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 .
Response to Amendment
The amendment filed August 7th, 2025 is acknowledged. Regarding the office action mailed May 7th, 2025:
The objection set forth under Objection to the Specification is withdrawn as set forth below.
The rejection set forth under 35 U.S.C. 112(b) is maintained as set forth below.
The rejections set forth under 35 U.S.C. 103 have been modified in view of the amendments.
New prior art has been introduced as necessitated by the amendments.
Responses to arguments follow their respective rejection sections.
Withdrawn Objection to the Specification
The objection to the disclosure as set forth on page 2 of the Office Action of May 7th, 2025 is withdrawn in light of the amendment to the specification provided by the applicant in the reply of August 7th, 2025, which is entered.
Claim Status
Claims 1, 5-8, 14, 16, 18-20 has been amended. Claims 2-4 and 12-13 have been cancelled. Claims 21-22 have been added. Claims 1, 5-11, 14-22 are under examination and discussed in this Office Action.
Claim Rejections - 35 USC § 112(b) – Withdrawn
Any of the rejections of claims made under 25 USC 112 (b), as set forth in the Office Action of May 7th, 2025, which are not reiterated below have been withdrawn in light of the amendments to the claims.
Claim Rejections - 35 USC § 112(b) – Maintained
Claims 1, 5-11, 14-19, and 21-22 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
The term “near-instantly” in claim 1, upon which claims 5-11, 14-19, and 21-22 depend, remains in the amended claim and continues to render the claim indefinite. The term is a relative term that is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. The timing required to generate partitions remain unclear.
Applicant’s assertion that the amendments render the § 112(b) rejection moot is not persuasive. The term “near-instantly” has not been removed from claim 1 by the present amendment. Accordingly, the indefiniteness rejection is maintained.
Claims 5-11, 14-19, and 21-22 depend from claim 1, inherit these deficiencies, and are rejected on the same basis.
Claim Rejections - 35 USC § 103 – Modified as Necessitated by Amendments and
New – Necessitated by Addition of Claims
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.
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(s) 1, 15-19, 21, and 22 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang (WO 2018005691; previously cited) in view of Hatori et al (2018; previously cited), in further view Replogle et al (Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing. Nat Biotechnol 38, 954–961 (2020). https://doi.org/10.1038/s41587-020-0470-y).
In regards to instant claim 1, Zhang and Hatori, as applied in the previous Office Action, teach a method of droplet based single cell screening, wherein the cells are genetically modified using an RNA guide CRISPR/Cas9 system (Zhang, para 0008, 0114, 0115, 0125), coencapsulated as single cells with single template particles in uniform partitions (Hatori, Fig. 1, Fig 2; page 9818, right column, para 2; Fig. 3), in a tube without specialized microfluidic equipment (Hatori, Fig. 1; page 9815, column 1, para 1), with cells lysed within partitions to release RNA including RNA guides and RNA transcripts (Zhang, para 0131; Hatori, page 9813, right column, para 2).
Zhang and Hatori does not teach an RNA guide that is non-polyadenylated and that comprises a capture sequence in a constant region comprising a 3’ end or stem loop 2, nor the reverse transcription of RNA guide using a capture primer comprising a complement of RNA guide comprising the capture sequence and a barcode unique to the PIP, alongside reverse transcription of polyadenylated RNA transcripts using a polyT primer. However, these deficiencies are made up in the teachings of Replogle.
Replogle teaches the design of modified sgRNA constant regions containing capture sequences placed either within the stem loop 2 of the sgRNA constant region (sgRNA-CR1cs1) or appended at the 3’ end of the sgRNA constant region (sgRNA-CR1cs2) (Replogle, page 962, left column, para 2; Fig. 1b; Supplementary Fig. 1c). These sgRNAs are non-polyadenylated (Replogle, page 954, right column, para 2).
Replogle further teaches that target-specific RT primers, comprising sequences complementary to the capture sequences (CS1 and CS2) into sgRNA constant regions, are delivered concurrently with barcoded oligo-dT primers into single-cell droplet reactions (Replogle, page 955, left column, para 1; Fig. 1a,b). these capture sequence specific RT primers anneal to the capture sequences within the modified sgRNA constant regions and enable direct reverse transcription of the non-polyadenylated sgRNAs to produce indexed cDNA complements comprising cell barcodes unique to each partition and unique molecular identifiers (Replogle, Fig. 1b; page 954, right column, para 3 – page 955, left column, para 1). Polyadenylated mRNA transcripts are simultaneously reverse transcribed using the barcoded oligo-dT primers to produce second cDNA complements (Replogle, Fig. 1b; Supplementary Fig. 1a, b).
It would have been prima facie obvious before the effective filing date of the claimed invention for a person having ordinary skill in the art to to combine the direct capture method of Replogle with the single cell droplet method of Zhang and the near-instantaneous PIP generation method of Hatori. All three references are directed to the same field of single cell CRISPR screening with transcriptome, and Replogle identifies the limitation of polyadenylated indexing by teaching that recombination during lentiviral delivery can uncouple polyadenylated indices from their assigned sgRNAs and proposes direct capture of the non-polyadenylated sgRNA via constant region capture sequences as the solution (Replogle, page 954, right column, para 2). A person of ordinary skill in the art would have been motivated to apply Replogle direct capture method to the Zhang and Hatori platform to eliminate the recombination problem while maintaining compatibility with standard droplet based scRNA-seq, with a reasonable expectation of success given that Replogle demonstrates that method is fully functional in the 10x Chromium droplet platform (Replogle, page 960, right column, para 2).
Respond to the Argument
The applicant’s argument that Zhang teaches away from the claimed invention because Zhang’s stem loop contains a functional Tn5 target site is not persuasive. The combination does not require modification of Zhang’s stem loop. Instead, Replogle teaches a distinct sgRNA constant region design (CR1) with capture sequences into stem loop 2 or the 3’ end, which is not modification of Zhang’s gRNA scaffold. A person of ordinary skill in the art combining these references would use Replogle’s modified sgRNA design with Zhang’s screening framework, not modify Zhang’s existing stem loop.
In regards to instant claim 15, Zhang teaches the RNA guided endonuclease is a Cas9 endonuclease (Zhang, para 0125).
In regards to instant claim 16, Hatori teaches combining template particles with cells in a first aqueous fluid and adding an immiscible oil as a second fluid (Hatori, Fig. 1; page 9815, left column, para 1), and agitating by vertexing to generate PIPs that contain a single one of the template particles (Hatori, Fig. 1, 2; page 9817, left column, para 3) and a single one of the cells (Hatori, page 9818, right column, para 2), and releasing nucleic acid molecules from the cells by sysing each of the single cells contained within the PIPs (Hatori, page 9813, right column, para 2; Zhang, para 0131).
In regards to instant claim 17, Hatori teaches the first and second fluids are immiscible, specifically an aqueous solution and oil (Hatori, Fig. 1).
In regards to instant claim 18, Hatori teaches that combining cells and template particles and generating uniform partitions are performed in a PCR tube (Hatori, Fig. 1; page 9815, left column, para 1).
In regards to instant claim 19, Hatori teaches that the generated partitions include some that encapsulate a single template particle without encapsulating a cell (Hatori, page 9818, right column, para 2).
In regards to instant claim 21, Replogle teaches placement of the capture sequence at the 3’ end of the sgRNA constant region (sgRNA-CR1cs2) with capture sequence CS2 having reverse complement 5′-GCTCACCTATTAGCGGCTAAGG-3′ incorporated as RT primer (Replogle, page 962, left column para 2).
In regards to instant claim 22, Replogle teaches placement of the capture sequence within the stem loop 2 of the sgRNA constant region (sgRNA-CR1cs1) with capture sequence CS1 having reverse complement 5′-GCTCACCTATTAGCGGCTAAGG-3′ incorporated as RT primer (Replogle, page 962, left column, para 2). Replogle further teaches that stem loop 2 incorporation (CS1) is the preferred configuration, as 3’ end incorporation if CS1 comprises guide activity (Replogle, page 955, left column, para 1).
Claims 5-9 and 14 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang (WO 2018005691; previously cited) in view of Hatori et al (2018; previously cited) and Replogle et al (Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing. Nat Biotechnol 38, 954–961 (2020). https://doi.org/10.1038/s41587-020-0470-y), as applied to claim 1 above, and further in view of Adamson et al (2016; previously cited) and Datlinger (2017; previously cited).
In regards to instant claim 5, Adamson teaches that cDNA molecules are indexed with a unique molecular identifier during reverse transcription within each partition (Adamson, page 1868, right column, para 1; Fig. 1A).
In regards to instant claim 6, Adamson teaches that cDNA complements of RNA transcripts further comprise a cell barcode unique to the partition, affixed during reverse transcription (Adamson, page 1868, right column, para 1; Fig. 1A).
In regards to instant claim 7, Adamson teaches sequencing the cDNA complements to generate sequence reads associated with each RNA guide and each RNA guide and each RNA transcript (Adamson, page 1868, right column, para 1-2; Fig. 1C, 1D, 1E).
In regards to instant claim 8, Adamson teaches associating each RNA guide sequence read with each RNA transcript sequence read having the same cell barcode unique to the partitions (Adamson, page 1868, right column, para 1; Fig. 1A).
In regards to instant claim 9, Datlinger teaches that the RNA guided endonuclease and RNA guide modify the expression of RNA transcripts in the cell as compared to a cell in the absence of the RNA guide (Datlinger, page 297, right column, para 2).
In regards to instant claim 14, Replogle and Adamson teaches combining the capture and polyT primer with cells and template particles and encapsulating these primers into each partition (Replogle, Fig. 1B; page, 955, left column, para 1; Adamson, page 1868, right column, para 1-2; Fig. 1A, 1B).
It would have been prima facie obvious before the effective filing date of the claimed invention for a person having ordinary skill in the art to use the methods of Adamson and Datlinger with the method of Zhang, Hatori, and Replogle, for the same reasons set forth in the previous Office Action regarding Adamson and Datlinger.
Claims 10 and 11 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang (WO 2018005691; previously cited) in view of Hatori et al (2018; previously cited), Replogle et al (Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing. Nat Biotechnol 38, 954–961 (2020). https://doi.org/10.1038/s41587-020-0470-y), Adamson et al (2016; previously cited) and Datlinger (2017; previously cited), and Datlinger (2017; previously cited), as applied to claim 9 above, and further in view of Abate (US 20170022538; previously cited).
In regards to instant claim 10, Abate teaches a method of droplet based single cell sequencing where cells can be tested for genetic disorders (Abate, 0182).
In regards to instant claim 11, Abate teaches a method where the genetic disorder is cancer (Abate, 0074; 0182; 0193).
It would have been prima facie obvious before the effective filing date of the claimed invention for a person having ordinary skill in the art to use the method of Abate with the combined method of Zhang, Hatori, Replogle, Adamson, and Datlinger because Abate teaches that droplet based method enables detection of cancer at lower concentrations and with greater accuracy than existing commercial products and published droplet techniques (Abate, 0437). Thus, the addition of the method of Abate would be a combination of prior art elements to yield predictable results.
Claim 20 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang (WO 2018005691; previously cited) in view of Hatori et al (2018; previously cited), and in further view of Adamson et al (2016; previously cited).
In regards to instant claim 20, Zhang teaches a method of droplet based single cell screening, wherein cells are genetically modified using an RNA guided CRISPR/Cas9 system (Zhang, para 0008, 0114, 0115, 0125), and coencapsulated as single cells with single template particles with cells lysed within partitions to release RNA (Zhang, para 0131; Fig. 2) reading on the limitations of combining a plurality of cells with template particles and releasing nucleic acid molecules from encapsulated cells. Zhang teaches both polyadenylated RNA guide barcodes and non-polyadenylated copies of RNA guides (Zhang, Fig. 6; para 169), reading on the limitations of “a first RNA molecule comprising a non-polyadenylated copy of the RNA guide” and “a second RNA molecule comprising a polyadenylated RNA guide” of the instantly rejected claim 20.
Adamson teaches that the Perturb-seq contains both an RNA polymerase III-driven sgRNA expression cassette, which produces a non-polyadenylated RNA guide, and a separate RNA polymerase II-driven barcode (GBC) that produces a polyadenylated transcript encoding the guide barcode (Adamson, page 1868, right column, para 2). The polyadenylated GBC is captured alongside mRNA using polyT oligo-dT primers during single cell RNA-seq (Adamson, page 1868, right column, para 1). Reverse transcription of the polyadenylated guide barcodes and RNA transcripts using polyT capture primers to form cDNA complements further comprising a barcode unique to the partition (Adamson, Fig. 1A4-5, UMI, CBC, GBC).
Respond to the Argument
Applicant’s argument that Zhang, Hatori, and Adamson do not disclose an RNA molecule that includes a guide barcode provided with a non-polyadenylated RNA guide is directly contradicted by Adamson, Figure 1B and page 1868, right column, para 1-2, which shows both molecules, the non-polyadenylated sgRNA expressed from an RNA polymerase III cassette and the polyadenylated GBC expressed from an RNA polymerase II cassette present together within the same lentiviral and expressed within the same cell. The claim limitation of comprising “a first RNA molecule comprising a non-polyadenylated copy of the RNA guide and a second RNA molecule comprising a polyadenylated RNA guide barcode” is directly taught by this two-cassette.
It would have been prima facie obvious before the effective filing date of the claimed invention for a person having ordinary skill in the art to combine the two-molecule detection of Adamson with the single cell droplet based screening method of Zhang and the PIP generation method of Hatori. All three references are directed to the same problem of linking CRISPR perturbation identity to single-cell transcriptome readout in droplet based platforms, and a person of ordinary skill in the art would have been motivated to combine them with a reasonable expectation of success because Adamson teaches that the GBC provides rich biological insights and enables systematic dissection of complex biological responses (Adamson, page 1868, left column, para 3).
Applicant argues that Zhang and Hatori fails to teach a non-polyadenylated RNA guide comprising a capture sequence in a constant region comprising a 3’ end or stem loop 2, and that Zhang’s polyA approach teaches away from this limitation. The Examiner acknowledges that Zhang and Hatori alone do not teach this feature, and the rejection have been modified to include Replogle as an additional reference. Replogle teaches non-polyadenylated sgRNAs with capture sequences engineered into stem loop (sgRNA-CR1cs1) or the 3’ end (sgRNA-CR1cs2) of the sgRNA constant region, which are then captured by complementary RT primers to directly produce indexed cDNA complements alongside polyT primed mRNA cDNA complements. Applicant’s teaching away argument regarding Zhang’s stem loop is not persuasive because the combination uses Replogle’s sgRNA constant region design in Zhang’s framework.
Conclusion
All claims stand rejected.
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Nura Choudhury whose telephone number is (571)272-6148. The examiner can normally be reached M-F, 9-5 ET.
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/NURA M. CHOUDHURY/Examiner, Art Unit 1683
/ANNE M. GUSSOW/Supervisory Patent Examiner, Art Unit 1683
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