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 .
Please note: The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim Status
Claims 1-4 and 6-10 are pending.
Claims 6-10 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.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 without traverse in the reply filed on 8/6/2025.
Claims 1-4 are being examined on the merits.
Nucleotide and/or Amino Acid Sequence Disclosures
Applicant’s submission on 11/24/2025 of a substitute specification that includes an Incorporation by Reference paragraph required by 37 CFR 1.821(c)(1) is acknowledged.
Specification
The objection to the disclosure due to informalities and embedded hyperlinks is withdrawn in light of Applicant’s amendments to correct informalities and remove browser-executable code. It is also acknowledged that Applicant has made significant amendments to properly denote trade names and marks used throughout the specification.
Claim Objections
Claims 1 and 3 are objected to because of the following informalities:
Claim 1, line 17-19 reads “wherein evaluating the function of one ore more mutations includes classifying each mutation into at least two types based on changes in the frequency of the mutation during cell proliferation” and should read “wherein evaluating the function of the one [[ore]]or more mutations includes classifying each of the one or more mutations into at least two types based on changes in the frequency of the one or more mutations during cell proliferation”.
Claim 3, line 5, reads “and analyzing the validated measured data to evaluate one or more functions” and should read “and analyzing the validated measured data to evaluate [[one or more]]the functions”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112b - Indefiniteness
Withdrawn 112b Rejections: The rejections of claims 1-5 under 35 U.S.C. 112(b) in the Office Action of 8/25/2025 are withdrawn in light of Applicant’s amendments to the claims and cancellation of claim 5. The rejection of claim 1 under 112b for the lack of antecedent basis regarding “the base editors”, “the function”, and “the mutation” is withdrawn in light of Applicant’s amendments to claim 1.
New 112b Rejections (Necessitated by Amendments):
Claims 1-4 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.
Claim 1 is directed to a method that comprises “generating a cell library including a nucleotide sequence encoding a guide RNA, a unique molecular identifier (UMI) nucleotide sequence, and an oligonucleotide including a target nucleotide sequence targeted by the guide RNA”. Claim 1 has been amended to include the limitation “wherein the UMI nucleotide sequence and the oligonucleotide including a target nucleotide sequence targeted by the guide RNA are directly or indirectly linked together”. It is unclear by the language of the claim whether the guide RNA sequence element is also linked together with the UMI and the target sequence, or whether the cell library contains separate sequences: one encoding a guide RNA and one encoding a UMI and a sequence targeted by the guide RNA. For the purposes of examination, the examiner is interpreting all sequences to be connected, as is the case for the plasmid libraries depicted in Figure 2 and as described in a potential embodiment of the oligonucleotide in paragraph [0039] of the instant specification.
Additionally, claim 1 is directed to a method that contains the step of “transducing the cell library into cells expressing the base editors and culturing the cells”. It is unclear how a “cell library” is transduced into “cells”. Further clarification is required. The claim has been amended to include the limitation “wherein the cell library is transduced into the cells in the form of a viral or plasmid library”. This does not clarify the situation. It is not possible for it to be both a cell library and a viral or plasmid library as these are not the same thing.
Due to the above reasons, claim 1 is rendered indefinite.
Claims 2-4 depend from claim 1, inherit these deficiencies, and are rejected on the same basis.
Claim Interpretation
The method of claim 1 comprises a step of “transducing the cell library into cells expressing the base editors and culturing the cells…wherein the cell library is transduced into cells in the form of a viral or plasmid library”. For purposes of examination the examiner is interpreting a viral library as being transduced into the cells to create a cell library as depicted in the working example described in paragraphs [0104-0107].
Claim Rejections - 35 USC § 103
Modified as Necessitated by Amendments
Claims 1-4 are rejected under 35 U.S.C. 103 as being unpatentable over Hanna (Hanna et al., Cell Feb 18, 2021; cited on IDS of 8/15/2022) in view of Michlits (Michlits et al., Nature Methods 2017; cited on IDS of 3/21/2025), Arbab (Arbab et al., Cell 2020; cited on IDS of 3/21/2025), and Grünewald (Grünewald et al., Nature Biotechnology 2020).
Regarding claims 1 and 2: Hanna teaches a method of evaluating the function of mutations by creating a pooled cell library that comprises a library of sgRNAs and that expresses a cytosine base editor (claims 1 and 2; Abstract and Graphical Abstract). “sgRNA” reads on guide RNA as defined by the instant specification in paragraph [0035] (“The guide RNA may be, for example, a single guide RNA (sgRNA)”). Hanna teaches generating a lentiviral library with sgRNAs tiled across BRCA1 and BRCA2 which are two proteins known to be involved in cancer (claim 1; “Identification of known loss-of-function variants in BRCA1 and BRCA2” and “Validation of BRCA1 and BRCA2 loss-of-function alleles”). Hanna teaches transducing the sgRNA library into cells that express a cytosine base editor, culturing the cells, harvesting the cells and performing deep sequencing (claim 1; Figure 2a). Hanna teaches measuring the base editing conversion efficiency and the frequency levels of protein mutations due to base editing (claim 1; Figures 2BC and S2EF). Hanna teaches classifying mutated alleles as “depletion” alleles by examining the frequency of protein mutation of the transduced cells over time, in which a loss of function of BRCA1 or BRCA2 decreases cell viability and thus can be used as negative selection to determine the function of mutations as “loss-of-function” when “depletion” is measured (claim 1; “Validation of BRCA1 and BRCA2 loss-of-function alleles” and Figures S2 and 3FG). Hanna additionally teaches that this screening methodology can be used to classify alleles as enriched (reads on outgrowing; Figure 4F, Functional screens of 52,034 variants in ClinVar).
Hanna does not teach the inclusion of a UMI sequence with the gRNA in the lentiviral library that is transduced into cells. However, the inclusion of UMI sequences with a gRNA in CRISPR-based screening methods is known in the art, as taught by Michlits.
Michlits teaches a UMI-based pooled CRISPR screening approach in which a UMI sequence is associated with each sgRNA (Abstract, Figure 2C, and “Generating a complex CRISPR library with random barcodes”). Michlits teaches that the UMI and the sgRNA are within the same vector molecule (Figure 2C).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Hanna to include the UMI with the sgRNA, as taught by Michlits. One would be motivated to include the UMI given the assertion by Michlits that this addition “overcomes the current limitations of screen analyses, improves hit identification and quantification, and enables phenotypic assessment at the single-cell level” (Main, paragraph 2). One would have a reasonable expectation of success given that Michlits demonstrates the use of the UMI for tracking individual sgRNAs in a lentiviral pool that is transduced into cells for a CRISPR-based pooled screen, which is a similar technology as to that taught by Hanna.
Hanna in view of Michlits does not teach including the target sequence targeted by the guide RNA in the nucleotide sequence that is transduced into the cell library. However, inclusion of a target sequence with a gRNA (gRNA-target sequence pair) is known in the art, as taught by Arbab.
Arbab teaches a gRNA-target sequence pair library that is used to assess the base editing efficiency of various cytosine base editors (CBEs) and adenine base editors in mammalian cells (ABEs; Introduction, paragraph 3 and Figure 1A). Arbab teaches that the gRNA and the target sequence are within the same vector (Figure 1A).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Hanna in view of Michlits to include the target sequence of the sgRNA with the sgRNA, as taught by Arbab. One would be motivated to include the target sequence of the sgRNA in order to perform a parallel, unbiased assessment of the base editing efficiency of whichever base editor is being employed, as taught by Arbab (Results – Development of a Genome-Integrated Target Site Library Assay for Base Editors). One would have a reasonable expectation of success given that Arbab demonstrates the construction of a library of sgRNA-target sequence pairs that can be transduced into cells and subsequently sequenced and analyzed after base conversion by CBEs or ABEs.
Hanna in view of Michlits and Arbab teaches transduction of sgRNA libraries in cells that are expressing either CBEs or ABEs. Hanna in view of Michlits and Arbab does not teach transducing a lentiviral library into cells expressing multiple types of base editors (claims 1 and 2). However, expression of both CBEs and ABEs in base editor screening libraries is known in the art, as taught by Grünewald.
Grünewald teaches transducing 28 different gRNAs into cells coexpressing both ABE and CBE (‘ABE & CBE mix’) and determining summed frequencies of dual editing (Main, paragraph 3).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Hanna in view of Michlits and Arbab to coexpress both ABE and CBE in the cells, as taught by Grünewald. One would be motivated to express both base editors given the assertion by Grünewald that inclusion of both base editors allows one to “concurrently introduce A-to-G and C-to-T substitutions” and “expands the range of possible DNA sequence alteration, broadening the research applications of CRISPR base editors” (Abstract). One would have a reasonable expectation of success given that Grünewald demonstrates successful conversion frequencies of an sgRNA library that is transduced into cells expressing both ABE and CBE (Extended Data Fig. 5 and 6).
Regarding claims 3 and 4: Hanna teaches analyzing the function of mutations in which the frequency of target mutations within the cancer-associated target genes (BRCA1 and BRCA2) are 75% or more (Figure 2BC). Hanna teaches determining the base conversion frequency as well and notes target editing efficiencies of over 60% (Figure S2E). Hanna does not explicitly teach only analyzing functions of mutations that meet these specific criteria, however Hanna does indicate that the most interesting sgRNAs are those that produce strong hits in their analysis of the function of target mutations and “indicate that an initial editing efficiency of ~50% at day 7 was sufficient to detect depletion in the primary screen” (“Validation of BRCA1 and BRCA2 loss-of-function alleles” paragraph 4). While Hanna does not explicitly teach excluding data that does not meet these criteria, one would be motivated to do so given that Hanna teaches that editing efficiencies over a threshold are sufficient to allow determination of function of mutations. One skilled in the art would, through routine optimization, be able to determine the criteria that are most suited for the best analysis according to the cell types used, the sgRNAs designed, and the specific base editors employed. It is noted that the courts have found that “where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). See MPEP 2144.05 II.
Response to Remarks
Applicant has traversed the rejection of claims 1-5 as being obvious under 35 USC 103 over Hanna in view of Michlits, Arbab, and Grunewald (pages 9-18 of Remarks of 11/24/2025). Applicant's arguments have been fully considered but they are not persuasive for the following reasons.
Applicant’s summary of the claimed invention on pages 9-11 with emphasis being placed on the working examples provided in the specification is acknowledged.
Applicant asserts that Hanna employs an sg-RNA-based base-editor screening for the purpose of examining whether specific mutations introduced into the BRCA1 or BRCA2 gene result in loss of function (pg 12 of Remarks). While Hanna does acknowledge that some mutations such as nonsense and splice site mutations, result in loss of function, Hanna also specifically acknowledges that these are the mutations that are depleted relative to silent mutations. Hanna is merely pointing out the correlation between depleting alleles and a function of said mutations (namely, loss of function as confirmed by their impact on cellular proliferation). Hanna is emphasizing the depleting mutations rather than the enriched mutations in this particular context (BRCA1 and BRCA2 mutations), but Hanna also acknowledges that enrichment of alleles can also be determined (Method details: Pooled screens). Applicant then argues that Hanna differs from the claimed method due to the fact that the claimed method introduces mutations into “arbitrary target sequences” and analyzes function based on “mutation-frequency change” (pg 12 of Remarks). In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., that mutations are introduced into arbitrary sequences) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). Hanna is introducing a library of sgRNAs tiled across the BRCA1 and BRCA2 genes and analyzing mutation frequency with classification into depleted or enriched (Figure 2). There is no language within the claim that the library has to be genome wide. Additionally, the pre-determined answer labeling in the case of the current application is outgrowing or depleting, followed by association of this classification with proliferation of the cells. This is exactly what Hanna is doing, classifying sgRNAs as depleting sgRNAs and the associating this with cell proliferation. Applicant acknowledges that Hanna examines mutations as “depleted” (page 12 of Remarks) but contends that this was done solely to classify the mutation as pathogenic, which is what is associated with proliferation of the cells. Applicant argues that the validation performed by Hanna to confirm pathogenicity sets it apart from the current claimed invention. However, Applicant performs a similar validation following a screening of mutations (paragraphs [0133 and 200] of instant specification). Hanna classifies certain sgRNAs as depleting, and following this, takes the strongest hits and performs a functional validation to confirm that these mutations do indeed exhibit functional phenotypic effects on the cells (Figures 2 and 3).
As stated by Applicant on page 12 of Remarks, Hanna performs validation experiments following these classifications of sgRNAs as depleting merely to confirm that said sgRNAs do in fact induce mutations that have a phenotypic effect. The validation experiments are to confirm depleting phenotypes, not to classify said phenotypes in the initial screening. Hanna provides a reasoning for doing so, as acknowledged by applicant (“sgRNAs may deplete due to reproducible but unanticipated effects” – Validation of BRCA1 and BRCA2 loss-of-function alleles). Hanna still discloses classifying mutations as depleting, contrary to Applicant’s assertion on page 13 of Remarks that Hanna does not disclose this classification at all.
Applicant asserts on page 13 of Remarks that Hanna only uses sgRNAs predicted to induce mutations at particular loci of the BRCA1 or BRCA2 gene. This is incorrect, as Hanna employs a BE tiling library for both genes to cover the entire locus of each, not at specific loci within each gene (Identification of known loss-of-function variants in BRCA1 and BRCA2).
Applicant then argues that Hanna does not include the target sequence of each gRNA or a unique UMI corresponding to each gRNA (page 13 of Remarks). In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Applicant asserts that in the method of Hanna “it is not possible to determine (1) whether a gRNA correctly edited a specific target sequence, (2) whether base editing was efficiently achieved, or (3) what phenotype arises from the mutation introduced by base editing, all within a single integrated analysis”. Hanna demonstrates that with their set up, they are able to determine that a gRNA correctly edited a specific target sequence at a sufficient efficiency (“We assessed BE activity using the day 7 time point and calculated the editing efficiency for each C near the sgRNA (positions −10 to 20, where position 1 is the first nucleotide of the guide and positions 21–23 are the PAM sequence). We observed substantial C > T editing in the target window (positions 4 to 8; median = 46.6% C > T editing), with low levels of C > R editing and deletions”) and what phenotype arises from said mutation introduced by base editing (depletion/loss of function; citations above). Hanna performs validation experiments but they’re not essential for evaluating mutation function, they’re just a way to validate certain hits and confirm that their classification into a phenotype such as depleting is consistent.
Applicant argues that Michlits teaches a nuclease-type CRISPR system, which differs fundamentally from the base-conversion type system of the present claims and that Michlits does not disclose classifying mutations into depletion or outgrowth phenotypes or determining the effect on cellular proliferation (pages 13-14 and 16 of Remarks). In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Applicant argues on page 14 and 16 of Remarks that Michlits uses a “1 target gene – 5 sgRNAs” approach rather than the “1 target sequence – 1 gRNA” approach of the present claims. While there were 5 sgRNA per gene, each sgRNA had a distinct target sequence and a unique molecular identifier. The sgRNAs each had their own unique UMI and did not overlap (“We also avoided sgRNAs that were less than 4 nt away from another”, Guide Selection).
Applicant argues on page 14 of Remarks that Arbab does not disclose examining the function of a target sequence or target gene using a cell library that includes both a target sequence and its corresponding sgRNA or classifying mutations into depletion or growth phenotypes. In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Applicant argues on page 14 of Remarks that Grunewald does not disclose any method for evaluation mutation function using a base editor. In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Applicant argues on page 15 of Remarks that the amended claims differ from the cited art in the following ways:
Constructs a cell library including a gRNA, its corresponding target sequence, and a unique UMI sequence. The combination of Hanna in view of Michlits and Arbab teach this, as described in the 103 rejection above.
Transduces the gRNA and its target sequence into cells as a single library and are analyzed simultaneously. Hanna in view of Michlits and Arbab teaches transducing the gRNA and its target sequence into cells as a single library (citations above). In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., analyzing simultaneously) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
Evaluates function of base-editing-induced mutations by classifying the mutation into one of two types (outgrowing or depletion) according to changes in mutation frequency. Hanna teaches classifying mutations into a depletion type or an enrichment (outgrowing) type based on changes in mutation frequency during cell proliferation.
Improves accuracy of gRNA functional evaluation by utilizing UMI count for each gRNA as a third parameter to enable precise determination of mutations introduces into target sequences. This is an advantage of including UMIs in the gRNA library, as taught by Michlits and acknowledged in the 103 rejection above.
Enables all-in-one performance of gRNA-target sequence selection and phenotypic analysis of mutations to identify significant mutations from a large-scale cell-library data set without requiring additional individual validation steps. While individual validation steps are not required by the instant claims, the claims do not prohibit additional validation upon identification of a mutation as outgrowing or depletion, which is taught by Hanna.
Applicant argues on page 15 of Remarks that one or ordinary skill in the art would not have been able to derive or predict from Hanna, Michlits, Arbab, or Grunewald the method of the present invention. In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., analyzing simultaneously) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
Applicant argues on page 16 of Remarks that the previous Office Action “fails to explain why a person of skill in the art would have turned to Michlits if seeking to improve the very different method disclosed in Hanna”. As cited by Applicant on page 16, the previous Office Action points out that the CRISPR-based pooled screen used by Michlits is similar technology to the employed by Hanna. Both methodologies involve transducing a lentiviral library of sgRNAs into cells and screening for editing efficiency and mutation functions. Michlits teaches the benefits of including a UMI associated with each sgRNA in a mutation screen, such as that employed by Hanna. Applicant asserts that there is a missing motivation to combine, and that the Office Action “simply assumes a standing motivation to combine every feature of every reference in combination with every other reference”. The Examiner respectfully disagrees. A clear motivation to include a UMI in a screening analysis is provided in the Office Action, namely that Michlits teaches that including a unique UMI for each sgRNA in a screen analysis “improves hit identification and quantification, and enables phenotype assessment at the single cell level” (Main, paragraph 2).
Applicant points out on page 17 of Remarks that “to properly support a rejection on the basis that an invention is the result of ‘routine optimization’, the examiner must make findings of relevant facts and present the underpinning reasoning in sufficient detail." MPEP § 2144.II.B. The MPEP explains that detail is sufficient only if the Office provides both 1) "an explanation of why it would have been routine optimization to arrive at the claimed invention" and 2) an explanation of "why a person of ordinary skill in the art would have had a reasonable expectation of success to formulate the claimed range." MPEP § 2144.II.B.”. Applicant asserts that the Office Action of 8/25/2025 satisfies neither of these requirements. The Examiner respectfully disagrees. In the rejection of claims 3 and 4, it is noted that it would be routine to determine what the most useful threshold/criteria is depending on factors such as cell type used, base editor employed, or mutations analyzed. Any one of these factors may change what the effective threshold is for accurate analysis and evaluation. One of skill in the art would know that these parameters would change based on these factors. As noted in the rejection, one would have a reasonable expectation of success to formulate the claimed range given that Hanna teaches that an editing efficiency of “~50% at day 7 was sufficient to detect depletion in the primary screen” and indicates that the depleting mutations (low z score) are associated with mutations in the screen that are at around 70-80% frequency in the intended protein mutation (Figure 2BC).
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). 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 KAILEY E CASH whose telephone number is (571)272-0971. The examiner can normally be reached Monday-Friday 8:30am-6pm ET.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Anne Gussow can be reached at (571)272-6047. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/KAILEY ELIZABETH CASH/Examiner, Art Unit 1683
/ANNE M. GUSSOW/Supervisory Patent Examiner, Art Unit 1683