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/Restriction
Applicant’s election, without traverse, of Group I (claims 1-4, 6, 8-11, 14-15, 17-20, 25-26, and 28) in the reply filed on 06/08/2026 is acknowledged.
Status of the Claims
Claims 1-4, 6, 8-11, 14-15, 17-20, 25-26, 28, 30, and 36 are pending.
Claims 30 and 36 have been withdrawn.
Claims 1-4, 6, 8-11, 14-15, 17-20, 25-26, and 28 are examined in this Office action.
Information Disclosure Statement
Initialed and dated copy of Applicant’s Information Disclosure Statement (IDS) filed on 02/04/2025 is attached to the instant Office action. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the IDS is being considered by the examiner.
Specification
The Abstract of the disclosure does not commence on a separate sheet in accordance with 37 CFR 1.52(b)(4) and 1.72(b). A new abstract of the disclosure is required and must be presented on a separate sheet, apart from any other text.
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code (page 27, lines 17, 18, and 20; page 28, line 13). Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Claim Objections
Claim 20 is objected to because of the following informalities: in line 2, the word “facilitate” should be amended to read ---facilitates---. Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Indefiniteness
Claim 28 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention. All dependent claims are included in these rejections unless they include a limitation that overcomes the deficiencies of the parent claim.
Claim 28 is rendered indefinite by the recitation of “a genetic manipulation intentionally introduced into the plant population”. This requires prior knowledge of the inventor’s intention, which renders the metes and bounds of the claim unclear.
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.
Claims 1, 6, 8, 11, 18, 19, 25, 26, and 28 are rejected under 35 U.S.C. 102(a) as being anticipated by GERSBACH (Gersbach et al., Pub. No.: US 2018/0291370 A1; Pub. Date: Oct. 11, 2018).
Claim 1 recites “[a] method for identifying multiple members within at least one gene set underlying a phenotype, the method comprising:
clustering coding sequences within genetic data of a plant species to sequence clusters, each cluster representing a gene set;
producing a CRISPR library comprising a plurality of polynucleotides, wherein each polynucleotide encodes one or more unique sgRNAs, wherein each of the sgRNAs targets a plurality of gene members comprised within the gene set;
transforming the library into a plurality of plants, thereby producing a plant population wherein each plant of the population comprises at least one sgRNA targeting multiple gene members;
screening the plant population for at least one selected phenotype;
selecting at least one plant showing the at least one selected phenotype; and
identifying in the selected plant the at least one sgRNA targeting the multiple-gene members;
thereby identifying said multiple gene members underlying said selected phenotype.”
In regard to claims 1 and 26, GERSBACH teaches and claims a method of high-throughput screening for one or more putative gene regulatory elements in a genome that modulate a phenotype (i.e., a method for identifying multiple members within at least one gene set underlying a phenotype). The method includes a) contacting a plurality of modified target cells with a library of single guide RNAs (sgRNAs) that target a plurality of gene regulatory elements within the genome (i.e., clustering coding sequences within genetic data of a plant species to sequence clusters, each cluster representing a gene set; producing a CRISPR library comprising a plurality of polynucleotides, wherein each polynucleotide encodes one or more unique sgRNAs, wherein each of the sgRNAs targets a plurality of gene members comprised within the gene set), thereby generating a plurality of test cells (i.e., transforming the library into a plurality of plants, thereby producing a plant population wherein each plant of the population comprises at least one sgRNA targeting multiple gene members; wherein the library or the plurality of libraries is transformed into a plurality of plants to form a plurality of transformed plants, each transformed plant expressing at least one sgRNA, each sgRNA targeting multiple members of a gene set (instant claim 26)), b) selecting a population of test cells or an organism having a modulated phenotype (i.e., screening the plant population for at least one selected phenotype; selecting at least one plant showing the at least one selected phenotype) c) quantitating the frequency of the sgRNAs within the population of selected cells or the organism, wherein the sgRNAs that target gene regulatory elements that modulate the phenotype are overrepresented or underrepresented in the selected cells (i.e., identifying in the selected plant the at least one sgRNA targeting the multiple-gene members); and d) identifying and characterizing the sgRNAs within the population of selected test cells or the organism thereby identifying the gene regulatory elements that modulate the phenotype (i.e., thereby identifying said multiple gene members underlying said selected phenotype) (Gersbach, Summary, page 1, paragraph 0007; claim 1).
GERSBACH teaches that the CRISPR/Cas9-based epigenomic editing system may be used with any type of cell. In some embodiments, the cell is a plant cell (Gersbach, page 17, paragraph 0174).
In regard to claim 6, GERSBACH teaches and claims that the method involves the design and synthesis of libraries of gRNAs targeted to all candidate gene regulatory elements in a genomic region or whole or entire genome. Lentiviral vectors encoding the gRNA library can be used to deliver the gRNA library to cell lines expressing a CRISPR/Cas9-based epigenomic editing system; the sgRNA is encoded by a polynucleotide sequence and packaged into a lentiviral vector, thereby generating a gRNA library lentiviral pool (i.e., wherein the library comprises at least one polynucleotide encoding for two different sgRNAs targeting the same gene members) (Gersbach, page 10, paragraph 123; claim 69) .
In regard to claim 8, GERSBACH teaches and claims that the disclosed methods can include screening for differential endogenous gene expression using genome-wide gene expression by RNA-seq (i.e., wherein the genetic data are RNA sequencing data selected from total RNA-seq and transcriptomics) (Gersbach, page 10, paragraph 0122; claim 63).
In regard to claim 11, GERSBACH teaches methods for high-throughput screening of regulatory element activity that involves the design and synthesis of libraries of gRNAs, using custom array synthesis, targeted to all active gene regulatory elements in a particular genomic region (targeted screen), or the entire genome (genome-wide screen) (i.e., wherein producing the CRISPR library comprises designing the plurality of sgRNAs following an analysis of the genetic data of the plant) (Gersbach, page 10, paragraph 0120).
In regard to claims 18 and 19, GERSBACH teaches that two libraries of gRNAs targeting DNase I hypersensitivity sites (DHSs) were developed. For each DHS, the top 50 gRNAs ranked based on predicted off-targets were selected, which resulted in 10,739 gRNAs in a 4.5 megabase region surrounding the 3-globin locus (HBE1 locus) and 10,137 gRNAs in a 5 megabase region surrounding the HER2 gene (i.e., wherein said method comprises producing a plurality of libraries, each library comprising a plurality of polynucleotides, wherein each polynucleotide encoding one or more unique sgRNAs targeting a plurality of gene members comprised within a gene set, wherein each library comprises a different gene set; wherein said method comprises producing 2 or more libraries) (Gersbach, page 21, paragraph 0201).
In regard to claim 25, GERSBACH teaches and claims that between 1 and 100 gRNAs per regulatory element can be generated, depending on the scope of the screen, to compensate for variation in individual gRNA activity; wherein between 5 and 50 gRNAs are generated per gene regulatory element (i.e., wherein the one or more unique sgRNAs comprises at least 10, at least 50, at least 100 sgRNAs) (Gersbach, pages 12-13, paragraph 0148; claim 57).
In regard to claim 28, GERSBACH teaches that by selecting the cells with modulated phenotype or gene expression, the regulatory elements are identified that are responsible for these characteristics directly in the cell type of interest (i.e., wherein the selected phenotype is attributed to a genetic manipulation intentionally introduced into the plant population) (Gersbach, page 10, paragraph 0120).
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.
Claims 2-4, 9, 14-15, and 17 are rejected under 35 U.S.C. 103 as being unpatentable over GERSBACH (Gersbach et al., Pub. No.: US 2018/0291370 A1; Pub. Date: Oct. 11, 2018) in view of HYAMS (Hyams, et al., 2018, J Mol Biol, Vol. 430(15), pages 2184-2195; included on IDS dated 02/04/2025).
Claim 2 recites “[t]he method of claim 1, wherein at least two of the unique sgRNAs target a single gene member”.
GERSBACH teaches the method of claim 1.
GERSBACH does not explicitly teach wherein at least two of the unique sgRNAs target a single gene member.
Examiner’s Comment: It is noted that Applicants used the CRISPys algorithm to design their sgRNA library, as taught by HYAMS in the following rejection.
HYAMS teaches that the development of the CRISPR–Cas9 system in recent years has made eukaryotic genome editing, and specifically gene knockout for reverse genetics, a simple and effective task. The system is directed to a genomic target site by a programmed single-guide RNA (sgRNA) that base-pairs with it, subsequently leading to site-specific modifications. However, many gene families in eukaryotic genomes exhibit partially overlapping functions, and thus, the knockout of one gene might be concealed by the function of the other. In such cases, the reduced specificity of the CRISPR–Cas9 system, which may lead to the modification of genomic sites that are not identical to the sgRNA, can be harnessed for the simultaneous knockout of multiple homologous genes (Hyams, Abstract).
HYAMS teaches CRISPys, an algorithm for the optimal design of sgRNAs that would potentially target multiple members of a given gene family. CRISPys first clusters all the potential targets in the input sequences into a hierarchical tree structure that specifies the similarity among them. Then, sgRNAs are proposed in the internal nodes of the tree by embedding mismatches where needed, such that the efficiency to edit the induced targets is maximized (Hyams, Abstract). CRISPys detects highly similar sequences among the set of all potential CRISPR–Cas9 targets located within the genes of interest, and designs sgRNAs that would target the gene set with highest efficacy (Hyams, page 2185, right column, first full paragraph).
HYAMS teaches that evaluation of the sgRNAs designed by CRISPys should enable its utilization for designing sgRNAs libraries to screen for phenotypes whose expression is dictated by genes that backup each other in human, plants, and other organisms (Hyams, page 2192, left column, first full paragraph).
Below is the Graphical Abstract of the CRISPys system taught by HYAMS.
PNG
media_image1.png
200
432
media_image1.png
Greyscale
In regard to claims 2, 3, and 4, HYAMS teaches four strategies for sgRNA design:
I A single sgRNA that could best target the entire gene set G.
II A single sgRNA that is optimized to target each of the input genes (i.e., wherein at least two of the unique sgRNAs target a single gene member (instant claim 2).
III Multiple sgRNAs, each directed toward subgroup of homologous genes (i.e., wherein at least two of the unique sgRNAs target at least two same gene members out of a plurality of gene members targeted by the at least two unique sgRNAs (instant claim 3); wherein at least two of the unique sgRNAs target the same plurality of gene members (instant claim 4)).
IV The minimal set of sgRNAs that could target the entire gene set with high efficiency (Hyams, pages 2185-2186) .
It is noted that Applicants used the same CRISPys algorithm as taught by HYAMS; thus, it would be inherent to the CRISPys algorithm to produce the unique sgRNAs as claimed in the instant application.
At the time the instant application was filed, it would have been obvious and within the scope of one of ordinary skill in the art to utilize the CRISPys algorithm in a plant as taught by HYAMS, in the high-throughput screening method as taught by GERSBACH. Based on the teachings of HYAMS, one of ordinary skill in the art would be motivated to use the CRISPys algorithm in a method such as the high-throughput screening method as taught by GERSBACH, knowing that CRISPys outperforms simpler alignment-based techniques. Thus, one of ordinary skill in the art would have a high expectation of success by following the teachings of HYAMS and GERSBACH.
The method of knocking out all genes related to a specific phenotype using sgRNAs and CRISPR technology is a technique that was routine in the art at the time the application was filed, as taught by the cited references and the state of the art in general.
In regard to claim 9, HYAMS teaches that CRISPys detects highly similar sequences among the set of all potential CRISPR–Cas9 targets, located within the genes of interest, and designs sgRNAs that would target the gene set with highest efficacy (i.e., wherein the gene set comprises members of a gene family, and wherein clustering the coding sequences comprises clustering coding sequences encoding polypeptides having at least 30% sequence identity) (Hyams, page 2185, right column, first full paragraph). Although HYAMS does not explicitly teach “at least 30% sequence identity”, one of ordinary skill in the art could appreciate “highly similar sequences” as taught by HYAMS as encompassing those sequences with at least 30% sequence identity.
In regard to claim 14, HYAMS teaches that the sgRNA design by CRISPys depends not only on the scoring function but also on the criterion by which one chooses to select the optimal sgRNA. Given the costly (and timely) experimental resources that are needed to validate a successful assay, researchers are most often interested in focusing their efforts in validating those targets whose targeting probability is high. This is implemented using the sΩ design, but necessitates the use of a pre-specified threshold above which targets are considered (i.e., wherein designing the plurality of sgRNA comprises using a computational algorithm determining the probability that a genomic target is cleaved by a given sgRNA) (Hyams, page 2191, left column, last paragraph).
In regard to claim 15, HYAMS teaches that the CRISPys algorithm accepts as input a set of (potentially homologous) sequences for which sgRNA candidates should be designed. In order to avoid targeting the designed sgRNA at intron–exon junctions, users may provide each gene as a set of exon sequences (i.e., wherein the computational algorithm computes all possible sgRNA target sites within the exonic regions on both DNA strands) (Hyams, page 2193, Program Availability).
In regard to claim 17, HYAMS teaches given the set of genes G, all potential targets are first extracted for each gene gi ∈ G. We also denote by Ga the subgroup of the input genes to which the targets belong (i.e., wherein said method comprises a step of further sub-grouping the gene set based on their sequence similarity) (Hyams, page 2186, left column, last paragraph).
Claims 10 and 20 are rejected under 35 U.S.C. 103 as being unpatentable over GERSBACH (Gersbach et al., Pub. No.: US 2018/0291370 A1; Pub. Date: Oct. 11, 2018) and HYAMS (Hyams, et al., 2018, J Mol Biol, Vol. 430(15), pages 2184-2195; included on IDS dated 02/04/2025) as applied to claims 2-4, 9, 14-15, and 17 as outlined in the rejection above, and in further view of INZÉ (EP 3521436 A1; 08/07/2019; included on IDS dated 02/04/2025).
Claim 10 recites “[t]he method of claim 1, wherein the gene set comprises members of a pathway, and wherein clustering the coding sequences is based on the functional or molecular characteristics of the pathway”.
GERSBACH and HYAMS teach the method of claim 1.
GERSBACH and HYAMS do not explicitly teach wherein the gene set comprises members of a pathway, and wherein clustering the coding sequences is based on the functional or molecular characteristics of the pathway.
However, INZÉ teaches a new method, BREEDIT, which bridges the gap between classical genetics (involving the entire genome) and genetic engineering of single genes by combining multiplex CRIPSR-mediated genome editing with breeding for selecting favorable phenotypes (Inzé, page 3, paragraph 0005).
INZÉ teaches compositions and methods employing multiple single guide RNAs/Cas endonuclease system in plants for genome modification of selected target sequences in the genome of a plant or plant cell, for selecting plants, for gene editing, and for modifying polynucleotides of interest present in the genome of a plant. The methods and compositions employ a multiple guide RNAs/Cas endonuclease system to provide for an effective system for modifying or altering target sites and nucleotides of interest within the genome of a plant, plant cell or seed (Inzé, page 3, paragraph 0006).
In regard to claim 10, INZÉ teaches that in the last four decades, there has been a tremendous progress in understanding the molecular basis of many different plant processes. The use of model organisms such as Arabidopsis and rice has been driving these increments in knowledge. Similarly, tremendous amount of research delivered insights in the molecular pathways steering seed development, root growth and branching, plant architecture, tolerance to severe stress, cold tolerance and many more agronomic traits. All this information enforced the idea that by altering the expression of these genes, plant growth and possibly crop yield could be improved (i.e., wherein the gene set comprises members of a pathway, and wherein clustering the coding sequences is based on the functional or molecular characteristics of the pathway) (Inzé, page 2, paragraph 0002).
At the time the instant application was filed, it would have been obvious and within the scope of one of ordinary skill in the art to utilize the method as taught by GERSBACH and HYAMS, to alter the expression of gene sets of a pathway, as taught by INZÉ. Based on the teachings of GERSBACH and HYAMS, one of ordinary skill in the art would be motivated to use the method as taught by GERSBACH and HYAMS to alter the expression of gene sets of a pathway knowing that by altering the expression of these genes, plant growth and possibly crop yield could be improved, as taught by INZÉ. Thus, one of ordinary skill in the art would have a high expectation of success by following the teachings of GERSBACH, HYAMS, and INZÉ.
In regard to claim 20, INZÉ teaches using multiplex amplicon sequencing to assay for the efficiency of genome editing in the different generations of maize plants. The frequency of mutations is followed in a subset of plants. To this end, multiplex amplicon sequencing of all target genes is used. In short, primer pairs are designed to generate amplicons of the target genes. Next a highly multiplexed PCR is conducted and the amplicons are end-repaired. Subsequently, sample specific adapters are ligated, the library PCR amplified and sequenced. With this method hundreds of genes can be followed simultaneously in up to 96 individual plants (i.e., wherein the one or more sgRNAs further comprise at least one adaptor nucleotide, wherein the adaptor nucleotide facilitate amplification of the at least one library) (Inzé, page 11, paragraph 0061).
Double Patenting
The non-statutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A non-statutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on non-statutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a non-statutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claim 1 is provisionally rejected on the ground of non-statutory double patenting as being unpatentable over claims 20-22 of co-pending Application No. 19/526209 (reference application) (Shani et al., filed 10/10/2023 as PRO 63/589255). Although the claims at issue are not identical, they are not patentably distinct from each other because all of the claims in question are directed to a method of identifying a gene(s) underlying a phenotype by targeting said gene(s). Instant claim 1 and 19/526209 claim 22 share virtually identical wording, except for the addition of the phrase “wherein the expression of each said sgRNA sequences is controlled by at least one tissue-specific and/or developmental stage-specific promoter” in 19/526209 claim 22.
This is a provisional non-statutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Summary
No claim is allowed.
Correspondence
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CHRISTINA MEADOWS whose telephone number is (703)756-1430. The examiner can normally be reached Monday - Friday 9:00 am - 5:00 pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Amjad Abraham can be reached at 571-270-7058. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
CHRISTINA MEADOWS
Examiner
Art Unit 1663
/CHRISTINA L MEADOWS/Examiner, Art Unit 1663
/Amjad Abraham/SPE, Art Unit 1663
Prosecution Insights