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 .
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 07-03-2025 has been entered.
Applicant's amendments to the claims filed on 06-03-2025 have been received and entered. Claims 3, 5, 7, 10-11, 13-14, 18, 20-21, 22-24, 26-49, 51, 53-56 have been canceled. Claim 57-62 have been added. Claims 1-2, 4, 6, 8-9, 12, 15-17, 19, 25, 50, 52, 57-62 are pending.
Election/Restrictions
Applicant’s election without traverse of Group I (claims 1-2, 4, 6, 8-9, 12, 15-17, 19, 22, and 25) in the reply filed on 08-09-2024 is acknowledged. In view of applicant amendments and upon further consideration, the restriction requirement is hereby withdrawn and all the withdrawn claims are hereby rejoined with the elected invention.
Claims 1-2, 4, 6, 8-9, 12, 15-17, 19, 25, 50, 52, 57-62 are under consideration.
Priority
This application is a 371 of PCT/US2020/015680 filed on 01/29/2020 which claims priority from US provisional application no. 62/798,078 filed on 01/29/2019.
New-Claim Rejections - 35 USC § 112 (b)
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 1-2, 4, 6, 9, 12, 16-17, 19, 25, 50, 52, 57-62 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.
The base claim 1 is directed to “A nucleic acid composition”; however, claim 1 requires “wherein the first nucleic acid molecule and the second nucleic acid molecule are stably integrated into the genome of a transgenic organism”. With the claim currently written, the claim requires a transgenic organism; however, the specification of the claimed invention does not define the term “composition” and/or the phrase “A nucleic acid composition”. It is unclear how “a nucleic acid composition” can be equivalent to “a transgenic organism”, or it is unclear how “A nucleic acid composition” can comprise “a transgenic organism” without being the transgenic organism itself. It is unclear if the nucleic acid composition is itself other than transgenic animal itself. It is unclear if the first and the second nucleic acid are (possibly being within one or different vectors) necessarily integrated in the same spot or different spots of the transgenic organism (different cell types, different organs, or even different chromosomes in the same cell). If the first and the second nucleic acid are integrated in different spots such as different cells, it is unclear how they work together. Thus, it is unclear exactly what embodiments contemplated by the disclosure that comprise the first and second nucleic acid molecules of claim 1 necessarily constitute a ‘nucleic acid composition’ and which embodiments are excluded by the claim as written.
Claims 2, 4, 6, 9, 12, 16-17, 19, 25, 50, 52, 57-62 are included in the rejection because they directly or indirectly depend from the rejected base claim. Appropriate correction is required.
Maintained and New-Claim Rejections - 35 USC § 103- necessitated by amendments
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1-2, 4, 6, 9, 12, 16-17, 19, 25, 50, 52, 57-62 were rejected under 35 U.S.C. 103 as being unpatentable over Cheng et al (Pub. No.: US 2019/0002889 A1, Pub. Date: Jan. 3, 2019) in view of Zhang et al (WO 2019/005884 A1, International Publication Date: 03 January 2019)
Claim interpretation:
The specification of the claimed invention teaches that the spacers can be arranged in tandem and interspersed by direct repeats. For example, a spacer can be positioned between two direct repeats. The guide RNA can include, e.g., as part of its sequence, [direct repeat 1 - spacer 1 - direct repeat 2 - spacer 2 - direct repeat 3 - spacer 3 - direct repeat 4 - spacer 4 - direct repeat 5]. In some instances, the guide RNA includes n spacers and n+1 direct repeats, where n ≥1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) (last para. of page 13-14). Therefore, the position of spacers and direct repeat is interpreted as being arranged in tandem and interspersed by direct repeats such as "repeat-spacer-repeat" sequences.
The specification of the claimed invention teaches a conserved 5'-AAAAC motif (Page 18, lines 28-29), For the sake of compact prosecution, “5' end” is interpreted as 5' end of AAAAC motif.
Regarding to claims 1 and 50, Cheng et al teach engineered, non-naturally occurring (CRISPR)-associated (Cas) systems that include ([0025], Page 2):
a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a direct repeat sequence and a spacer sequence capable of hybridizing to a target nucleic acid (a first nucleic acid molecule) ([0025], Page 2).
a nucleic acid encoding the effector protein, wherein the effector protein is capable of binding to and of targeting the target nucleic acid sequence complementary to the RNA guide spacer sequence (a second nucleic acid molecule) ([0025], Page 2).
In some embodiments, the Type VI-D CRISPR-Cas effector protein is Rsp Cas13d ([0019], page 2). Cheng et al also teaches FIG. 2A depicts a schematic tree comparing the different type VI subtype locus structures ([0114], page 8). Cheng et al stated that “in tandem with the effector gene synthesis, we first computationally designed an oligonucleotide library synthesis (OLS) pool containing "repeat-spacer-repeat" sequences, where "repeat" represents the consensus direct repeat sequence found in the CRISPR array associated with the effector, and "spacer" represents sequences tiling the pACYC184 plasmid” ([0338], page 40).
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Cheng et al teach plasmid compositions: the delivery is via plasmids. The dosage can be a sufficient number of plasmids to elicit a response ([0296], page 26). The eukaryotic expression vector can be a lentiviral plasmid backbone, adeno-associated viral (AAV) plasmid backbone. Notably, the small size of Type VI-D CRISPR Cas effector proteins, e.g., Cas13d effector proteins, make them ideally suited for packaging along with its crRNA and appropriate control sequences into a single adeno-associated virus particle; the packaging size limit of 4.7 kb for AAV may preclude the use of larger Cas13 effectors [0393], page 51 and example 7).
Cheng et al teach a cell comprising the system that is a eukaryotic cell or a prokaryotic cell, and an animal model or a plant model comprising the cell (see claims 119-120 and 125, page 188) (For the claimed: A transgenic organism of claim 50).
Although Cheng et al teach the use of lentiviral plasmid backbone, adeno-associated viral (AAV) plasmid backbone that have ability to integrate into the genome of target cells, Cheng et al do not specifically teach wherein the first nucleic acid molecule and the second nucleic acid molecule are stably integrated into the genome of a transgenic organism. However, Zhang et al cure the deficiency.
Zhang et al provide a eukaryotic host cell comprising:
(a) a first regulatory element operably linked to a direct repeat sequence and one or more insertion sites for inserting one or more guide RNA sequences up- or downstream (whichever applicable) of the direct repeat sequence, wherein when expressed, the guide sequence(s) direct(s) sequence specific binding of the Cas13 CRISPR complex to the respective target sequence(s) in a eukaryotic cell, wherein the Cas13 CRISPR complex comprises a Cas13 enzyme complexed with the one or more guide sequence(s) that is hybridized to the respective target sequence(s) ([0466], page 113);
(b) a second regulatory element operably linked to an enzyme-coding sequence encoding said Casl3 enzyme comprising preferably at least one nuclear localization sequence and/or NES ([0466], page 113).
In some embodiments, the host cell comprises components (a) and (b). Where applicable, a tracr sequence may also be provided. In some embodiments, component (a), component (b), or components (a) and (b) are stably integrated into a genome of the host eukaryotic cell ([0466], page 113).
Therefore, it would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the rejected claims to combine the teachings of prior art to modify the method of Cheng et al by stably integrating guide RNA sequences with direct repeat sequence and enzyme-coding sequence encoding Cas13 enzyme into a genome of the host eukaryotic cell as taught by Zhang et al as instantly claimed, with a reasonable expectation of success. Said modification amounting to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated to do so because Zhang et al teach the host cell with CRISPR Cas13 components stably integrated into a genome of the host eukaryotic cell would result in the base change that does not rely on endogenous repair machinery and is permanent for as long as the RNA molecule exists in the cell ([0110], page 19-20). One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Zhang et al were successful in RNA base editing with the use of Cas13 enzyme with detailed instructions and working examples.
Regarding to claims 2 and 52, Cheng et al teach that in some embodiments, the effector protein is RspCas13d (SEQ ID NO: 2) or EsCas13d (SEQ ID NO: 1) ([0043], page 4).
Regarding to claim 4, Cheng et al teach that the CRISPR-associated proteins include at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) Nuclear Localization Signal (NLS) attached to the N-terminal or C-terminal of the protein ([0205], page 15).
Regarding to claim 6, Cheng et al teach the Cas13d ortholog and NES combination that yielded the highest endogenous mammalian RNA knockdown activity…. ([0399], page 51).
Regarding to claim 9, Cheng et al teach that in some embodiments, the spacer length is from about 15 to about 42 nucleotides ([0226], Page 18).
Regarding to claim 12, Cheng et al teach that in some embodiments, the direct repeat length of the guide RNA is at least 16 nucleotides, …. ([0226], Page 18). The CRISPR array includes direct repeat sequences typically 36 nucleotides in length, which are generally well conserved ([0177], page 12).
Regarding to claim 16-17, Cheng et al teach that in some embodiments, the CRISPR systems include multiple RNA guides (e.g., two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, thirty, forty, or more) RNA guides. In some embodiments, the CRISPR systems described herein include a single RNA strand or a nucleic acid encoding a single RNA strand, wherein the RNA guides are arranged in tandem. The single RNA strand can include multiple copies of the same RNA guide, multiple copies of distinct RNA guides, or combinations thereof. The processing capability of the Type VI-D CRISPR-Cas effector proteins described herein enables these effectors to be able to target multiple target nucleic acids (e.g., target RNAs) without a loss of activity…. ([0225], page 17).
Regarding to claim 19, Cheng et al teach that “in some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a cell-specific promoter …..” ([0209], page 16)
Regarding to claim 25, Cheng et al teach that the disclosure also provides a cell (e.g., a eukaryotic cell or a prokaryotic cell (e.g., a bacterial cell)) comprising a system described herein. In some embodiments, the eukaryotic cell is a mammalian cell (e.g., a human cell) or a plant cell. The disclosure also provides animal models (e.g., rodent or rabbit models) and plant model that include the cells ([0057], page 4).
Regarding to claim 57-62, Zhang et al teach “an efficient multiplexed system employing Cas13 has been demonstrated in Drosophila employing gRNAs processed from an array containing inventing tRNAs” ([0624], page 166). Zhang et al also teach the use of the adenosine deaminase protein or catalytic domain thereof is a … Drosophila adenosine deaminase protein or catalytic domain thereof. ([0015], page 4). Also, Zhang et al teach “In some embodiments, the Cas13 enzyme is a type V or VI CRISPR system enzyme ……… in other aspects, the invention provides a eukaryotic organism; preferably a multicellular eukaryotic organism, comprising a eukaryotic host cell according to any of the described embodiments …… the organism may be an arthropod such as an insect” (see [0467] page 113-114).
Claims 8, 15 are rejected under 35 U.S.C. 103 as being unpatentable over Cheng et al (Pub. No.: US 2019/0002889 A1, Pub. Date: Jan. 3, 2019) in view of Zhang et al (WO 2019/005884 A1, International Publication Date: 03 January 2019) as applied to claims 1-2, 4, 6, 9, 12, 16-17, 19, 25, 50, 52, 57-62 above, and further in view of Konermann et al (Cell 173, 665–676, April 19, 2018, Doi: 10.1016/j.cell.2018.02.033).
The teachings of Cheng et al and Zhang et al above are incorporated here in their entirety.
Cheng et al does not specifically teach RfxCas13d and AAAAC motif at 5' end of the guide RNA. However, Konermann et al cure the deficiency.
Regarding to claim 8, Konermann et al teach transcriptome engineering with RNA-targeting
type VI-D crispr effectors (title). Konermann et al teach that proceeding with RfxCas13d and AdmCas13d as lead candidates, Konermann et al compared their ability to knock down endogenous transcripts …… (Page 669, right column, 2nd para.). Konermann et al also teach RfxCas13d in figure 1A (see below)
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Regarding to claim 15, Konermann et al teach that Cas13d direct repeats (DRs) are highly conserved in length and predicted secondary structure (Figure S2C), with a 36 nt length, an 8–10 nt stem with A/U-rich loop, and a 5’-AAAAC motif at the 3’ end of the direct repeat (Figure S2D) (Page 667, left column, 2nd para.).
Therefore, it would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the rejected claims to combine the teachings of prior art to modify the method of the nucleic acid molecule comprising a sequence encoding a Cas13 polypeptide and transgenic animal as taught by Cheng et al and Zhang et al by using RfxCas13d and 5’-AAAAC motif as taught by Konermann et al. Said modification amounting to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated to do so because Konermann et al provide explicit advantage of generating a programmable RNA-binding module for efficient targeting of cellular RNA, enabling a general platform for transcriptome engineering and future therapeutic development (see Abstract). One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Konermann et al provid detailed instructions with working example and data to generate such programmable RNA-binding module.
Response to Arguments
Applicant's arguments filed 03-06-2025 have been fully considered but they are not persuasive.
Applicant argues that:
“Based on the combined teachings of Cheng and Zhang, when Zhang is considered in its entirety, a person of ordinary skill in the art would have no expectation of successfully arriving at the present claims based on the teachings of the combined references. ……. When considered as a whole, neither Cheng nor Zhang, either alone or in combination, teach or suggest a first nucleic acid molecule comprising a sequence encoding a Casl3 polypeptide and a second nucleic acid molecule comprising a sequence encoding a guide RNA comprising one or more spacers and at least two Casl3-specific direct repeats, wherein the first nucleic acid molecule and the second nucleic acid molecule are stably integrated into the genome of a transgenic organism, wherein the transgenic organism is an invertebrate, an insect, or a Drosophila fly.” (Remarks, page 2-3).
Response to Arguments:
In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case,
As explained above, Cheng et al teach engineered, non-naturally occurring (CRISPR)-associated (Cas) systems that include ([0025], Page 2): a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a direct repeat sequence and a spacer sequence capable of hybridizing to a target nucleic acid (a first nucleic acid molecule) ([0025], Page 2). a nucleic acid encoding the effector protein, wherein the effector protein is capable of binding to and of targeting the target nucleic acid sequence complementary to the RNA guide spacer sequence (a second nucleic acid molecule) ([0025], Page 2). Cheng et al stated that “in tandem with the effector gene synthesis, we first computationally designed an oligonucleotide library synthesis (OLS) pool containing "repeat-spacer-repeat" sequences, where "repeat" represents the consensus direct repeat sequence found in the CRISPR array associated with the effector, and "spacer" represents sequences tiling the pACYC184 plasmid” ([0338], page 40).Cheng et al teach a cell comprising the system that is a eukaryotic cell or a prokaryotic cell, and an animal model or a plant model comprising the cell (see claims 119-120 and 125, page 188).
Although Cheng et al teach the use of lentiviral plasmid backbone, adeno-associated viral (AAV) plasmid backbone that have ability to integrate into the genome of target cells, Cheng et al do not specifically teach wherein the first nucleic acid molecule and the second nucleic acid molecule are stably integrated into the genome of a transgenic organism. However, Zhang et al cure the deficiency.
Zhang et al provide a eukaryotic host cell comprising: (a) a first regulatory element operably linked to a direct repeat sequence and one or more insertion sites for inserting one or more guide RNA sequences up- or downstream of the direct repeat sequence, ; (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said Casl3 enzyme comprising preferably at least one nuclear localization sequence and/or NES ([0466], page 113). In some embodiments, the host cell comprises components (a) and (b). Where applicable, a tracr sequence may also be provided. In some embodiments, component (a), component (b), or components (a) and (b) are stably integrated into a genome of the host eukaryotic cell ([0466], page 113).
One of ordinary skill in the art would have been motivated to do so because Zhang et al teach the host cell with CRISPR Cas13 components stably integrated into a genome of the host eukaryotic cell would result in the base change that does not rely on endogenous repair machinery and is permanent for as long as the RNA molecule exists in the cell ([0110], page 19-20). One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Zhang et al were successful in RNA base editing with the use of Cas13 enzyme with detailed instructions and working examples.
It is noted that there is no requirement for limitation of “wherein the transgenic organism is an invertebrate, an insect, or a Drosophila fly” in claims 1-2, 4, 6, 9, 12, 16-17, 19, 25, 50, 52.
Regarding to claims 57-62. Zhang et al teach “an efficient multiplexed system employing Cas13 has been demonstrated in Drosophila employing gRNAs processed from an array containing inventing tRNAs” ([0624], page 166). Zhang et al also teach the use of the adenosine deaminase protein or catalytic domain thereof that is a Drosophila adenosine deaminase protein or catalytic domain thereof. ([0015], page 4). Also, Zhang et al teach “the organism may be an arthropod such as an insect” (see [0467] page 113-114)
New-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)(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.
(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 1-2, 4, 6, 8, 9, 12, 15, 16, 17, 19, 25, 50, 52, 57-62 are rejected under 35 U.S.C. 102 (a)(1) and (a)(2) as being anticipated by Hsu et al (Pub. No.: US 2019/0207890 A1, Pub. Date: Jul. 4, 2019, Division of application No. 15/937,669, filed on Mar. 27, 2018).
Regarding to claims 1 and 50, Hsu et al teach “Provided herein are CRISPR/Cas methods and compositions for targeting RNA molecules, which can be used to detect, edit, or modify a target RNA” (Abstract). Hsu et al teach “such a CRISPR-Cas system can include (1) at least one Cas13d protein or at least one Cas13d nucleic acid coding sequence (such as a mRNA or a vector encoding the at least one Cas13d protein) and (2) at least one CRISPR-Cas system guide nucleic acid molecule (such as a guide RNA, gRNA) that hybridizes with the one or more target RNA molecules, or at least one nucleic acid molecule encoding the gRNA” ([0008], page 1), and “the gRNA that hybridizes with the one or more target RNA molecules in an Cas13d mediated manner includes one or more direct repeat (DR) sequences, one or more spacer sequences, such as one or more sequences comprising an array of DR-spacer-DRspacer.” ([0012], page 2).
Hsu et al teach “the Cas13d protein can be expressed from injected plasmid DNA, injected mRNA, or stably integrated copies into the animal genome. The gRNA can be directly injected or expressed from a vector or stably integrated copies into the animal genome.” ([0449], page 26) (For the claimed: wherein the first nucleic acid molecule and the second nucleic acid molecule are stably integrated into the genome of a transgenic organism).
Regarding to claims 2 and 52, Hsu et al teach “This disclosure relates to a CRISPR/Cas system for modifying (including detecting) RNA, which utilizes novel Cas13d proteins (also referred to a CasR and nCas1) and guide RNAs.” ([0003], page 1).
Regarding to claim 4, Hsu et al teach “in one example, an Cas13d protein provided herein (such as a native Cas13d or an Cas13d with mutated HEPN domain(s)) includes a subcellular localization signal” ([0384], page 19) and “SEQ ID NOs: 256 and 257 are exemplary Cas13d nuclear localization signal amino acid and nucleic acid sequences, respectively.” ([0279], page 9).
Regarding to claims 6 and 8, Hsu et al teach “FIGS. 8A-8D: RNA knockdown activity screen of engineered Cas13d orthologs in human cells ……. (D) Comparison of Adm and Rfx Cas13d ortholog constructs for knockdown of endogenous B4GALNT1 mRNA reveals RfxCas13d-NLS (CasRx) to be most effective for both guide RNA architectures” ([0030], page 4), and “Engineered Type VI-D CRISPR effectors can be used to efficiently knockdown endogenous RNAs in human cells and manipulate alternative splicing, paving the way for RNA targeting applications and further effector domain fusions as part of a transcriptome engineering toolbox” ([0007], page 1).
Regarding to claims 9 and 12, Hsu et al teach “Guide molecules generally exist in various states of processing. In one example, an unprocessed guide RNA is 36 nt of DR followed by 30-32 nt of spacer …. In some embodiments, an unprocessed spacer is about 28-32 nt long …. while the mature (processed) spacer can be about 10 to 30 nt, 10 to 25 nt, 14 to 25 nt, 20 to 22 nt, or 14-30 nt …. In some embodiments, an unprocessed DR is about 36 nt …..” ([0416], page 24). Note: DR is direct repeat.
Regarding to claim 15, Hsu et al teach Cas13d direct repeats (DRs) are highly conserved in length and predicted secondary structure (FIG. 3C), with a 36 nt length, an 8-10 nt stem with A/U-rich loop, and a 5'-AAAAC motif at the 3' end of the direct repeat (FIG. 3D) ([0520], page 34).
Regarding to claims 16-17, Hsu et al teach methods of targeting one or more target RNA molecules are provided ([0014], page 2). In some examples, at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 different gRNAs are used. For example, such a method could include targeting at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 different target RNA molecules, targeting at least 2, at least 3, at least 4, at least 5, at least 10, or at least 20 different regions of one or more RNA molecules, or combinations thereof ([0353], page 16).
Regarding to claim 19, Hsu et al teach “many inducible promoters are of use, such as GALl-10 (induced by galactose), PHO5 (induced by low extracellular inorganic phosphate)….” ([0406], page 23).
Regarding to claim 25, Hsu et al teach “Also provided are recombinant cells that include any Cas13d protein (or nucleic acid molecule encoding Cas13d), any gRNA, any RNP complex, or any vector, provided herein” ([0019], page 3).
Regarding to claims 57-62, Hsu et al teach “The Cas13d nucleic acid coding sequence …. can be inserted into an expression vector including, but not limited to a plasmid, virus or other vehicle that can be manipulated to allow insertion or incorporation of sequences and can be expressed in either prokaryotes or eukaryotes. Hosts can include microbial, yeast, insect, plant and mammalian organisms” ([0404], page 22). Hsu et al teach “Such recombinant cells (e.g., which can be used to generate a cell-free system) can be eukaryotic or prokaryotic. Examples of such cells include, but are not limited to bacteria, archaea, plant, fungal, yeast, insect, and mammalian cells, such as …. Drosophila cells,” ([0426], page 25). Hsu et al teach “In some examples, the subject is a laboratory animal/organism, such as a zebrafish, Xenopus, C. elegans, Drosophila, mouse, rabbit, or rat” ([0327], page 13).
Thus, claims 1-2, 4, 6, 8-9, 12, 15-17, 19, 25, 50, 52, 57-62 are anticipated by Hsu et al.
Conclusion
No claim is allowed.
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/KHOA NHAT TRAN/Examiner, Art Unit 1632
/PETER PARAS JR/Supervisory Patent Examiner, Art Unit 1632