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 without traverse of Group II in the reply filed on January 7, 2026 is acknowledged. Claims 1, 2, 25, 29-34 and 43-46 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to nonelected Groups 1 and 3, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 1/7/2026. Thus, claims 5, 6, 8, 9 and 35-42 are under examination (9/19/2025).
Claim Status
Claims 5, 6, 8, 9 and 35-42 were previously presented and are under examination (9/19/2025).
Priority
Claims 5, 6, 8, 9 and 35-42 receive a priority date of 4/26/2021, the effective filing date of US Provisional 63180005.
Information Disclosure Statement
The listing of references in the specification is not a proper information disclosure
statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information
submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be
incorporated into the specification but must be submitted in a separate paper." Therefore, unless
the references have been cited by the examiner on form PTO-892, they have not been
considered.
The information disclosure statement (IDS) submitted on 10/11/2023 is being considered by the examiner.
Specification
The disclosure is objected to because of the following informalities (see MPEP § 608.01):
The use of the terms “Zymo Research” (p.91), “Thermo Fisher” (p. 91-92), “Sigma Aldrich” (p. 92), “Worthington” (p. 92), “10X Genomics” (p. 92-93), and “Illumina” (p. 92-93) and which are trade names or marks used in commerce, have been noted in this application. The terms should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Claim Objections
Claim 5 is objected to as a result of the following informality:
Claim 5, step (x) at line 4 “promoter operably linked toa nucleic acid” should be replaced with “promoter operably linked to a nucleic acid” to correct a minor spelling issue.
Claim 6 is objected to as a result of the following informality:
Claim 6 at line 2 “or an organoid a library” should be replaced with “or an organoid or a library” to correct a minor grammatical issue.
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 5, 6, 8, 9 and 35-42 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 5 is rejected. Claim 5 recites the limitation "the one or more first expression vectors" in step (vii) at line 11. There is insufficient antecedent basis for this limitation in the claim.
Claim 5 is further rejected. Claim 5 recites the limitation " the first plurality of expression vectors" in step (ix) at line 10. There is insufficient antecedent basis for this limitation in the claim.
Claims 6, 8, 9 and 35-42 are included in this rejection due to their dependency on claim 5.
Claim 42 is further rejected. Claim 42 recites the limitation "the expression cassette " in line 2. There is insufficient antecedent basis for this limitation in the claim.
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 5, 6, 8, 9, 35-36, 38 and 42 are rejected under 35 U.S.C. 102 (a)(1) and (a)(2) as being anticipated by Zhang et al. (WO 2018/035387 A1; published 2/22/2018).
Regarding claim 5, Zhang teaches embodiments directed to engineered CRISPR-Cas effector proteins that comprise at least one modification compared to an unmodified CRISPR-Cas effector protein that enhances binding of the of the CRISPR complex to the binding site and/or alters editing preference as compared to wild type and in certain example embodiments, the CRISPR-Cas effector proteins a Type II effector protein (Abstract). Further, Zhang teaches that the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue- specific regulatory elements are used to express the nucleic acid) (Paragraph 161, lines 1-5).
Zhang also teaches that one or more vectors driving expression of one or more elements of a nucleic acid-targeting system are introduced into a host cell such that expression of the elements of the nucleic acid-targeting system direct formation of a nucleic acid-targeting complex at one or more target sites and for example, a nucleic acid-targeting effector enzyme and a nucleic acid-targeting guide RNA could each be operably linked to separate regulatory elements on separate vectors (Paragraph 162, lines 1-5). Zhang further teaches that RNA(s) of the nucleic acid-targeting system can be delivered to a transgenic nucleic acid-targeting effector protein animal or mammal, e.g., an animal or mammal that constitutively or inducibly or conditionally expresses nucleic acid-targeting effector protein; or an animal or mammal that is otherwise expressing nucleic acid-targeting effector proteins or has cells containing nucleic acid-targeting effector proteins, such as by way of prior administration thereto of a vector or vectors that code for and express in vivo nucleic acid-targeting effector proteins and alternatively, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the nucleic acid-targeting system not included in the first vector, nucleic acid-targeting system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5' with respect to ("upstream" of) or 3' with respect to ("downstream" of) a second element (Paragraph 162, lines 5-10). Zhang also teaches that the coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction and in some embodiments, a single promoter drives expression of a transcript encoding a nucleic acid-targeting effector protein and the nucleic acid-targeting guide RNA, embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron), and further in some embodiments, the nucleic acid- targeting effector protein and the nucleic acid-targeting guide RNA may be operably linked to and expressed from the same promoter where delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for expression of one or more elements of a nucleic acid-targeting system are as used (Paragraph 162, lines 10-15).
Specifically, Zhang teaches that in certain embodiments, sequencing-based DSB detection assay comprises labeling a site of a DSB with an adapter comprising a primer binding site, labeling a site of a DSB with a barcode or unique molecular identifier, or combination thereof, as described herein elsewhere (Paragraph 209, lines 5-10). Zhang also teaches that in certain embodiments, the number of (sub)selected target sites needed to treat a population is estimated based on based low frequency sequence variation, such as low frequency sequence variation captured in large scale sequencing datasets and further in certain embodiments, the number of (sub)selected target sites needed to treat a population of a given size is estimated (Paragraph 214, lines 1-5).
Additionally, Zhang teaches that the guide RNA may comprise a sequence complementary to a target miRNA or an siRNA, which may or may not be present within a cell and in this way, only when the miRNA or siRNA is present, for example through expression (by the cell or through human intervention), is there binding of the RNA sequence to the miRNA or siRNA which then results in cleavage of the guide RNA an RNA-induced silencing complex (RISC) within the cell and therefore, in some embodiments, the guide RNA comprises an RNA sequence complementary to a target miRNA or siRNA, and binding of the guide RNA sequence to the target miRNA or siRNA results in cleavage of the guide RNA by an RNA-induced silencing complex (RISC) within the cell (Paragraph 297, lines 1-5).
Further, Zhang teaches that additional guide RNA can be delivered via a vector, e.g., a separate vector or the same vector that is encoding the CRISPR complex and when provided by a separate vector, the CRISPR RNA that targets Cas9 expression can be administered sequentially or simultaneously and when administered sequentially, the CRISPR RNA that targets Cas9 expression is to be delivered after the CRISPR RNA that is intended for e.g. gene editing or gene engineering (Paragraph 336, lines 1-5). Zhang also teaches that in this fashion, the Cas enzyme associates with a first gRNA capable of hybridizing to a first target, such as a genomic locus or loci of interest and undertakes the function(s) desired of the CRISPR-Cas system (e.g., gene engineering); and subsequently the Cas9 enzyme may then associate with the second gRNA capable of hybridizing to the sequence comprising at least part of the Cas9 or CRISPR cassette, and where the gRNA targets the sequences encoding expression of the Cas9 protein, the enzyme becomes impeded and the system becomes self-inactivating (Paragraph 336, lines 10-15).
Zhang also teaches that the invention provides a CRISPR-Cas system comprising one or more vectors for delivery to a eukaryotic cell, wherein the vector(s) encode(s): (i) a CRISPR enzyme; (ii) a first guide RNA capable of hybridizing to a target sequence in the cell; (iii) a second guide RNA capable of hybridizing to one or more target sequence(s) in the vector which encodes the CRISPR enzyme; (iv) at least one tracr mate sequence; and (v) at least one tracr sequence. The first and second complexes can use the same tracr and tracr mate, thus differing only by the guide sequence, wherein, when expressed within the cell: the first guide RNA directs sequence-specific binding of a first CRISPR complex to the target sequence in the cell; the second guide RNA directs sequence-specific binding of a second CRISPR complex to the target sequence in the vector which encodes the CRISPR enzyme; the CRISPR complexes comprise (a) a tracr mate sequence hybridized to a tracr sequence and (b) a CRISPR enzyme bound to a guide RNA, such that a guide RNA can hybridize to its target sequence; and the second CRISPR complex inactivates the CRISPR-Cas system to prevent continued expression of the CRISPR enzyme by the cell. The CRISPR enzyme can be Cas9, particularly SpCas9, SaCas9, or StCas9 (Paragraph 338, lines 1-15).
Specifically, Zhang teaches that in some embodiments, the system comprises the tracr sequence under the control of a third regulatory element, such as a polymerase III promoter and in some embodiments, the tracr sequence exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. Determining optimal alignment is within the purview of one of skill in the art (Paragraph 700, lines 20-25).
Zhang also teaches that the application provides methods for developing the therapeutic use of a nucleic acid-targeting system where the nucleic acid-targeting complex an effective means for modifying a target DNA or RNA (single or double stranded, linear or super-coiled) and the nucleic acid- targeting complex has a wide variety of utility including modifying (e.g., deleting, inserting, translocating, inactivating, activating) a target DNA or RNA in a multiplicity of cell types, which as such the nucleic acid-targeting complex has a broad spectrum of applications in, e.g., gene therapy, drug screening, disease diagnosis, and prognosis (Paragraph 767, lines 1-10). Zhang also teaches that an exemplary nucleic acid- targeting complex comprises a DNA or RNA-targeting effector protein complexed with a guide RNA hybridized to a target sequence within the target locus of interest (Paragraph 767, lines 1-10).
Additionally, Zhang teaches that the first regulatory element is a polymerase III promoter and, in some embodiments, the second regulatory element is a polymerase II promoter, where the guide sequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides, or between 10-30, or between 15-25, or between 15-20 nucleotides in length (Paragraph 707, lines 35-40). Further, Zhang teaches that in some embodiments one or more guide(s) edit the one or more target(s) while one or more self-inactivating guides inactivate the CRISPR/Cas9 system and thus, for example, the described CRISPR-Cas9 system for repairing expansion disorders may be directly combined with the self-inactivating CRISPR-Cas9 system described herein and such a system may, for example, have two guides directed to the target region for repair as well as at least a third guide directed to self-inactivation of the CRISPR-Cas9 (Paragraph 347, lines 1-5).
Specifically, Zhang teaches that the invention provides a vector system comprising one or more vectors and in some embodiments, the system comprises: (a) a first regulatory element operably linked to a tracr mate sequence and one or more insertion sites for inserting one or more guide sequences upstream of the tracr mate sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a AAV-CRISPR complex to a target sequence in a eukaryotic cell, wherein the CRISPR complex comprises a AAV-CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence; and (b) said AAV-CRISPR enzyme comprising at least one nuclear localization sequence and/or at least one NES; wherein components (a) and (b) are located on or in the same or different vectors of the system (Paragraph 700, lines 1-10). Further Zhang teaches that in some embodiments, component (a) further comprises the tracr sequence downstream of the tracr mate sequence under the control of the first regulatory element where component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences direct sequence specific binding of a AAV-CRISPR complex to a different target sequence in a eukaryotic cell and the system comprises the tracr sequence under the control of a third regulatory element, such as a polymerase III promoter (Paragraph 700, lines 10-20).
Regarding claim 6, Zhang teaches that the previously described method can be applied to an organism in some embodiments of these aspects which may be an animal; for example, a mammal. Also, the organism may be an arthropod such as an insect (Paragraph 706, lines 40-45).
Regarding claim 8, Zhang teaches that the expression system for stable integration into the genome of a plant cell may contain one or more of the following elements: a promoter element that can be used to express the RNA and/or CRISPR-Cas enzyme in a plant cell; a 5' untranslated region to enhance expression ; an intron element to further enhance expression in certain cells, such as monocot cells; a multiple-cloning site to provide convenient restriction sites for inserting the guide RNA and/or the CRISPR-Cas gene sequences and other desired elements; and a 3' untranslated region to provide for efficient termination of the expressed transcript (Paragraph 870, lines 1-10).
Regarding claim 9, Zhang teaches that the guide RNA may comprise a sequence complementary to a target miRNA or an siRNA, which may or may not be present within a cell and in this way, only when the miRNA or siRNA is present, for example through expression (by the cell or through human intervention), is there binding of the RNA sequence to the miRNA or siRNA which then results in cleavage of the guide RNA an RNA-induced silencing complex (RISC) within the cell (Paragraph 297, lines 1-5). Further, Zhang teaches that addition of non-targeting nucleotides to the 5' end (e.g. 1 - 10 nucleotides, preferably 1 - 5 nucleotides) of the "self-inactivating" guide RNA can be used to delay its processing and/or modify its efficiency as a means of ensuring editing at the targeted genomic locus prior to CRISPR-Cas9 shutdown (Paragraph 344, lines 1-5).
Regarding claim 35, Zhang teaches that in some general embodiments, the Cas9 enzyme used for multiplex targeting is associated with one or more functional domains and in some more specific embodiments, the CRISPR enzyme used for multiplex targeting is a deadCas9 as defined herein elsewhere and ach of the guide sequence is at least 16, 17, 18, 19, 20, 25 nucleotides, or between 16-30, or between 16-25, or between 16-20 nucleotides in length (Paragraph 765, lines 15-20). Specifically, Zhang teaches that in some embodiments, a loop in the guide RNA is provided and this may be a stem loop or a tetra loop where the loop is preferably GAAA, but it is not limited to this sequence or indeed to being only 4bp in length (Paragraph 115, lines 1-5). Further Zhang teaches that preferred loop forming sequences for use in hairpin structures are four nucleotides in length, and most preferably have the sequence GAAA, however, longer or shorter loop sequences may be used, as may alternative sequences (Paragraph 115, lines 1-5).
Regarding claim 36, Zhang teaches that the single cell sequencing comprises unique molecular identifiers (UMI), whereby the capture rate of the measured signals, such as transcript copy number or probe binding events, in a single cell is determined (Paragraph 1369, lines 15-20).
Regarding claim 38, Zhang teaches that in particular embodiments, the CRISPR-Cas system may be used as a generic nucleic acid binding protein with fusion to or being operably linked to a functional domain for activation and/or repression of endogenous plant genes where exemplary functional domains may include but are not limited to translational initiator, translational activator, translational repressor, nucleases, in particular ribonucleases, a spliceosome, beads, a light inducible/controllable domain or a chemically inducible/controllable domain; where typically in these embodiments, the Cas9 protein comprises at least one mutation, such that it has no more than 5% of the activity of the Cas9 protein not having the at least one mutation; the guide RNA comprises a guide sequence capable of hybridizing to a target sequence (Paragraph 915, lines 1-10).
Regarding claim 42, Zhang teaches that the first regulatory element is a polymerase III promoter and, in some embodiments, the second regulatory element is a polymerase II promoter, where the guide sequence is at least 15, 16, 17, 18, 19, 20, 25 nucleotides, or between 10-30, or between 15-25, or between 15-20 nucleotides in length (Paragraph 707, lines 35-40). Further, Zhang teaches that in some embodiments one or more guide(s) edit the one or more target(s) while one or more self-inactivating guides inactivate the CRISPR/Cas9 system and thus, for example, the described CRISPR-Cas9 system for repairing expansion disorders may be directly combined with the self-inactivating CRISPR-Cas9 system described herein and such a system may, for example, have two guides directed to the target region for repair as well as at least a third guide directed to self-inactivation of the CRISPR-Cas9 (Paragraph 347, lines 1-5).
Zhang also teaches that one or more vectors driving expression of one or more elements of a nucleic acid-targeting system are introduced into a host cell such that expression of the elements of the nucleic acid-targeting system direct formation of a nucleic acid-targeting complex at one or more target sites and for example, a nucleic acid-targeting effector enzyme and a nucleic acid-targeting guide RNA could each be operably linked to separate regulatory elements on separate vectors (Paragraph 162, lines 1-5). Zhang further teaches that RNA(s) of the nucleic acid-targeting system can be delivered to a transgenic nucleic acid-targeting effector protein animal or mammal, e.g., an animal or mammal that constitutively or inducibly or conditionally expresses nucleic acid-targeting effector protein; or an animal or mammal that is otherwise expressing nucleic acid-targeting effector proteins or has cells containing nucleic acid-targeting effector proteins, such as by way of prior administration thereto of a vector or vectors that code for and express in vivo nucleic acid-targeting effector proteins and alternatively, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the nucleic acid-targeting system not included in the first vector, nucleic acid-targeting system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5' with respect to ("upstream" of) or 3' with respect to ("downstream" of) a second element (Paragraph 162, lines 5-10).
Zhang teaches each and every limitation of claims 5, 6, 8, 9, 35-36, 38 and 42, and therefore Zhang anticipates claims 5, 6, 8, 9, 35-36, 38 and 42.
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 text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
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.
Claim 37, 40-41 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. (WO 2018/035387 A1; published 2/22/2018), as applied to claims 1, 6, 8, 9, 35-36, 38 and 42 above, in view of Bent et al. (US PGPub 2019 / 0249226 A1, filed 8/15/2019), and in view of Zhong et al. (US 2002/0064771 A1; published 5/30/2002), and in further view of Stuurman et al. (2013/0004951 A1; published 1/3/2013).
As previously shown, Zhang teaches embodiments directed to engineered CRISPR-Cas effector proteins that comprise at least one modification compared to an unmodified CRISPR-Cas effector protein that enhances binding of the of the CRISPR complex to the binding site and/or alters editing preference as compared to wild type and in certain example embodiments, the CRISPR-Cas effector proteins a Type II effector protein (Abstract). Further, Zhang teaches that the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue- specific regulatory elements are used to express the nucleic acid) (Paragraph 161, lines 1-5).
Zhang also teaches that one or more vectors driving expression of one or more elements of a nucleic acid-targeting system are introduced into a host cell such that expression of the elements of the nucleic acid-targeting system direct formation of a nucleic acid-targeting complex at one or more target sites and for example, a nucleic acid-targeting effector enzyme and a nucleic acid-targeting guide RNA could each be operably linked to separate regulatory elements on separate vectors (Paragraph 162, lines 1-5). Zhang further teaches that RNA(s) of the nucleic acid-targeting system can be delivered to a transgenic nucleic acid-targeting effector protein animal or mammal, e.g., an animal or mammal that constitutively or inducibly or conditionally expresses nucleic acid-targeting effector protein; or an animal or mammal that is otherwise expressing nucleic acid-targeting effector proteins or has cells containing nucleic acid-targeting effector proteins, such as by way of prior administration thereto of a vector or vectors that code for and express in vivo nucleic acid-targeting effector proteins and alternatively, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the nucleic acid-targeting system not included in the first vector, nucleic acid-targeting system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5' with respect to ("upstream" of) or 3' with respect to ("downstream" of) a second element (Paragraph 162, lines 5-10). Zhang also teaches that the coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction and in some embodiments, a single promoter drives expression of a transcript encoding a nucleic acid-targeting effector protein and the nucleic acid-targeting guide RNA, embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron), and further in some embodiments, the nucleic acid- targeting effector protein and the nucleic acid-targeting guide RNA may be operably linked to and expressed from the same promoter where delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for expression of one or more elements of a nucleic acid-targeting system are as used (Paragraph 162, lines 10-15).
Regarding claims 37, 40-41, Zhang teaches Zhang teaches that the invention provides a vector system comprising one or more vectors and in some embodiments, the system comprises: (a) a first regulatory element operably linked to a tracr mate sequence and one or more insertion sites for inserting one or more guide sequences upstream of the tracr mate sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a AAV-CRISPR complex to a target sequence in a eukaryotic cell, wherein the CRISPR complex comprises a AAV-CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence; and (b) said AAV-CRISPR enzyme comprising at least one nuclear localization sequence and/or at least one NES; wherein components (a) and (b) are located on or in the same or different vectors of the system (Paragraph 700, lines 1-10). Further Zhang teaches that in some embodiments, component (a) further comprises the tracr sequence downstream of the tracr mate sequence under the control of the first regulatory element where component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences direct sequence specific binding of a AAV-CRISPR complex to a different target sequence in a eukaryotic cell and the system comprises the tracr sequence under the control of a third regulatory element, such as a polymerase III promoter (Paragraph 700, lines 10-20).
Zhang teaches that the single cell sequencing comprises unique molecular identifiers (UMI), whereby the capture rate of the measured signals, such as transcript copy number or probe binding events, in a single cell is determined (Paragraph 1369, lines 15-20).
However, Zhang does not teach or suggest the specific sequence set forth in SEQ ID NOs: 1-2 for capture or guide sequences, SEQ ID NOs: 5-84 for the enrichment sequences or SEQ ID NO: 85 for the UMI or UGI.
Bent teaches methods of generating supports (e.g., beads) comprising barcode molecules coupled, which may comprise a barcode sequence and a functional sequence (Abstract). Further, Bent teaches SEQ ID NO: 156, which has a 100% similarity match to SEQ ID NO: 1, of the instant claim, as shown in Figure 1 below.
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[AltContent: textbox (Figure 1: Bent teaches SEQ ID NO: 156, which has a 100% similarity match to SEQ ID NO: 1 of the instant application. )]
Bent also teaches SEQ ID NO: 155, which has a 100% similarity match to SEQ ID NO: 2, of the instant claim, as shown in Figure 2 below.
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[AltContent: textbox (Figure 2: Bent teaches SEQ ID NO: 155, which has a 100% similarity match to SEQ ID NO: 2 of the instant application. )]
Zhong teaches efficient HCV replicase complexes comprising novel RNA template and
primer pair, including assay systems which use such complexes, for detecting replicase activity, quantitatively studying the kinetics and mechanism of HCV NS5B-catalyzed nucleotide incorporation, and identifying inhibitors of HCV replicase (Abstract). Further, Zhong teaches SEQ ID NO: 32, which has a 100% similarity to SEQ ID NO: 6, of the instant claim, as shown in Figure 3 below.
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[AltContent: textbox (Figure 3: Zhong teaches SEQ ID NO: 32, which has a 100% similarity match to SEQ ID NO: 6 of the instant application. )]
Stuurman teaches an invention relating to a new strategy for identification, and optional isolation, of a nucleic acid sequence that is expressed in an organism and that is causally related to a particular phenotype (trait of a character) of said organism (Abstract). Further, Stuurman teaches SEQ ID NO: 10 which has a 100% Query Match to SEQ ID NO: 85, as shown in Figure 4 below.
[AltContent: textbox (Figure 4: Stuurman teaches SEQ ID NO: 10, which has a 100% query match to SEQ ID NO: 85 of the instant application. )]
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It would have been obvious to a person of ordinary skill in the art at the time of the invention to use such known short synthetic oligonucleotide sequences, including degenerate or variable sequences, as spike-in or capture oligonucleotides in a nucleic acid library system, since short synthetic sequences were routinely used as tags, barcodes, or control sequences in molecular assays. Therefore, modifying the library of the reference to include a spike oligonucleotide as claimed would have been an obvious design choice with a reasonable expectation of success.
More so, it would have been obvious to a person of ordinary skill in the art at the time of the invention to incorporate the known barcode, enrichment, or capture sequences taught by Bent, Zhong and Stuurman into the CRISPR-based vector system of Zhang in order to facilitate identification, enrichment, or tracking of nucleic acid constructs during sequencing or screening assays. Such modification would have been motivated by the well-known advantages of using barcode or identifier sequences to distinguish and quantify nucleic acid molecules in pooled experiments, and a skilled artisan would have had a reasonable expectation of success because these short synthetic oligonucleotide sequences were routinely incorporated into nucleic acid constructs and sequencing libraries using established molecular biology techniques.
Allowable Subject Matter
Regarding claim 39, the prior art of record, including Zhang in view of Bent, Zhong, and Stuurman, teaches nucleic acid libraries comprising capture sequences, enrichment sequences, barcodes, and related oligonucleotide elements useful for sequencing and identification of nucleic acid constructs. However, the prior art of does not teach or suggest the specific spike oligonucleotide sequence recited in SEQ ID NOs: 3-4.
Further, the closest prior art of record, Sims et al. (US PGPub 2021/0254143 A1; filing date 11/27/2018), teaches sequences that are the closest teachings to the claimed sequences, the sequences taught by Sims exhibit only approximately 80% similarity (SEQ ID NO: 8068 and SEQ ID NO: 4986) to the sequences corresponding to SEQ ID NOs: 3-4, and therefore does not disclose or suggest the claimed sequences, as shown below in Figures 5-6.
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[AltContent: textbox (Figure 5: Sims teaches SEQ ID NO: 8068, which has an 80% similarity match to SEQ ID NO: 3 of the instant application. )]
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[AltContent: textbox (Figure 6: Sims teaches SEQ ID NO: 4986, which has a 78% similarity match to SEQ ID NO: 4 of the instant application. )]
As discussed above, the cited references, even when considered in further view of Sims, simply disclose various barcode, capture, and identifier sequences, but none teach or suggest, alone or in combination, the particular sequences corresponding to SEQ ID NOs: 3-4 as recited in the instant claim.
Accordingly, the prior art fails to teach or render obvious the specific spike oligonucleotide sequence limitations of claim 39. However, claim 39 is objected to as being dependent upon a rejected based claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
Conclusion
No claim is allowed; however, SEQ ID NO: 5 set forth in instant claim 40 is free of the prior art.
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/ELIZABETH ROSE LAFAVE/ Examiner, Art Unit 1684
/HEATHER CALAMITA/ Supervisory Patent Examiner, Art Unit 1684