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 currently pending and under examination.
Priority/Effective Filing Date
The present application was filed on October 25, 2023, and is a CON of PCT/US2022/026183, filed on April 25, 2022 and claims benefit of U.S. Provisional Patent Application No: 63/179,921, filed on April 26, 2021.
Drawings
The drawings filed on October 25, 2023 are acceptable.
Specification
Applicant is reminded of the proper language and format for an abstract of the disclosure.
The abstract should be in narrative form and generally limited to a single paragraph on a separate sheet within the range of 50 to 150 words in length. The abstract should describe the disclosure sufficiently to assist readers in deciding whether there is a need for consulting the full patent text for details.
The language should be clear and concise and should not repeat information given in the title. It should avoid using phrases which can be implied, such as, “The disclosure concerns,” “The disclosure defined by this invention,” “The disclosure describes,” etc. In addition, the form and legal phraseology often used in patent claims, such as “means” and “said,” should be avoided.
In the present case, the abstract uses phrases which can be implied “The disclosure provides…” and “…in some embodiments…”
The use of the terms: SPRI, KAPA, HiFi, Illumina, Geneious, Affymetrix, 454 Life Sciences, Solexa, Helicos Biosciences, Applied Biosystems, SMRT, Ion Torrent, Pacific Biosciences, Oxford Nanopore Technologies, Roche, Nextera, XVIVO, Lonza, Gemini Bio, Sigma, Milteny, Qiagen, QuBit, Agilent, Tapestation, and MiSeq, which are each a trade name or a mark used in commerce, has been noted in this application. The term 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.
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.
Claim Rejections - 35 USC § 112(b) - Indefiniteness
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.
Claims 1-20 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor, regards as the invention.
Claims 1, 18, and 20 require “a custom template switch oligo (TSO)”. It is unclear what is meant by “custom”. The specification does not define a particular TSO sequence, and the specification does not describe any customizations of a particular TSO sequence known in the art.
Claims 1, 2, 18, and 20 recite the phrase: “lysing the cells in the presence of dNTPs, a well-specified barcoded oligoDT primer comprising a unique molecular identifier (UMI), and a PCR handle”. It is unclear whether the “PCR handle” is intended to be a component of the well-specified oligoDT primer comprising a unique molecular identifier, or is intended to be a distinct component in the lysis system. As presently claimed, the claim phrase appears to recite the “PCR handle” as a distinct oligonucleotide in the reaction system. However, in step e (claim 1), the cDNA produced in step d is “incubat[ed]… with cDNA amplification primers that specifically bind the PCR handle and the TSO”, which appears to require that the PCR handle is at an end of the cDNA molecule corresponding to the 3’ oligo-dT sequence (i.e. the PCR handle is a component of the barcoded-oligo-dT primer) and the TSO is at the opposite end of the cDNA molecule corresponding to the 5’ end of the mRNA molecule to which the TSO binds during reverse transcription.
Claims 1 and 20 recite “genomic primers that specifically bind a region of interest” (step e) and “nested primers that specifically bind a region of interest” (claim 1, step f; claim 20, step g). It is unclear whether “a region of interest”, recited in step e of claims 1 and 20, is the same “a region of interest” recited in steps f and g of claims 1 and 20, respectively. In contrast, claims 2 and 18 clearly recite “further amplification of the genomic [region of interest] with nested primers” (claim 2, step d) or “nested primers capable of specific amplification of a region within the genomic [region of interest]” (claim 18, step g).
The recitations of “a region of interest” and “nested primers… a region of interest” in claims 1 and 20 introduce uncertainty as to whether the claim requires: a) a first step of amplifying a genomic region of interest using a pair of primers followed by a second step of amplifying a “nested” amplicon within the amplicon produced by the first step using a second pair of primers that bind to sequences within the first amplicon OR b) a first step of amplifying a genomic region of interest, “I”, followed by a second step of amplifying a genomic region of interest “II” using a set of nested PCR primers (i.e. 2 external primers and 2 internal primers for the region “II”).
Claim 1 and 2 recite “separating at least two of the cDNA, ADT libraries, and gDNA libraries” (claim 1 step g, claim 2 step e). It is unclear whether “separating at least two” of the three recited library types requires separating all of the library types from each of the others (i.e. A+B+C [Wingdings font/0xE0] A, B, C) or whether “separating at least two” requires separating any pair of two library types from the third library type (i.e. A+B+C [Wingdings font/0xE0] A, B+C (“A and B” and “A and C” are separated, etc.)).
Claims 1, 2, and 18 require incubating at least a portion of the libraries “with an exonuclease” (claims 1 and 2)… “to degrade unconsumed primers” (claim 18). It is unclear whether the claims are intended to encompass incubating the libraries with any exonuclease, or whether the claims are intended to be limited to exonucleases that selectively degrade single stranded DNA (ssDNA). Commercially available, non-selective exonucleases with activity on dsDNA and ssDNA are known in the art and appear to be encompassed by the claims as presently written (e.g. ExoV, T5 Exonuclease, etc.).
Claim 15, as presently written, is dependent upon claim 4 and recites the claim term “the alteration” in line 1. Neither claim 4, nor claim 1, upon which claim 4 depends, recites “an alteration”. Therefore, there is insufficient antecedent basis for this limitation in the claim. However, claim 14 recites “one or more of the cells comprises an alteration…”. It is unclear if the recited dependence of claim 15 upon claim 4 is due to a typographical error omitting the “1” in “claim 14”, or if the claim is meant to depend from claim 4 and is lacking antecedent basis for the claim term. In the interest of compact prosecution, claim 15 has been addressed in the 103 rejection(s) which follow as requiring “an alteration”.
Claims 3-17 and 19 are additionally indefinite because they depend from, and thus include the indefinite limitations of the independent claims 1 or 18.
Claim Rejections - 35 USC § 103
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.
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.
Claims 1-5, 8-14, 17-18, and 20 are rejected under 35 U.S.C. 103 as being unpatentable over Wei et al., “Towards a Single Cell Portrait of Rheumatoid Arthritis- Development of a Single Cell Multiomics Pipeline for Phase 2 of the Accelerating Medicine Partnership (AMP) – RA Network” [Abstract] Arthritis Rheumatol. 2019; 71 (suppl 10) November 10, 2019 in view of Stoeckius et al., “Simultaneous epitope and transcriptome measurement in single cells” Nature Methods Vol. 14, No. 9, pp. 865-871, July 31, 2017, Velten et al., “Identification of leukemic and pre-leukemic stem cells by clonal tracking from single-cell transcriptomics” Nature communications (2021) 12:1366 (published March 1, 2021), Foley et al., “Gene expression profiling of single cells from archival tissue with laser-capture microdissection and Smart-3SEQ” Genome Research 29:1816-1825 and supplementary information, and Kim et al., US 10,622,094 B2 (Issued April 14, 2020).
Regarding claims 1, 2, 18, and 20, Wei et al. teach methods for concurrently characterizing single cell genomic DNA and mRNA comprising: (a) labeling isolated cells with fluorescently (i.e. detectably) labeled antibodies that bind cell surface markers of interest, (b) labeling the detectably labeled cells with a panel of CITE-seq antibodies (i.e. oligo-conjugated antibodies), (c) index sorting the cells into single compartments, characterizing the cell surface marker expression of each cell (i.e. flow cytometry), and performing CITE-seq and single cell (sc) ATAC-seq (i.e. a genomic DNA assay) on the single cells (Wei et al., Figure 1 (reproduced below) and abstract)
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Wei et al. do not teach the methodological details of CITE-seq.
However, Stoeckius et al. teach the CITE-seq method comprises labeling single cells with antibodies coupled to barcoded and 3’ polyadenylated oligonucleotide tags “Antibody Derived Tags (ADTs)”, sorting single cells into individual compartments, lysing the individual cells in said compartments in the presence of dNTPs, barcoded oligo-dT primers comprising a PCR handle, and reagents for reverse transcription (i.e. a template switch oligo) (Stoeckius et al., page 865, column 2, paragraph 1 and figure 1A (reproduced below)).
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Stoeckius et al. further teach that the oligodT primers (and/or the antibody barcodes) may comprise unique molecular identifiers (Stoeckius et al, page 869, columns 1-2 bridging paragraph).
Furthermore, while Stoeckius et al. demonstrate CITE-seq using droplet-based compartmentalization of single cells, they further teach that CITE-seq is readily adaptable to microwell-based methods with no or minor customizations (Stoeckius et al., page 867, column 2, paragraph 2).
Stoeckius et al. further teach that amplified cDNAs and ADTs from the single cells in individual compartments comprising unique barcoding sequences are pooled together and the pooled cDNAs and ADTs can be separated by size and converted into Illumina libraries separately [or together] and may further be pooled after library synthesis to adjust their relative proportions to ensure that the required sequencing depth is obtained for each library (i.e. pooling samples from each well and subsequently separating the cDNAs from the ADT libraries prior to preparing the cDNA and ADT libraries for sequencing.
Additionally, Wei et al. do not teach amplifying a genomic (i.e. DNA) region of interest in addition to amplifying the cDNAs and ADTs produced during CITE-seq (i.e. genomic primers that specifically bind a region of interest… and nested primers [within the region of interest] wherein at least one of the nested primers comprises a barcode, a UMI, and a PCR handle or a capture sequence).
However, Velten et al. teach methods comprising single cell RNA-seq and targeted sequencing of genomic sites of interest (i.e. regions of interest) using targeting outer primers and nested inner primers comprising sequencing adapters (i.e. barcodes, PCR handle) (Velten et al., page 10, column 1, paragraphs 4-6).
Additionally, Foley et al. teach methods comprising measuring mRNAs in single cells using template switching oligonucleotides that are advantageously blocked with biotin (i.e. one member of a binding pair) at their 5’ ends to discourage concatenation of additional adapters (Foley et al., Supplemental file 1).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have combined the methods of Wei et al., comprising mRNA and CITE-seq in the same single cells and parallel ATAC-seq with the teachings of Stoeckius et al. comprising detailed CITE-seq methodology, Foley et al. comprising guidelines for optimal design of TSOs comprising biotinylated 5’ ends, and Velten et al., comprising mRNA and targeted genomic DNA sequencing in the same single cells. The ordinary artisan would have been motivated to combine these methods because: a) Wei et al. describe performing CITE-seq for simultaneous measurement of mRNA and antibody derived tags (i.e. proteins) in the same single cells and Stoeckius et al. is the publication disclosing the development of CITE-seq comprising the well-known methodological considerations disclosed in said publication, b) Foley et al. teach biotinylated TSOs reduce unwanted adapter concatenation, and c) Velten et al. teach that other single cell droplet- or microwell- based protocols for single cell RNA-seq and clonal tracking, such as single cell RNA-seq and single cell ATAC-seq do not provide adequate coverage on nuclear genomic sites of interest for clonal tracking and that their methods comprising targeted cDNA and genomic DNA sequencing using nested PCR primers provides increased coverage at sites of relevant genomic mutations (i.e. regions of interest) necessary for clonal tracking of cell lineage during aging, oncogenesis, and hematopoiesis (Velten et al., page 9, column 1-2 bridging paragraph).
Regarding the alternative embodiment recited by claim 1 (and required by claim 18) wherein the nested primers for targeted genomic sequencing comprise a capture sequence comprising a barcode, a UMI, and an exonuclease blocking agent and requiring treatment of the nested PCR product on the capture oligo with an exonuclease, Kim et al. teach methods for quantifying low abundance genomic DNA sequences comprising nested PCR comprising primers with unique tag sequences (i.e. barcodes, unique molecular identifiers), capturing tagged products, and digesting non-captured sequences with an enzyme that degrades ssDNA (Kim et al., column 16-18 and column 24, paragraph 1). In particular, Kim et al. teach such enzymes for removing unhybridized probes (i.e. capture probes, primers) include Exonuclease I (Kim et al., column 35).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have combined the methods described above with the teachings of Kim et al. comprising nested PCR amplification of low abundance DNA molecules (such as those from the single cells taught by Velten et al.) with primers comprising unique identifiers, sequencing barcodes, and capture sequences followed by degradation of unhybridized ssDNA (i.e. non-target molecules, unconsumed primers) with an exonuclease. The ordinary artisan would have been motivated to combine the teachings of Kim et al. with the methods of Wei et al. because Kim et al. teaches that such sequence capture methods are useful for separating target sequences from background signal (Kim et al., columns 16-18).
Regarding claim 3, Wei et al. teach sequencing the libraries (Wei et al., figure 1).
Regarding claim 4, Kim et al. teach that sequence selection (i.e. capture oligonucleotide addition) can be performed on genomic regions directly (i.e. before amplification) (Kim et al., column 19, paragraph 3).
Regarding claim 5, Kim et al. teach the exonuclease can be ExoI (Kim et al., column 35).
Regarding claims 8 and 9, Stoeckius et al. teach that amplified cDNAs and ADTs from the single cells in individual compartments comprising unique barcoding sequences are pooled together and the pooled cDNAs and ADTs can be separated by size and converted into Illumina libraries separately [or together] and may further be pooled after library synthesis to adjust their relative proportions to ensure that the required sequencing depth is obtained for each library (i.e. pooling samples from each well and subsequently separating the cDNAs from the ADT libraries prior to preparing the cDNA and ADT libraries for sequencing. Alternatively, Chen et al. teach that cDNA and gDNA can be separately prepared (Chen et al., page 1458, column 1, paragraph 6).
Regarding claims 10-12, Stoeckius et al. teach separating the cDNAs from the ADTs using SPRI beads wherein the cDNAs are greater than 300bp in length and the ADTs are ~180bp in length (i.e. less than 500bp in length) (Stoeckius et al., supplementary CITE-seq protocol). Similarly, Chen et al. teach separating cDNA and genomic DNA using bead size selection (i.e. SPRI beads) (Chen et al., page 1458, column 1, paragraph 6).
Methods for selectively purifying populations of nucleic acids based upon their different lengths are well known to the ordinary artisan, and modification of protocols to select for particular sizes of nucleic acids comprising particular size cutoffs are routine optimization steps within Next Generation Sequencing library kits widely known and available to the ordinary artisan.
Regarding claim 13, Wei et al. teach separation of cDNA and ADT libraries prior to or in parallel with preparation of a gDNA library (Wei et al., figure 1).
Regarding claim 14, Velten et al. teach methods comprising amplifying genomic regions of interest for clonal tracking comprising targeted cDNA and genomic DNA sequencing using nested PCR primers targeting sites of relevant genomic mutations (i.e. regions of interest, alterations of genomic DNA sequence relative to a reference genome sequence) necessary for clonal tracking of cell lineage during aging, oncogenesis, and hematopoiesis (Velten et al., page 9, column 1-2 bridging paragraph).
Regarding claim 17, Wei et al. teach sorting cells by detectable cell surface markers wherein the cell surface marker is CD45 (Wei et al., figure 1).
Claims 6, 7, and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Wei et al. in view of Stoeckius et al., Velten et al., Foley et al., and Kim et al., as applied to claims 1-5, 8-14, 17-18, and 20 above, and further in view of Li et al., “Substrate specificity-enabled terminal protection for direct quantification of circulating MicroRNA in patient serums” Chem. Sci. 2019, 10, 5616.
Regarding claims 6, 7, and 19, Wei et al. in view of Stoeckius et al., Velten et al., Foley et al., and Kim et al. teach methods for enriching nucleic acid subpopulations having a particular target sequence involving protecting target sequences from exonuclease digestion by hybridizing the target nucleic acid with primers specific to the target sequence, generating a double stranded product, and digesting single stranded nucleic acids with an exonuclease (Kim et al., column 16-17 bridging paragraph). Kim et al. further teach that the exonuclease may be Exo I (Kim et al., column 35). The references above do not teach a mechanism by which the target-sequence hybridized probe blocks the activity of Exonuclease I.
However, Li et al. teach that Exonuclease I is a 3’-5’ Exonuclease dependent on the presence of a 3’-OH on the (deoxy)ribose sugar ring of the 3’ terminal nucleotide in a polynucleotide. Li et al. tested a variety of 3’ modifications to a set of polynucleotides and found that 3’ phosphorylation of the terminal nucleotide demonstrated the strongest resistance to exonuclease I digestion (Li et al., abstract).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention of one of ordinary skill in the art to have modified the methods taught by Wei et al. in view of Stoeckius et al., Velten et al., Foley et al., and Kim et al. comprising a step of selective enrichment of a targeted genomic region of interest amplified by nested PCR using a capture oligonucleotide that protects the target amplicon from Exonuclease I digestion with the teachings of Li et al. that 3’ phosphorylation of polynucleotides confers excellent exonuclease I resistance. The ordinary artisan would have been motivated to modify the capture oligonucleotide taught by Wei et al. in view of Stoeckius et al., Velten et al., Foley et al., and Kim et al. with the 3’ phosphate modification taught by Li et al. because Kim et al. teaches that hybridization-dependent protection from exonuclease cleavage advantageously enriches target amplicons relative to non-specific sequences.
Claim 15 is rejected under 35 U.S.C. 103 as being unpatentable over Wei et al. in view of Stoeckius et al., Velten et al., Foley et al., and Kim et al., as applied to claims 1-5, 8-14, 17-18, and 20 above, and further in view of Papalexi et al., “Characterizing the molecular regulation of inhibitory immune checkpoints with multi-modal single-cell screens” bioRxiv (published June 28, 2020).
Regarding claim 15, Wei et al. in view of Stoeckius et al., Velten et al., Foley et al., and Kim et al. teach methods for concurrently characterizing genomic DNA regions of interest, mRNA, and antibody-derived tags in single cells that were sorted into individual wells using fluorescent antibodies against particular cell surface markers of interest wherein the genomic DNA regions of interest comprise genomic DNA sequence alterations (i.e. mutations) relative to a reference genome. Wei et al. in view of Stoeckius et al., Velten et al., Foley et al., and Kim et al. do not teach that the genomic DNA sequence alterations were introduced using a genome editing technique.
However, Papalexi et al. teach a method that combines pooled CRISPR screens (i.e. cells comprising genomic DNA sequence alterations introduced using a genome editing technique) with single cell mRNA and surface protein measurements with CITE-seq (Papalexi et al., Abstract).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have combined the methods taught by Wei et al. in view of Stoeckius et al., Velten et al., Foley et al., and Kim et al. comprising concurrently characterizing genomic DNA regions of interest, mRNA, and antibody-derived tags in single cells that were sorted into individual wells using fluorescent antibodies against particular cell surface markers of interest wherein the genomic DNA regions of interest comprise genomic DNA sequence alterations (i.e. mutations) relative to a reference genome with the method taught by Papalexi et al. comprising assaying panels of CRISPR-modified immune cells by the CITE-seq methodology taught by Stoeckius et al.
The ordinary artisan would have been motivated to combine the methods taught by Wei et al. in view of Stoeckius et al., Velten et al., Foley et al., and Kim et al. with the method taught by Papalexi et al. because of the teaching of Papalexi that leveraging multi-modal data (i.e. CITE-seq) in CRISPR screening allows for identification of transcriptional and post-transcriptional modes of regulation of complex cell phenotypes (i.e. suppression of T cell-mediated immune responses frequently observed in human cancers).
Furthermore or alternatively, the ordinary artisan would have been motivated to incorporate the nested PCR based measurement of specific genomic regions of interest taught by Wei et al. in view of Stoeckius et al., Velten et al., Foley et al., and Kim et al. with the methods of Papalexi et al. to confirm which particular CRISPR edits occurred in a particular single cell having a cell-surface protein and/or mRNA expression phenotype of interest.
Claim 16 is rejected under 35 U.S.C. 103 as being unpatentable over Wei et al. in view of Stoeckius et al., Velten et al., Foley et al., Kim et al., and Papalexi et al., as applied to claim 15 above, and further in view of Hanna et al., “Massively parallel assessment of human variants with base editor screens” Cell 184, 1064-1080 (published February 18, 2021).
Regarding claim 16, Wei et al. in view of Stoeckius et al., Velten et al., Foley et al., Kim et al., and Papalexi et al. teach methods comprising concurrently characterizing genomic DNA regions of interest, mRNA, and antibody-derived tags in single cells that were sorted into individual wells using fluorescent antibodies against particular cell surface markers of interest wherein the genomic DNA regions of interest comprise genomic DNA sequence alterations (i.e. mutations) relative to a reference genome and the alteration(s) was/were introduced by a genomic editing technique (i.e. CRISPR screening).
Wei et al. in view of Stoeckius et al., Velten et al., Foley et al., Kim et al., and Papalexi et al. do not teach that the genomic editing technique involves base-editing or homology-directed recombination editing.
However, Hanna et al. teach methods comprising screening CRISPR base-edited cells for point mutations that confer specific phenotypes including drug sensitivity and resistance, allowing for the identification of mutations that sensitize cells to drug treatments, which may be particularly useful for guiding the design of future inhibitors (Hanna et al., abstract and page 1075, column 1, paragraph 3).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have combined the methods taught by Wei et al. in view of Stoeckius et al., Velten et al., Foley et al., Kim et al., and Papalexi et al. comprising concurrently characterizing genomic DNA regions of interest, mRNA expression, and antibody-derived tags in single cells comprising genomic alterations introduced by genomic editing as part of a strategy for screening libraries of edited cells for genomic alterations and corresponding changes in mRNA and protein abundance with the methods taught by Hanna et al. comprising CRISPR screening using precise base-editing Cas9 enzymes for point-mutation level resolution of particular genomic alterations that are causal for particular phenotypes underlying responsiveness of particular cell types to particular drug treatments (Hanna et al., page 1077, column 1, paragraph 2), wherein “[base editing] libraries that introduce specific mutations into catalytic domains of potential drug targets may be a better surrogate for small molecule inhibition than either knockout or knockdown approaches” (e.g. the CRISPR-screens taught by Papalexi et al.) (Hanna et al., page 1077, column 1, paragraph 2). Furthermore, Hanna et al. teach that, in contrast to knockout screens which typically include multiple sgRNAs per gene (e.g. the CRISPR-screens taught by Papalexi et al.), base editing screens can typically create a given edit using a single or small number of sgRNAs (Hanna et al., page 1077, column 1, paragraph 2), reducing the requirement for sgRNA design and increasing the need for validation of particular genomic alterations introduced by said genomic editing.
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the methods taught by Wei et al. in view of Stoeckius et al., Velten et al., Foley et al., Kim et al., and Papalexi et al. comprising concurrently characterizing mRNA, ADT, and genomic regions of interest in single CRISPR edited cells sorted into individual wells based upon their expression of particular cell surface markers to comprise the improved CRISPR editing techniques (i.e. base-editing) taught by Hanna et al. The ordinary artisan would have been motivated to modify the CRISPR-screening methods taught by Wei et al. in view of Stoeckius et al., Velten et al., Foley et al., Kim et al., and Papalexi et al. with the base editing methods taught by Hanna et al. because of the teaching of Hanna et al. that base editing screens require fewer sgRNAs to obtain a given edit and can provide higher resolution genetic information in screens linking genetic alterations to particular phenotypes such as drug responsiveness (i.e. single nucleotide polymorphism rather than knockout of an entire gene) (Hanna et al., page 1077).
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure. Mimitou et al., “Multiplexed detection of proteins, transcriptomes, clonotypes and CRISPR perturbations in single cells” Nature Methods Vol. 16, pp 409-412 (published May 2019). Mimitou et al. is an earlier publication with similar authorship describing similar methods to those taught by Papalexi et al. Mimitou et al. teach methods wherein RNA-seq and CITE-seq were concurrently performed in single cells from a panel of CRISPR-screened cells, referred to as “ECCITE-seq” by the authors.
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ZACHARY MARK TURPIN whose telephone number is (703)756-5917. The examiner can normally be reached Monday-Friday 8:00 am - 5:00 pm.
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/Z.M.T./Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682