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 .
Priority
This application is a 371 of PCT/US2021/034984 filed on 05/28/2021, claims the priority under 35 U.S.C. §119(e) to U.S. provisional patent application No. 63/031,203, filed May 28, 2020.
Restriction/Election
Applicant's election with traverse of Group I, claims 1-11, drawn to a system for integrating a donor sequence into genome of a target cell, comprising: a first guide RNA (gRNA); a second gRNA; and a third gRNA, wherein the first and second gRNA flank the donor sequence and are independently capable of guiding a nuclease to a respective region of the donor sequence flanked by the first and second gRNA, and wherein the third gRNA binds a locus of interest in the genome of the target cell and is capable of guiding a nuclease thereto; and for species election, Applicant elected a/the donor sequence in a nucleic acid vector for initial examination, and claims 1-11 read on the elected species, in the reply filed on 09/02/2025 is acknowledged. Notwithstanding the foregoing election, Applicant respectfully traverses. The traversal is on the ground(s) that for clarity, the term “a donor sequence” in claim 1 is in the context of “for integrating into genome of a target cell" and it does not indicate a source from the genome. As elected, the donor sequence in claim 1 would be in a nucleic acid vector. This is not found persuasive because Applicant did not specifically traverse lack of unity regarding restriction between Group I and Group II, and clarification regarding the term “a donor sequence” in claim 1 is acknowledged.
Claims 1-25 are pending.
Claims 12-25 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to nonelected inventions, Groups II-IV, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 09/02/2025.
Claims 1-11 are currently under examination to the degree that “a donor sequence” in claim 1 is in a nucleic acid vector.
The requirement is still deemed proper and is therefore made FINAL.
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 1-11 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.
Claim 1 recites “A system for integrating a donor sequence into genome of a target cell,
comprising: a first guide RNA (gRNA); a second gRNA; and a third gRNA, wherein the first and second gRNA flank the donor sequence and are independently capable of guiding a nuclease to a respective region of the donor sequence flanked by the first and second gRNA, and wherein the third gRNA binds a locus of interest in the genome of the target cell and is capable of guiding a nuclease thereto”.
Claim 2 recites “The system of claim 1, wherein the donor sequence has a sense strand and an anti-sense strand, and the first gRNA is single-stranded guide RNA (sgRNA) and binds upstream of the donor sequence on the sense strand”.
Claim 7 recites “The system of claim 1, further comprising the donor sequence in a nucleic acid vector, wherein ---”.
Claim 1 recites limitation “for integrating a donor sequence into genome of a target cell” in preamble and the limitation “wherein the first and second gRNA flank the donor sequence and are independently capable of guiding a nuclease to a respective region of the donor sequence flanked by the first and second gRNA”. It is noted that “for integrating a donor sequence into genome of a target cell” is the intended use of claimed system. The limitation “wherein the first and second gRNA flank the donor sequence and are independently capable of guiding a nuclease to a respective region of the donor sequence flanked by the first and second gRNA” is the characteristics of claimed gRNAs. Accordingly, the relationship between “a first/second/third gRNAs” and “a/the donor sequence” is unclear regarding whether the limitation “a/the donor sequence” is part of claimed system,
Claim 2 recites “the donor sequence has a sense strand and an anti-sense strand”, which is a double stranded nucleic acid molecule, whereas “the first gRNA is single-stranded guide RNA (sgRNA)”. Claim 2 depends from independent claim 1. It is unclear whether the limitation “a second gRNA” and “a third gRNA” are “single-stranded guide RNA (sgRNA)” or “double stranded nucleic acid molecule”, especially in the context of (i) limitation “the donor sequence flanked by the first and second gRNA” recited in instant claim 1, (ii) limitation “the donor sequence in a nucleic acid vector” recited in instant claim 7. In other words, it is unclear what molecular structures are encompassed by the limitation “the first and second gRNA flank the donor sequence” recited in in claim 1 when “the first and second gRNA” are single-stranded guide RNA, and “the donor sequence” is a double stranded nucleic acid molecule. Claims 3-11 depend from claim 1.
For examination purpose, the claimed system is interpreted as a product comprising cited gRNAs, and the limitation “a/the donor sequence” is directed to intended use, and is not part of claimed system/product; whereas the plurality of “guide RNA (gRNA)” recited in claim 1 is interpretated as single-stranded guide RNA (sgRNA) that is transcribed from the sense/coding strand of its corresponding double-stranded DNA molecule.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 1-6 are rejected under 35 U.S.C. 101 because the claimed invention is directed to an abstract idea, a law of nature and a natural phenomenon without significantly more.
Step 1: The claims are directed to the statutory judicial category of a composition of matter.
Step 2A, prong one: The claims recite judicial exceptions, products of nature.
Claim 1 recites “A system for integrating a donor sequence into genome of a target cell,
comprising: a first guide RNA (gRNA); a second gRNA; and a third gRNA, wherein the first and second gRNA flank the donor sequence and are independently capable of guiding a nuclease to a respective region of the donor sequence flanked by the first and second gRNA, and wherein the third gRNA binds a locus of interest in the genome of the target cell and is capable of guiding a nuclease thereto”.
Claim 2 recited “The system of claim 1, wherein the donor sequence has a sense strand and an anti-sense strand, and the first gRNA is single-stranded guide RNA (sgRNA) and binds upstream of the donor sequence on the sense strand”.
Claim 3 recited “The system of claim 1, wherein the locus of interest in the genome of the target cell has a sense strand and an anti-sense strand, and the third gRNA is sgRNA and binds the locus of interest on the anti-sense strand in the genome of the target cell.
Claim 4 recited “The system of claim 1, wherein the donor sequence has a sense strand and an anti-sense strand, the locus of interest has a sense strand and an anti-sense strand, and wherein the first gRNA is sgRNA and binds upstream of the donor sequence on the sense strand, the third gRNA is sgRNA and binds the locus of interest on the anti-sense strand in the genome of the target cell, and the second gRNA is sgRNA and binds downstream of the donor sequence on the anti- sense strand”.
Claim 5 recited “The system of claim 2, further comprising the donor sequence, one or more nucleases, or a combination of the donor sequence and the one or more nucleases”.
Claim 6 recited “The system of claim 5, comprising the donor sequence and one or more Cas nucleases, wherein the first gRNA and a first of the Cas nucleases form a complex with the donor sequence; the second gRNA and a second of the Cas nucleases form a complex with the donor sequence; the third gRNA and a third of the Cas nucleases form a complex; or a combination of forming any two or three of the complexes”.
The claimed “gRNAs”, “donor sequence”, and “Cas nucleases” recited in claims 1-6 are directed to products of nature.
Step 2A, prong two: The judicial exceptions are not integrated into a practical
application. These judicial exceptions are not integrated into a practical application because claims 1-6 collectively as written are directed three guide RNA (gRNA) molecules with intended use for integrating a donor sequence into genome of a target cell, whereas the claimed guide RNA (gRNA) molecules are product of nature identified in bacteria as an immune system responsive to bacteriophage infection. It is noted that the limitation “wherein the first and second gRNA flank the donor sequence and are independently capable of guiding a nuclease to a respective region of the donor sequence flanked by the first and second gRNA, and the limitation “wherein the third gRNA binds a locus of interest in the genome of the target cell and is capable of guiding a nuclease thereto” recited in claim 1 are directed to a natural phenomenon without significantly more.
Step 2B: The claims do not provide an inventive concept.
MPEP 2106.05(d)):
The courts have recognized the following laboratory techniques as well-understood,
routine, conventional activity in the life science arts when they are claimed in a merely generic
manner (e.g., at a high level of generality) or as insignificant extra-solution activity:
Determining the level of a biomarker in blood by any means, Mayo, 566 U.S. at 79, 101
USPQ2d at 1968; Cleveland Clinic Foundation v. True Health Diagnostics, LLC, 859
F.3d 1352, 1362, 123 USPQ2d 1081, 1088 (Fed. Cir.2017);
Using polymerase chain reaction to amplify and detect DNA, Genetic Techs. Ltd. v.
Merial LLC, 818 F.3d 1369, 1376, 118 USPQ2d 1541, 1546 (Fed. Cir. 2016); Ariosa
Diagnostics, Inc. v. Sequenom, Inc., 788 F.3d 1371, 1377, 115 USPQ2d 1152, 1157 (Fed.
Cir. 2015);
Detecting DNA or enzymes in a sample, Sequenom, 788 F.3d at 1377-78, 115 USPQ2d at
1157); Cleveland Clinic Foundation 859 F.3d at 1362, 123 USPQ2d at 1088 (Fed. Cir.
2017);
Immunizing a patient against a disease, Classen Immunotherapies, Inc. v. Biogen IDEC,
659 F.3d 1057, 1063, 100 USPQ2d 1492, 1497 (Fed. Cir. 2011);
Analyzing DNA to provide sequence information or detect allelic variants, Genetic
Techs. Ltd., 818 F.3d at 1377, 118 USPQ2d at 1546;
Freezing and thawing cells, Rapid Litig. Mgmt. 827 F.3d at 1051, 119 USPQ2d at 1375;
Amplifying and sequencing nucleic acid sequences, University of Utah Research
Foundation v. Ambry Genetics, 774 F.3d 755, 764, 113 USPQ2d 1241, 1247 (Fed. Cir.
2014); and
Hybridizing a gene probe, Ambry Genetics, 774 F.3d at 764, 113 USPQ2d at 1247.
Regarding claims 1-6 being directed to products of nature and natural phenomenon, which are well documented in the prior arts. In this regard, for instance, Zhu et al. (2016) (Zhu et al., Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR-Cas9 library, Nat Biotechnol., 2016 Dec;34(12):1279-1286. doi: 10.1038/nbt.3715. Epub 2016 Oct 31) teaches that “The CRISPR–Cas system seen in bacteria and archaea1 has been devel-oped into a genome editing tool with wide-ranging applications. Functional screens of coding genes have been widely adopted, in which pooled libraries of single-guide RNAs (sgRNAs) that target the coding regions of genes associated with specific phenotypes can be selected using cell growth or specific markers as a readout” (See Introduction, left column, page 1279). Similarly, Jun et al. teaches that “CRISPR/Cas9 for genome editing requires delivery of a guide RNA sequence and donor DNA for targeted homologous recombination. Typically, single-stranded oligodeoxynucleotide, serving as the donor template, and a plasmid encoding guide RNA are delivered as two separate components”. (See Abstract).
Claim Rejections - 35 USC § 102
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)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1-6 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Jun et al. (2018) (Jun et a., Straightforward Delivery of Linearized Double-Stranded DNA Encoding sgRNA and Donor DNA tor the Generation of Single Nucleotide Variants Based on the CRISPR/Cas9 System, ACS Synth Biol., 2018 Jul 20;7(7):1651-1659. doi: 10.102 1/acssynbio. 7600345. Epub 2018 Jul 2.).
Regarding claims 1-6, Jun et al. teaches that “CRISPR/Cas9 for genome editing requires delivery of a guide RNA sequence and donor DNA for targeted homologous recombination. Typically, single-stranded oligodeoxynucleotide, serving as the donor template, and a plasmid encoding guide RNA are delivered as two separate components. However, in the multiplexed generation of single nucleotide variants, this two-component delivery system is limited by difficulty of delivering a matched pair of sgRNA and donor DNA to the target cell. Here, we describe a novel codelivery system called "seR-DNA" that uses a linearized double-stranded DNA consisting of donor DNA component and a component encoding sgRNA. Our sgR-DNA-based method is simple to implement because it does not require cloning steps. We also report the potential of our delivery system to generate multiplex genomic substitutions in Escherichia coli and human cells.” (See Abstract).
Jun et al. teaches that “We reasoned that development of a system that simultaneously delivers sgRNA and donor DNA would allow high-throughput generation of substitution mutations. Unlike gene knockout, which requires only a guide RNA vector to induce indels at a target sequence by nonhomologous end joining (NHEJ) in mammalian cells, substitution of a nucleotide requires an additional component (i.e., donor DNA); the substitution mutation can be generated by homologous recombination (HR) when sgRNA and donor DNA that target the same gene are co-delivered. (bridging paragraph, left to right columns, page 1651).
Jun et al. teaches that “Here, we describe the development of a matched sgRNA-donor DNA pair (sgR-DNA) by assembly of ss RNA-encoding DNA and donor DNA (Figure 1B and Supporting Figure S1) to facilitate scalable production of matched sgRNA-donor DNA conjugates by a standard polymerase chain reaction (PCR) protocol. We used our system to engineer Escherichia coli (E. coli) lacking the non-NHEJ pathway and then extended our system to mammalian cell engineering. We observed that a pool of sgR-DNAs could be used to generate
substitution mutations in both E. coli (11-plex) and human cells (10-plex). (bridging paragraph, left to right columns, page 1652).
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Figure 1. Strategy for introducing substitution mutations using sgR-DNA. (A) Efficient multiplex mismatch modification via the sgR-DNA-based genome editing. Comparison of the method based on a vector expressing sgRNA together with ssODNs as donors (left) and our sgR- DNA-based mismatch modification method (right). Spacers targeting the three different regions are shown in orange, blue, and purple, and matched donors are indicated by slashed orange, blue, and purple lines, respectively. (B) Schematic diagram of the sgR-DNA-based method.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
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 1-9 are rejected under 35 U.S.C. 103 as being unpatentable over Jun et al. (2018) (Jun et a., Straightforward Delivery of Linearized Double-Stranded DNA Encoding sgRNA and Donor DNA tor the Generation of Single Nucleotide Variants Based on the CRISPR/Cas9 System, ACS Synth Biol., 2018 Jul 20;7(7):1651-1659. doi: 10.102 1/acssynbio. 7600345. Epub 2018 Jul 2.) in view of Trinh et al. (2011) (Trinh et al. A versatile gene trap to visualize and interrogate the function of the vertebrate proteome, Genes Dev. 2011 Nov 1;25(21):2306-20. doi: 10.1101/gad.174037.111; This reference is cited as NPL #3 in the IDS filed by Applicants on 09/02/2025).
The teachings of Jun et al. (2018) have been documented above in the rejection of claims 1-6 under 35 U.S.C. 102(a)(1).
Jun et al. (2018) does not explicitly teach the limitations regarding “a splice acceptor sequence”, “a splice donor sequence” and “recombinase” recited in claim 7.
Regarding claims 7-11, Trinh et al. (2011) teaches that “We report a multifunctional gene-trapping approach, which generates full-length Citrine fusions with endogenous proteins and conditional mutants from a single integration event of the FlipTrap vector. We identified 170 FlipTrap zebrafish lines with diverse tissue-specific expression patterns and distinct subcellular localizations of fusion proteins generated by the integration of an internal citrine exon. Cre-mediated conditional mutagenesis is enabled by heterotypic lox sites that delete Citrine and "flip" in its place mCherry with a polyadenylation signal, resulting in a truncated fusion protein. Inducing recombination with Cerulean-Cre results in fusion proteins that often mislocalize, exhibit mutant phenotypes, and dramatically knock down wild-type transcript levels. FRT sites in the vector enable targeted genetic manipulation of the trapped loci in the presence of Flp recombinase. Thus, the FlipTrap captures the functional proteome, enabling the visualization of full-length fluorescent fusion proteins and interrogation of function by conditional mutagenesis and targeted genetic manipulation (See Abstract, Trinh et al. 2011).
Trinh et al. (2011) teaches in Figure 4. Cre-lox-mediated recombination of FlipTrap lead to knockdown of trap genes.
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Figure 4. Cre-lox-mediated recombination of FlipTrap lead to knockdown of trap genes.
(A) Schematic of Cre-mediated recombination. Cre recombination of the lox sites lead
to two intermediates: (1) Recombination of the loxP sites lead to flipping of the mCherry and polyA sequences into the forward orientation and citrine and the splice donor into the reverse orientation. (2) Recombination of the loxPV sites lead to flipping of the mCherry and polyA sequences into the forward orientation. Further recombination of either intermediate lead to the excision of the citrine and splice donor, resulting in a mutant gene trap allele that contains a splice acceptor, followed by a 3’ exon encoded by mCherry. Expression of the Cre-induced mutant allele leads to the production of a truncated protein fused to mCherry. (B–E) Confocal images of the otic vesicle of a Gt(hmga2-citrine)ct29a embryo injected with cre and membrane-cerulean mRNA. (F–I) Confocal images of the otic vesicle in embryos from a cross between Gt(hmga2-citrine)ct29a and Tg(bactin2:cerulean-cre)ct5000 adults. (B,D) Citrine expression after Cre-lox recombination. (C,G) Expression of mCherry upon Cre-lox recombination. (D) Membrane-cerulean counterstain. (H) Expression of Cerulean-cre localized to the nucleus Tg(bactin2:cerulean-cre)ct5000 in embryos. (E,I) Merges of B–D and F–H, respectively, demonstrate that expression of either Cre (D) or Cerulean-Cre fusion protein (I) leads to the recombination and conversion of the Citrine to mCherry fusion protein. The arrowhead points to Citrine-positive nuclei that have not undergone Cre-lox recombination in the somatic tissue of progenies from a Gt(hmga2-citrine)ct29a adult crossed to a Tg(bactin2:cerulean-cre)ct5000 adult. Bar, 20mm. (J) Quantification of relative wildtype transcripts in wild-type (blue) and homozygous Cre-induced mutant embryos as determined by RT-qPCR. Relative levels of transcripts in wild-type siblings and homozygous mutant embryos were normalized to glyceraldehyde 3-phosphate dehydrogenase (gapdh) transcripts. Transcript levels of the trap gene in wild-type siblings are expressed as 100%, while transcript levels in homozygous mutant embryos are represent as a percentage relative to wild-type siblings. Error bars represent standard deviations from triplicate RT-qPCR experiments performed on five to 10 embryos per line. The trapped gene and designated alleles are listed in the X-axis. (K) Bright-field image of wild-type sibling (top of image) and homozygous mutant embryos (bottom of image) for Gt(tpm4a-mCherry)ct31aR at 48 hpf. Homozygous mutant embryos exhibit defects in cardiac contraction and pericardial edema (arrow). See also Supplemental Figures S5 and S6.
Trinh et al. (2011) further teaches that “Gene trapping offers an alternative approach to the systematic tagging of known genes, relying instead on the random insertion of a reporter throughout the genome to create fusion proteins with a marker such as lacZ or GFP. Traditional gene-trapping vectors, with a splice acceptor site immediately upstream of a promoterless reporter construct, provide expression data by creating a fusion transcript between a splice donor of the endogenous gene and the splice acceptor of the reporter gene (Gossler et al. 1989; Skarnes et al. 1992; International Gene Trap Consortium 2004). The reporter sequence reveals the endogenous gene expression during development, while the splice fusion of the 5’ end of the truncated endogenous gene creates a defined mutation. Gene trapping has become a
powerful approach for studying developmental-regulated genes because it can provide information on both the spatiotemporal expression and the function of the gene (See bridging paragraph, pages 2306-2307).
It would have been prima facia obvious for a skilled artisan to incorporate the teachings of Trinh et al. (2011) into the teachings of Jun et al. (2018) to reach methods recited in instant claims 1-9 with reasonable expectation of success because both Trinh et al. (2011) and Jun et al. (2018) are directed to methodology of regulated gene expression involving a site-specific molecular integration event of a donor sequence into a genomic locus of interest via targeted homologous recombination.
A skilled artisan would have been motivated to incorporate the Trinh et al. (2011) into the teachings of Jun et al. (2018) because (i) Jun et al. (2018) teaches that “In summary, we have demonstrated a new delivery method, which has the potential for multiplex HR-mediated genome modification through delivery of linearized double-stranded sgRNA-encoding DNA and donor DNA (i.e., sgR-DNA). Construction of sgR-DNA is straightforward, involving the production of small linear DNA fragments by PCR, avoiding the laborious cloning step (See right column, page 1655); and (ii) Trinh et al. (2011) specifically teaches that “The FlipTrap is a versatile and multifunctional vector, enabling both the visualization of the functional proteome and the interrogation of the proteome’s function through a single integration event. To visualize the functional proteome, the FlipTrap creates fluorescent tags of full-length proteins at their endogenous loci (See Discussion, left column, page 2315).
Claims 10-11 are rejected under 35 U.S.C. 103 as being unpatentable over Jun et al. (2018) (Jun et a., Straightforward Delivery of Linearized Double-Stranded DNA Encoding sgRNA and Donor DNA tor the Generation of Single Nucleotide Variants Based on the CRISPR/Cas9 System, ACS Synth Biol., 2018 Jul 20;7(7):1651-1659. doi: 10.102 1/acssynbio. 7600345. Epub 2018 Jul 2.) in view of Trinh et al. (2011) (Trinh et al. A versatile gene trap to visualize and interrogate the function of the vertebrate proteome, Genes Dev. 2011 Nov 1;25(21):2306-20. doi: 10.1101/gad.174037.111, as applied to claims 1-9 above, and further in view of Vaschetto (2018) (Vaschetto, Modulating signaling networks by CRISPR/Cas9-mediated transposable element insertion, Curr Genet., 2018 Apr;64(2):405-412. doi: 10.1007/s00294-017-0765-9. Epub 2017 Oct 14).
The teachings of Jun et al. (2018) have been documented above in the rejection of claims 1-6 under 35 U.S.C. 102(a)(1) or 102(a)(2).
The teachings of Trinh et al. (2011) have been documented in the rejection of claims 1-9 under 35 U.S.C. 103.
Regarding “a visible marker” recited in instant claim 10 and “a fluorescent protein” recited in instant claim 11, it is noted that Trinh et al. (2011) teaches that “Gene trapping offers an alternative approach to the systematic tagging of known genes, relying instead on the random insertion of a reporter throughout the genome to create fusion proteins with a marker such as lacZ or GFP (See bridging paragraph, pages 2306-2307).
The combined teachings of Jun et al. (2018) Trinh et al. (2011) do not explicitly teach the limitations regarding “transposable elements” recited in instant claim 10.
Vaschetto (2018) teaches that “In recent past, transposable elements (TEs) were referred to as selfish genetic components only capable of copying themselves with the aim of increasing the odds of being inherited. Nonetheless, TEs have been initially proposed as positive control elements acting in synergy with the host. Nowadays, it is well known that TE movement into host genome comprises an important evolutionary mechanism capable of increasing the adaptive fitness. As insights into TE functioning are increasing day to day, the manipulation
of transposition has raised an interesting possibility of setting the host functions, although the lack of appropriate genome engineering tools has unpaved it. Fortunately, the emergence of genome editing technologies based on programmable nucleases, and especially the arrival of a multipurpose RNA-guided Cas9 endonuclease system, has made it possible to reconsider this challenge. For such purpose, a particular type of transposons referred to as miniature inverted repeat transposable elements (MITEs) has shown a series of interesting characteristics for designing functional drivers. Here, recent insights into MITE elements and versatile RNA-guided CRISPR/Cas9 genome engineering system are given to understand how to deploy the potential of TEs for control of the host transcriptional activity” (See Abstract, Vaschetto 2018).
It would have been prima facia obvious for a skilled artisan to incorporate the teachings of Vaschetto (2018) into the combined teachings of Jun et al. (2018) and Trinh et al. (2011) to reach methods recited in instant claims 10-11 with reasonable expectation of success because
Vaschetto (2018) specifically teaches “Transposable elements and the CRISPR/Cas9
genome engineering system: control of the host genome” (See left column, page 407) whereas June (2018) teaches CRISPR/Cas9 for genome editing requires delivery of a guide RNA sequence and donor DNA for targeted homologous recombination (See Abstract).
A skilled artisan would have been motivated to incorporate the Trinh et al. (2011) into the teachings of Jun et al. (2018) because (i) Jun et al. (2018) teaches that “In summary, we have demonstrated a new delivery method, which has the potential for multiplex HR-mediated genome modification through delivery of linearized double-stranded sgRNA-encoding DNA and donor DNA (i.e., sgR-DNA). Construction of sgR-DNA is straightforward, involving the production of small linear DNA fragments by PCR, avoiding the laborious cloning step (See right column, page 1655); (ii) Trinh et al. (2011) specifically teaches that “The FlipTrap is a versatile and multifunctional vector, enabling both the visualization of the functional proteome and the interrogation of the proteome’s function through a single integration event. To visualize the functional proteome, the FlipTrap creates fluorescent tags of full-length proteins at their endogenous loci (See Discussion, left column, page 2351); and (iii) Vaschetto (2018) concludes that “Moreover, nowadays, we are able to engineer CRISPR/Cas9-targeted loci to efficiently achieve site-specific TE integration, thereby allowing regulation of target genes by exploiting intrinsic TE capabilities for controlling transcription” (See Concluding remarks, right column, page 410).
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Wu-Cheng Winston Shen whose telephone number is (571)272-3157. The examiner can normally be reached Mon.-Fri. 8:00 AM-5:00 PM.
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/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
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