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 .
Claim Status
Claims 1-20 are pending on the application.
Claims 1-4 are examined on the merits.
Priority
This application is a national stage application, of International Patent Application No. PCT/US2020/043608, filed 07/24/2020, which claims priority from U.S. Provisional Application 62879348, filed 07/26/2019 is acknowledged.
Election/Restrictions
Claims 5-20 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected Group, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 10/15/2025.
Applicant’s election without traverse of Group I corresponding to claims 1-4, in the reply filed on 10/15/2025 is acknowledged.
.
Drawings
The drawings are objected to under 37 CFR 1.83(a) because they fail to show colors as described in the specification.
Fig. 10B, indicates the compact library (purple) in K562 cell (e.g., paragraph 0035).
Fig. 11A, indicates large-scale screen (dark purple), or predicted relative activities assigned by the CNN model ensemble (light purple) (e.g., paragraph 0036).
Any structural detail that is essential for a proper understanding of the disclosed invention should be shown in the drawing. MPEP § 608.02(d). Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Nucleotide and/or Amino Acid Sequence Disclosures
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiencies and the required response to this Office Action are as follows:
Specific deficiency – Nucleotide and/or amino acid sequences appearing in the drawings 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. See Fig. 5C.
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.
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 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.
Claims 1-4 are rejected under 35 U.S.C. 103 as being
unpatentable over Finnigan G. (“Finnigan” WO 2019/067011 A1, cited as reference 2 in IDS filed 01/25/2022) in view of Gilbert L. (“Gilbert” US 2017/0204407 A1, cited as reference 1 in IDS filed 01/25/2022).
Regarding claim 1-2, Finnigan teaches methods comprising
introducing into the eukaryotic cell a modified CRISPR-Cas gene drive
system, where the gene drive is expressed in the cell to produce functional CRISPR nuclease and single guide RNA (e.g., paragraph 2nd, lane 11, page 5; Fig. 1 [see below]). Finnigan teaches multiple independent approaches to regulate, control, titer, optimize, and/or inhibit gene drive systems, that can be used alone or in combination to provide a specific level of desired activity (e.g., paragraph 2nd, lane 11, page 10; Fig. 1). Finnigan teaches CRISPR/Cas9 gene editing or gene drive activity is modulated through modifications to the single guide RNA, such as through changing its length or creating a sequence "mismatch" to the proposed target sequence and reduce binding efficiencies, a 19 bp guide RNA with a single mismatch at the 5' end is demonstrated to reduce drive activity by about 50% when included in the CRISPR gene editing vector (haploid editing and diploid gene drive systems) (e.g., paragraph 2nd, lane 17, page 15). Finnigan teaches sgRNAs of lengths less than 20 bp, the 3' most segment of the target site was used. The mismatch(es) occur at the 5' end of the sgRNA guide sequence. G/C was (randomly) changed to A/T and vice versa. The penultimate bp at the 5' end of the sgRNA sequence was deleted (e.g., paragraph 5th, lane 19, page 31). Finnigan teaches guide lengths of 17 to 22 bp, we varied the number of 5' mismatches (one, two, or three consecutive mutations-A/T to G/C and vice versa, chosen at random) as well as deletion of the penultimate base at the 5' end (e.g., paragraph 2nd, lane 14, page 38; Fig. 4B [see bellow]). Finnigan teaches altering the 5' mismatch from G➔ A to either G➔ C or G➔ T resulted in a near loss of editing and a phenocopy of the 18 bp guide length sgRNA (e.g., paragraph 2nd, lane 2, page 39; Fig. 5). Finnigan teaches combinations of one or more of the foregoing approaches can also be used in to create a near limitless ability to finely tune the activity of the gene editing system in humans, animals and agricultural technology (e.g., paragraph 2nd , lane 3 , page 4). Finnigan teaches the editing drive system is tunable along a full spectrum of drive efficiencies (up to less than 99%) within a population, for example efficiencies, effectiveness, or activities that are less than about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91 %, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% and even less than about 5% as efficient, effective, and/or active than the same system without one or more of the modifications described herein. In other words, the efficiencies, effectiveness, or activities of the system are decreased by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% and even by at least 95% as compared to the same system without one or more of the modifications (e.g., paragraph 1st, lane 5, page 11).
Fig. 1
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Fig. 4B
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Finnigan does not teach CRISPR interference (CRISPRi) or CRISPR
activation (CRISPRa) and second and third sgRNA that targets the gene, as required by the instant claims 1-2. However, this is cured by Gilbert.
Regarding claim 1, Gilbert teaches sgRNAs are selected to inhibit
transcription of human target loci (e.g., targeted to optimized human
CRISPRi target sites), activate transcription of human target loci ( e.g.,
targeted to optimized human CRISPRa target sites) (e.g., paragraph 0013).
Gilbert teaches a library containing a plurality of sgRNAs, wherein the
plurality of sgRNAs comprise binding regions that are designed according
to one or more of the following criteria: designed to target a genetic
element that is at least 5 bp apart from a genetic element targeted by
another sgRNA of the library; designed to begin with a "G" nucleotide;
designed to have a mismatch scoring metric for off-target genetic elements of below a pre-designated thresh old (e.g., designed to have a mismatch scoring metric of less than or equal to 0); designed to target a region between, or between about, 0 and about 750 bp upstream of the
transcription start site (TSS) (e.g., paragraph 0106). Gilbert teaches method of optimizing an sgRNA, the method comprising: performing any of the foregoing methods, wherein the plurality of structurally distinct sgRNAs target different regions within or next to a single genetic element, wherein the most overrepresented or underrepresented sgRNAs in the selected cells are identified as optimized sgRNAs that target the genetic element (e.g., paragraph 0022). Gilbert teaches that the pair wise combinations of structurally distinct sgRNAs comprise a first member and a second member of the pair, and wherein the first member and second member target unlinked genetic elements (e.g., paragraph 0021). Gilbert teaches to evaluate the biological validity of the screen, genes were ranked by the average phenotype of their three strongest sgRNAs, and the 50 hits with the strongest protective effect and the 50 hits with the strongest sensitizing effect were defined as "top hits" (all of these are far outside of the range seen with otherwise matched negative control
sgRNAs) (e.g., paragraph 0209). Gilbert teaches rules for further improving CRISPRi library performance. (A) sgRNAs with longer
protospacers are less active. (B) sgRNAs with poly-U homotrimers, homotetramers or more have decreased activity. (C) Overall purine content of sgRNAs correlates with increased activity. (D) sgRNA activity varies depending on the base immediately following the PAM. (E) sgRNA activity
is optimal within a window from +25 to +100 bp (e.g., paragraph 0040; Fig. 14 [see below]).
Fig. 14:
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Regarding claim 2, Gilbert teaches that the pair wise combinations of structurally distinct sgRNAs comprise a first member and a second member of the pair, and wherein the first member and second member target unlinked genetic elements (e.g., paragraph 0021). Gilbert teaches that CRISPRi transcriptional silencing is highly sensitive to mismatches between target DNA site and the sgRNA (e.g., paragraph 0191). Gilbert teaches rules for CRISPRi off-target activity at endogenous genes, a set of highly active sgRNAs from the test library was selected. For each of these sgRNAs, the activity of a series of derivative sgRNAs with a variable number and position of mismatches was tested. This experiment allowed measurement of the amount of off-target gene repression for sgRNAs with mismatch base pairing relative to sgRNAs for which high on-target activity was established. Even a single mismatch at the 3' end of the protospacer decreased CRISPRi activity while sgRNAs that passed the bioinformatics off-target filter, showed very little activity (e.g., paragraph 0192; Fig. 2).
Fig. 2
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Gilbert teaches CRISPRi activity is highly sensitive to mismatches between the sgRNA and DNA sequence. The on- and off-target activity of dCas9, dCas9-KRAB and Cas9 was measured for a series of sgRNAs with a varying number and position of mismatches. Each sgRNA is a point with the related mismatch series connected by lines. The measured
off-target activity of each sgRNA with one or more mismatch is displayed as percent of the on-target activity for the corresponding sgRNA with 0 mismatches (e.g., paragraph 0036; Fig. 10 [see below]).
Fig.10:
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Regarding claims 3-4, Gilbert teaches sgRNAs are selected to inhibit
transcription of human target loci (e.g., targeted to optimized human
CRISPRi target sites), activate transcription of human target loci ( e.g.,
targeted to optimized human CRISPRa target sites) (e.g., paragraph 0013).
Finnigan and Gilbert are both directed to CRISPR-CAS transcriptional
modulation. It would have been prima facie obvious to one of ordinary skill in the art, at the time the invention was filed, to modify the method comprising -introducing into the eukaryotic cell a modified CRISPR-Cas gene drive system, where the gene drive is expressed in the cell to produce functional CRISPR nuclease and single guide RNA with mismatches in the single guide RNA to reduce specificity of the system to the target sequence taught by Finnigan with the teachings of Gilbert -sgRNAs selected to inhibit transcription or activate transcription of human target loci, to optimized containing a plurality of sgRNAs, where CRISPRi transcriptional silencing is highly sensitive to mismatches between target DNA site and the sgRNA, by varying number and position of mismatches- taught by Gilbert; considering the positional effects of sgRNA mismatches on the activity of CRISPR activation/inhibition, for a person of ordinary skill in the art it would have been obvious to achieve the predictable result of obtaining a set of rules to determine the positional effects of sgRNA mismatches on the second and third sgRNAs that target the genetic element suitable for modulation of gene transcription.
One of ordinary skill in the art would have been motivated to develop a method of CRISPR/Cas gene editing or gene drive activity modulated through modifications to the single guide RNAs, such as through changing its length or creating a sequence mismatch obtaining optimized sgRNAs to the proposed target DNA sequence and reducing binding efficiencies that target the genetic element for inhibition or activation of transcription of a human target loci for a treatment of a phenotype.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JULIO GOMEZ RODRIGUEZ whose telephone number is (571)270-0991. The examiner can normally be reached Monday - Friday 8:00 am - 5:00 pm.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Jennifer Dunston can be reached at 5712722916. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/JULIO WASHINGTON GOMEZ RODRIGUEZ/Examiner, Art Unit 1637
/J. E. ANGELL, Ph.D./Primary Examiner, Art Unit 1637