799). However, Kapitonov does not teach any experimental examples in which IscB and TnpA function as nucleases. Instead, Kapitonov discloses “the simple domain organization of these proteins, suggests that TnpB is the ancestral form to both IscB and Cas9. The relatively high sequence similarity between Cas9 and IscB2 and, even more important, the shared presence of two nuclease domains indicate that these families have a common ancestor” (pg. 806, left-column, third paragraph).
Applicant’s own work Strecker et al (Science, 2019, 365, 48-53) teaches a nickase Cas9 tethered to a single-stranded DNA transposase TnpA facilitated transposition of a donor construct into the genome of E. coli (pg. 1, middle-column, first paragraph; right-column, first paragraph). Strecker further teaches Cas effector protein binding to DNA generates an R-loop structure, exposing a substrate for TnpA (pg. 1, middle-column, first paragraph). Strecker also demonstrates the TnsD subunit of natural transposase Tn7, which functions to direct integrations into a unique attachment site on the chromosome of E. coli via DNA-protein interaction, is performed by a nuclease inactive Cas12K via an RNA-directed interaction (Fig. 5). In the same year, Bhatt et al (NAR, 2019, 47(15): 8126-8135) teaches a nuclease inactive dCas9 tethered to a mariner transposase Hsmar1 to facilitate transposition of a donor construct into the genome of E. coli (pg. 8130, right-column). Both Strecker and Bhatt teach that transposon recognizes specific sequences located at the left and right ends of donor construct and mediates a “cut and paste” transposition of the donor construct into target site. In this system, the integration of donor construct does not rely on double-stranded breaks or cellular repair pathway mechanism, but rather on transposase-mediated insertion. Both Strecker and Bhatt further teach their engineered fusion constructs in which Cas9 provides the nuclease activity (or inactive nuclease Cas12k taught by Strecker), while the transposase facilitates donor construct integration.
Thus, the state of the art prior to the filling date teaches nuclease activity and transposition of donor construct are distinct functions performed by different proteins.
It was not until after the effective filling date that Applicant’s own work, Altae-Tran et al (Science, 2021, 374, 57-65) disclosed IscB is an RNA-guided endonuclease, and Karvelis et al (Nature, 2021, 599, 692-696) also disclosed TnpB, which encodes TnpA, is also an RNA-guided endonuclease. However, these disclosures cannot be relied upon to establish enablement as of the filling date.
In summary, as of the filling date, there is no prior art disclosing any successful engineered composition comprising IscB or TnpA nuclease with a DNA polymerase and a VirD2 polypeptide. The state of the art did not provide guidance on making and using the claimed invention without substantial and unpredictable experimentation.
Experimentation Required
For example, it would be necessary for one of ordinary skill in the art to conduct the following experimentation in order to practice the claimed invention:
i) validate IscB and TnpA’s nuclease activity in vitro and in vivo
ii) engineering and validating functional fusion proteins including optimization on fusion orientation, linker composition, solubility, retention of nuclease activity, donor construct association, and polymerase activity.
iii) establishing a mechanism for donor construct integration as state of the art teaches transposase-mediated integration are unidirectional and located at a fixed distance to one side of the targeted DNA site. It is not known whether donor integration would be performed with the same mechanism if the transposase acts as a nuclease.
Taking into consideration the factors outlined above, including the nature of the invention, the breadth of the claims, the guidance provided by the applicant, and the state of the art, it is concluded that an unreasonable amount experimentation would be required to make and use the invention as claimed.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1-7, 9, 11-13, and 16 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Hummel et al (US 2021/0171947 A1; Filed Date: 6 Dec 2020; PRO 62/944,976 Filed Date: 6 Dec 2019; hereinafter as Hummel)
Regarding claim 1, Hummel teaches an engineered composition comprising all the components in the claimed invention: a nucleic acid binding protein (e.g., a CRISPR-Cas effector protein ), a guide nucleic acid, an Agrobacterium effector protein, a DNA-dependent DNA polymerase, and optionally a T-DNA contact a target nucleic acid to thereby modify the nucleic acid ([0102]). Hummel teaches a DNA-dependent DNA polymerase is either directly fused to the nucleic acid-binding protein or recruited via affinity tags, wherein the nucleic acid-binding protein directs the DNA-dependent DNA polymerase to be in vicinity of the target nucleic acid ([0102]). Hummel further teaches one of the exemplary Agrobacterium effector proteins is VirD2 ([0160]), and CRISPR Cas effector protein is either directly fused to Agrobacterium effector proteins ([0173]) or to an affinity polypeptide that is capable of interacting with the Agrobacterium effector protein ([0021]). Hummel also teaches a guide nucleic acid is capable of forming a complex with CRISPR-effector protein, thereby guiding the CRISPR-Cas fusion protein (Cas effector and Agrobacterium effector), and optionally T-DNA associated with Agrobacterium effector, to the target nucleic acid ([0021]).
Regarding claim 2, Hummel teaches a T-DNA (i.e., donor construct) is capable of forming a complex with the VirD2 protein which Tyr29 covalently attaches to the 5'-end of the T-DNA and nuclear localization signal transfers the T-DNA into the nucleus ([0157]). Hummel further teaches the T-DNA comprises a homology repair template that may be used for modifying a target nucleic acid [0161], and DNA-dependent DNA polymerase "may be utilized to incorporate the edit" ([0167] and [0240]).
Regarding claim 3, Hummel teaches wherein the T-DNAs are ssDNA molecules ([0155]), and contains left and right borders (i.e., binding sequences) for VirD1 and VirD2 proteins ([0157]).
Regarding claim 4, Hummel teaches that T-DNA is harbored as a double-stranded sequence encoding left and right borders (i.e., 5’ and 3’ boundary sequences) for VirD1 and VirD2 proteins to associate, nick, and release the T-DNA as ssDNA molecules, and VirD2 protein remains covalently attached at Tyr29 to the 5'-end of the T-DNA and guides the transfer of T-DNA into the nucleus ([0157]). Although Hummel does not explicitly teach a donor polynucleotide sequence located between the 5’ and the 3’ boundary sequences, such configuration is inherent in donor templates used for homology-directed repair (HDR) (see teachings discussed above in claim 2) and T-DNA natural transfer process in Agrobacterium ([0157]). As is well understood by a person ordinary skill in the art, T-DNA processed by VirD1 and VirD2 necessarily comprise the donor sequence positioned between the 5’ and 3’ borders. Thus, Hummel disclose inherently a donor sequence located between the 5’ and the 3’ boundary sequences.
Regarding claim 5, Hummel teaches the natural transfer process in Agrobacterium cells involves VirD1 and VirD2 functioning together (i.e., connected) by associating with each end of the T-DNA, nick it, and releasing it as a ssDNA molecule ([0157] and see discussion regarding the term “connected” under “Claim Interpretation” section).
Regarding claim 6, Hummel teaches wherein the T-DNA further comprises a 3' and/or 5' homology region with sequences complementarity to target nucleic acid ([0237]).
Regarding claim 7, Hummel teaches wherein the T-DNA may have a length of about 400 to 30,000 nucleotides ([0235]).
Regarding claim 9, Hummel teaches wherein the composition comprises a CRISPR-Cas effector fusion protein and a CRISPR guide nucleic acid "capable of forming a complex with the CRISPR-Cas effector protein", and which "spacer sequence is capable of hybridizing to a target nucleic acid, thereby guiding the CRISPR-Cas fusion protein to the target nucleic acid" ([0021]).
Regarding claim 11, it is noted that it recites wherein the Cas polypeptide in the composition of claim 8, which lacks antecedent basis. For compact prosecution, this instant is interpreted as referring to the composition of claim 9, which contains the first recitation for “Cas polypeptide”. Regarding claim 11, Hummel teaches wherein the CRISPR-Cas effector protein is a nickase that is configured to nick a site on the first or second strand of the target nucleic acid ([0211]).
Regarding claim 12, Hummel teaches the composition further comprises a second guide nucleic acid capable of forming a complex with another CRISPR-Cas effector protein that nicks a site on the first strand of the target nucleic acid (i.e., non-targeted strand) near a second site on the second strand that has been nicked by a different CRISPR-Cas effector protein (targeted strand) ([0213]).
Regarding claim 13, Hummel teaches wherein the VirD2 is derived from Agrobacterium tumefaceins ([0302]).
Regarding claim 16, Hummel teaches a single expression cassette encoding a CRISPR-Cas effector protein (i.e., site-specific nuclease), an Agrobacterium effector protein (i.e., VirD1 and VirD2), a DNA dependent polymerase or a 5'-3' exonuclease polypeptide, a T-DNA sequence (i.e., donor), and/or a guide nucleic acid ([0096]).
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 14 and 15 are rejected under 35 U.S.C. 102(a)(2) as anticipated by or, in the alternative, under 35 U.S.C. 103 as obvious over Hummel et al (US 2021/0171947 A1; Filed Date: 6 Dec 2020; PRO 62/944,976 Filed Date: 6 Dec 2019; hereinafter as Hummel), in view of Halperin et al (WO 2019/051097 A1; Published Date: 14 Mar 2019; hereinafter as Halperin)
Regarding claim 14 and 15, the teachings of Hummel regarding the composition of claim 1 is discussed and applied above to claim 1. Hummel further teaches wherein the DNA-dependent polymerase is a domain thereof or a 5'-3' exonuclease polypeptide or domain thereof ([0096]).
Hummel does not explicitly teach wherein the DNA-dependent polymerase is an DNA Poly I polymerase or an E. coli DNA polymerase, but DNA-dependent polymerase is a broad genus of enzymes including, not limited to, DNA polymerase I, polymerase III, and reverse transcriptase.
Halperin teaches a fusion protein of a Cas9 nickase (nCas9) and a E. coli DNA polymerase I mutant to introduce mutations during efficient homology-directed repair integration (Abstract and [00346]).
Thus, it would have been obvious to one of ordinary skill in the art before the effective filling date of the invention to have modified the DNA-dependent polymerase in the composition of Hummel with an E. coli DNA polymerase as taught by Halperin, because it would have merely amounted to a simple substitution of prior art elements according to known methods to yield predictable results. One would have been motivated to have done so for the advantage of 5’-3’ exonuclease function of E. coli DNA polymerase in addition to its nucleotide filling and proofreading capabilities during polynucleotide repair and synthesis. One would have had a reasonable expectation of success in doing so because both Hummel and Halperin teaches a composition of fusion proteins comprising CRISPR-Cas effector and DNA polymerase, and their applications in genome editing.
Conclusion
No claims are allowable.
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QIWEN SU-TOBON
Examiner
Art Unit 1636
/NEIL P HAMMELL/Supervisory Patent Examiner, Art Unit 1636