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 .
This office action has been formulated as second non-final rejection since the previous rejection has been modified using new arts and new objections has been added, see below.
Election/Restrictions
Applicant's amendments of claims in the reply filed on 03/16/2026 is acknowledged. Claims 103-104, 108-114, 128-131, 133, 135-153 are pending.
Claims 136-153 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made with traverse in the reply filed on 07/16/2024.
Applicant is reminded that upon the cancelation of claims to a non-elected invention, the inventorship must be corrected in compliance with 37 CFR 1.48(a) if one or more of the currently named inventors is no longer an inventor of at least one claim remaining in the application. A request to correct inventorship under 37 CFR 1.48(a) must be accompanied by an application data sheet in accordance with 37 CFR 1.76 that identifies each inventor by his or her legal name and by the processing fee required under 37 CFR 1.17(i).
Thus claims 103-104, 108-114, 128-131, 133, 135 are examined in this office action.
Nucleotide and/or Amino Acid Sequence Disclosures
This application contains sequence disclosures that are encompassed by the definitions for nucleotide and/or amino acid sequences set forth in 37 CFR § 1.821(a)(1) and (a)(2). However, this application fails to comply with the requirements of 37 CFR §§ 1.821 through 1.825 for the reason(s).
These requirements will not be held in abeyance and must be addressed in response to this Office action.
Following figures fail to comply with the sequence rules.
Drawing Figure 2 has 12 nucleotide sequences and 2 amino acid sequences that has more than 10 nucleotide and amino acid sequences but there are no sequence identifiers (SEQ ID NO: ).
Drawing Figure 4 has 1 nucleotide sequences that has more than 10 nucleotide sequences but there are no sequence identifiers (SEQ ID NO: ).
Drawing Figure 5 has 8 nucleotide sequences that has more than 10 nucleotide acid sequences but there are no sequence identifiers (SEQ ID NO: ).
Drawing Figure 5 has 3 nucleotide sequences that has more than 10 nucleotide acid sequences but there are no sequence identifiers (SEQ ID NO: ).
Drawing Figure 7 has 8 nucleotide sequences that has more than 10 nucleotide acid sequences but there are no sequence identifiers (SEQ ID NO: ).
Drawing Figure 8 has 2 nucleotide sequences that has more than 10 nucleotide acid sequences but there are no sequence identifiers (SEQ ID NO: ).
Drawing Figures 9 and 10 have 8 nucleotide sequences each that has more than 10 nucleotide acid sequences but there are no sequence identifiers (SEQ ID NO: ).
Drawing Figure 11 has 3 nucleotide sequences that has more than 10 nucleotide acid sequences but there are no sequence identifiers (SEQ ID NO: ).
Drawing Figure 14 has 2 nucleotide sequences that has more than 10 nucleotide acid sequences but there are no sequence identifiers (SEQ ID NO: ).
Drawing Figure 16, 20-21, and 23-24 have 2 nucleotide sequences each that has more than 10 nucleotide acid sequences but there are no sequence identifiers (SEQ ID NO: ).
Drawing Figure 29 has 3 nucleotide sequences that has more than 10 nucleotide acid sequences but there are no sequence identifiers (SEQ ID NO: ).
Specification fail to comply with the sequence rules.
Specification pages 41, 43-48, 50-54, 56-64 comprise nucleotides and amino acid sequences in multiple places but there are no sequence identifiers (SEQ ID NO: ).
Each sequences showed in figure and specification should include sequence identifiers (SEQ ID NOs:).
If any of these sequences are not included in sequence listings they must be included in sequence listings. Each sequence with the mutation must have an independent sequence ID NO and must be listed in the sequence listing as an independent sequence.
Applicant must include each of the sequences and variants of sequences described in the specification as the disclosure of invention in the listings of the sequences.
Applicant are advised that the issue will not be held in abeyance.
Specific deficiency – Nucleotide and/or amino acid sequences appearing in the drawings and specification are not identified by sequence identifiers in accordance with 37 CFR 1.821(d). Sequence identifiers for nucleotide and/or amino acid sequences must appear either in the drawings or in the Brief Description of the Drawings and specifications.
Required response – Applicant must provide:
Replacement and annotated drawings in accordance with 37 CFR 1.121(d) inserting the required sequence identifiers;
AND/OR
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required sequence identifiers into the Brief Description of the Drawings, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
New listings of the variants of sequences not currently in sequence listings.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code for example in page 44, second paragraph, and page 36, last paragraph. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
The use of the term “Addgene” in page 22, paragraph 3, page 37, line 1, “Bio-rad” in page 37, “Eppendorf” in page 37, “Tsingke” in page 37, which are a trade names or marks used in commerce, have been noted in this application. The terms should be accompanied by the generic terminology; furthermore the terms should be capitalized wherever they appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM, or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Drawings
Color photographs and color drawings are not accepted in utility applications unless a petition filed under 37 CFR 1.84(a)(2) is granted, see Figure submitted on 11/24/2020 under file name 83-97.pdf which includes multiple color drawings and photographs. Any such petition must be accompanied by the appropriate fee set forth in 37 CFR 1.17(h), one set of color drawings or color photographs, as appropriate, if submitted via the USPTO patent electronic filing system or three sets of color drawings or color photographs, as appropriate, if not submitted via the via USPTO patent electronic filing system, and, unless already present, an amendment to include the following language as the first paragraph of the brief description of the drawings section of the specification:
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Color photographs will be accepted if the conditions for accepting color drawings and black and white photographs have been satisfied. See 37 CFR 1.84(b)(2).
Claim Objections
Claims 110 and 133 are objected to because of the following informalities:
Regarding claims 110 and 133, since the phrase “RNP complex” has not been expressly defined in specification, applicants are advised to clearly defined the first time it appears in the claim as stated in page 22 paragraph 3 as “ribonucleoprotein (RNP) complexes”.
Appropriate correction is required.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(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.
Anticipated by McKenna et al.
Claims 103-104 and 108-114 are rejected under 35 U.S.C. 102 (a)(1) as being anticipated by McKenna et al. (Published: 2016, Journal: Science 353 (6298) pages 1-13).
Claims are drawn to a method of generating a mutation in an organism by sequentially generating two or more DNA breaks at a specific site of the genome of the organism, spontaneously repairing them and generating later DNA breaks wherein the DNA breaks are generated by a CRISPR/Cas system comprising nuclease and target RNA.
Regarding claims 103-104, McKenna et al. discloses a method of generating a mutation in Zebrafish D. rerio genome by sequentially generating two or more DNA breaks at 10 CRISPR/Cas9 unique targets edited by distinct sgRNAs to create diverse alleles (page 2, right paragraph 2, page 4, mid paragraph 2), the embryo captured in different time point showed that with increase in timepoint the average number of site edited increases over time (se Figure 3 below). McKenna et al. discloses their results showed the edits introduced to the barcode (i.e. specific site) during embryogenesis are inherited through development and tissue homeostasis and can be detected in adult organs (page 5, right paragraph 1). The mutation happened across many cell divisions (page 8, left paragraph 1).
McKenna et al. discloses their method uses CRISPR/Cas9 genome editing to accumulate combinatorial sequence diversity to a compact multitarget, densely informative barcode (i.e. target sites). (see Figure 1 below).
McKenna et al. discloses intertarget deletions involving sites 1, 3, and 5 or focal edits of sites 1 and 3 (Fig. 1, B and C, and table S1) showing combinatorial editing of specific site would give rise to large number of unique alleles (mutations) (page 2, right paragraph 3).
Therefore McKenna et al. discloses a method of generating mutation by sequentially generating two or more DNA breaks at a specific site in a genome of the Zebra fish.
The later DNA break in following generations of cells would have been generated based on new sequence since first DNA break would produce new sequence in the organismal genome when there will be either insertion and deletion caused by first DNA break.
Later DNA break would target a new sequence since a new sequence is generated by the previous DNA break. Since applicant has not specifically recited what is size of the new sequence, the previous DNA break would create new sequence by the insertions and deletions. Site of barcode in the Zebra fish would be the specific site where two or more DNA breaks has been created and again further modified with new mutation during the further development of embryo or replication during cell development.
McKenna et al. does not use template in their method.
Regarding claim 108, McKenna et al. discloses to generate mutant alleles they injected Cas9 and ten different sgRNAs that complement to the barcode target site wherein different generations were observed for 4.3, 9, 30 and 72 hpf (hours post fertilization) (see Figure 3).
Regarding claim 109, McKenna et al. disclose their method include clones derived from single cells (i.e. same recipient cell) (page 4, left paragraph 3, mid-paragraph 2).
Regarding claim 110, McKenna et al. Figure 3 discloses one cell zebrafish embryo were injected with complexed Cas9 RNPs containing sgRNAs that matched each of the 10 targets in the array (see Figure 3 below).
Regarding claim 111, Cas9 nuclease is capable of performing genome editing (see Figure 3 above).
Regarding claims 112-113, target nuclease Cas9 is in plasmid (i.e. DNA) (McKenna et al., Supplementary data, page 3, paragraph 3).
Regarding claim 114, McKenna et al. discloses the liposome mediated cell transfection (i.e. using Lipofectamine 3000 (Life Technologies)) (McKenna et al., Supplementary data, page 3, paragraph 2).
Therefore McKenna et al. anticipate the method of generating a mutation.
PNG
media_image1.png
1057
1771
media_image1.png
Greyscale
PNG
media_image2.png
864
1231
media_image2.png
Greyscale
Fig. 1. GESTALT. (A) An unmodified array of CRISPR/Cas9 target sites (i.e., a barcode) is engineered into a genome (gray cell). Editing reagents are introduced during expansion of cell culture or in vivo development of an organism, resulting in a unique pattern of insertions and deletions (right) that are stably accumulated in specific lineages (green cell lineage). The lineage relationships of alleles that differ in sequence can often be inferred on the basis of these accumulated edits. (B) The 25 most frequent alleles from the edited v1 barcode are shown. Each row corresponds to a unique sequence, with red bars indicating deleted regions and blue bars indicating insertion positions. Blue bars begin at the insertion site, with their width proportional to the size of the insertion, which will rarely obscure immediately adjacent deletions. The number of reads observed for each allele is plotted at the right (log10 scale; the green bar corresponds to the unedited allele). The frequency at which each base is deleted (red) or flanks an insertion (blue) is plotted at the top. Light gray boxes indicate the location of CRISPR protospacers, and dark gray boxes indicate protospacer adjacent motif (PAM) sites. For the v1 array, inter-target deletions involving sites 1, 3, and 5 or focal (single target) edits of sites 1 and 3 were observed predominantly. (C) A histogram of the size distribution of insertion (top) and deletion (bottom) edits to the v1 array is shown. The colors indicate the number of target sites affected. Although most edits are short and affect a single target, a substantial proportion of edits are inter-target deletions. (D) We tested three array designs in addition to v1, each comprising 9 to 10 weaker off-target sites for the same sgRNA (v2 to v4) (22). Editing of the v2 array is shown with layout as described in (B). Editing of the v3 and v4 arrays is shown in fig. S3, A and B. The weaker sites within these alternative designs exhibit lower rates of editing than the v1 array but also a much lower proportion of intertarget deletions. (E) A histogram of the size distribution of insertion (top) and deletion (bottom) edits to the v2 array is shown. In contrast with the v1 array, almost all edits affect only a single target.
Anticipated by Sadhu et al.
Claims 128-131, 133 and 135 are rejected under 35 U.S.C. 102 (a)(1) as being anticipated by Sadhu et al. (Published:2016, Journal: Science, 352 (6289): 1113-1116).
Claims are drawn to method of screening editing events by sequentially generating two more DNA breaks at a specific site of a first target gene and spontaneously repairing them wherein later DNA break is generated based on a new sequence generated from a repair of previous DNA break. The claims are drawn to generating mutant cell with phenotypic selectable trait and selection of the trait.
Regarding claims 128 and 131, Sadhu et al. discloses 95 gRNAs targeting the bacterial Streptococcus pyogenes Cas9 to sites distributed across the left arm of the yeast Saccharomyces cerevisiae chromosome 7 (Chr 7L). Sadhu et al. discloses the gRNAs targeted heterozygous sites wherein after cutting, repair and mitosis and CRISPR induced mutation, the four GFP- (i.e. GFP negative) lines per targeted site were picked of total 384 lines (~96 targeted sites) (page 113, middle last paragraph).
Sadhu et al. discloses 75% of LOH recombination events occurred within 20 kb of the targeted site (page 113, right first paragraph). Gene for manganese sensitivity was mapped in yeast strain BY wherein variants encodes a phenylalanine in BY and a leucine in yeast strain RM at position 548 of Pmrl, a manganese transporter gene (page 113, right last paragraph).
Therefore Sadhu et al. discloses a sequentially generating two or more breaks at a specific site of a first target gene of manganese sensitivity of the yeast cell.
Sadhu et al. further discloses introducing into the strain BY the alleles of RM of Pmr1-F548L and other neighboring polymorphism using a CRISPR-based variant replacement approach where in polymorphism F548L had significant manganese resistance (see Figure 4 below). The new introduction was based on the information of new sequence generated from a repair of previous DNA break.
PNG
media_image3.png
793
1080
media_image3.png
Greyscale
Therefore Sadhu et al. discloses the later DNA break was generated based on the information of recombination of BY and RM strains chromosome fragment due to the first DNA break int the chromosome showing F548L polymorphism causes the manganese resistance.
The method generated a mutant cell with a manganese resistance in BY strain after the specific site of the manganese sensitivity gene was cut and repaired to produce the recombinant fragment from strain RM producing phenotypic selectable trait of manganese resistance that was used to apply selection pressure to make selection for manganese resistant in recombinant lines of yeast (page 113, right last paragraph).
Regarding claim 129, the manganese resistance is phenotypic selectable trait and has growth advantage.
Regarding claim 130, The cutting and repair produced the substitution of many bases from strain RM to BY.
Regarding claim 133, Sadhu et al. discloses 95 gRNAs targeting the bacterial Streptococcus pyogenes Cas9 to sites distributed across the left arm of the yeast Saccharomyces cerevisiae chromosome 7 (Chr 7L) which would have produced DNA breaks.
Regarding claim 135, Sadhu et al. teaches their Cas9 was introduced on a plasmid (i.e. DNA) (page 2, last paragraph).
Therefore Sadhu et al. anticipate the claims .
Response to Applicant’s Argument
Applicant's arguments filed 03/16/2026 have been fully considered but since the claims are examined with new art rejection they are moot point.
Conclusion
No claim is allowed.
Examiner’s Contact Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to SANTOSH SHARMA whose telephone number is (571)272-8440. The examiner can normally be reached Mon-Fri 8:00 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 A. 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.
/SANTOSH SHARMA/Examiner, Art Unit 1663
/DAVID H KRUSE/Primary Examiner, Art Unit 1663
Prosecution Insights