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, 4, 7, 17, 19, 37, and 40 have been amended. Claims 16, 18, and 41 have been canceled. Claims 51-77 which were withdrawn previously, have now been canceled as well. Claims 1-15, 17, 19-40, and 42-50 are pending and under examination. Claims 1 and 37 are independent claims.
Response to Arguments
Objections Withdrawn
The objection to claim 2 has been withdrawn following the applicants’ amendments.
Rejections Withdrawn
The rejection of claims 4-13, 15-18, and 40-41 under 35 U.S.C. § 112(a) for failing
to comply with the enablement requirement regarding the non-specific binding of CRISPR/Cas9 is withdrawn following the applicants’ amendments.
The rejection of claims 18 and 41 under 35 U.S.C. § 112(a) for failing to comply with the enablement requirement pertaining to the use of chemical compounds for making CRISPR/Cas9 less sequence specific is withdrawn following the applicants’ amendments and cancelation of the claims.
The rejection of claim 16 under 35 U.S.C. § 112(a) for failing to comply with the written description requirement is withdrawn following the applicants’ amendments and cancellation of the claim.
The rejection of claims 1-50 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to particularly point out and distinctly claim the subject matter which the inventor regards as the invention is withdrawn following the applicants’ amendments.
The rejection of claim 19 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to particularly point out and distinctly claim the subject matter which the inventor regards as the invention is withdrawn following the applicants’ amendments.
Rejections Maintained
In regards to the rejected claims 6 and 37 under 35 U.S.C. § 112(a) for failing to comply with
the written description requirement, the applicants’ arguments filed 01/29/2026 have been fully considered by they are not persuasive. The applicants argue that paragraph 64 of the specification demonstrates possession of the claimed “CRISPR/Cas9 end sequence” as recited in claims 6 and 37. The Examiner respectfully disagrees.
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Although paragraph 64 attempts to define the term “CRISPR/Cas9 end sequence,” the disclosure reflects a fundamental misunderstanding of the structure required to form a functional CRISPR/Cas9-guide RNA complex. Specifically, the specification describes the CRISPR/Cas9 end sequence as a “double-stranded nucleic acid”. However this is not the case as crRNA and tracrRNA are both single stranded RNA molecules. While they may have a portion that hybridizes to bind the Cas9 protein, However, the claim requires a single-guide RNA, which is not a double stranded. Furthermore, the portion of the single-guide RNA that interacts with Cas9, commonly derived from the tracrRNA component, is not double-stranded, but rather a single-stranded RNA region that adopts specific secondary structures (e.g., stem-loop or hairpin conformations) required for Cas9 binding and activity (see Ann Ran, Nature Protocols, 2013, Fig. 1). Double-stranded nucleic acids, whether DNA or RNA, are not known to form functional Cas9 guide complexes, and are not
Accordingly, the specification fails to provide adequate written description support for claims 6 and 37, and the rejection under 35 U.S.C. § 112(a) is maintained.
In regards to the rejection of claims 17, 18, 40, and 41 under 35 U.S.C. § 112(a), the applicants arguments filed 01/29/2026 have been fully considered but they are not persuasive. The rejection has been partially withdrawn since claims 18 and 41 have been cancelled, however the applicants’ amendments to claims 17 and 40 do not enable the claims.
The claims recite a method of preparing an immobilized library of double-stranded nucleic acid fragments, in which applying the double-stranded nucleic acid to the solid support comprises treating the CRISPR/Cas9 enzymes with one or more reagents selected from betaine, dimethyl sulfoxide (DMSO), ethanol, ethylene glycol, dimethylacetamide (DMA), dimethylformamide (DMFA), and/or sulphalane (see Mattos et al., Current Opinion in Structural Biology, 2001, pg. 761 ¶3). The specification fails to teach how treatment of immobilized CRISPR/Cas9 enzymes with these reagents would result in a functional method for preparing an immobilized library of double-stranded nucleic acid fragments. It is well documented in the biochemical literature that water-miscible organic solvents, including DMA, DMFA, and DMSO, can significantly alter enzyme structure and catalytic activity by disrupting protein conformation and the hydration shell necessary for proper folding and function, leading to reduced enzyme activity. Unlike conventional buffer components or well-characterized Cas9 reaction additives, the claim recites a heterogenous group of organic solvents and small molecules, some of which are known protein denaturants or nucleic-acid destabilizing agents. The specification does not disclose any concentrations, exposure times, reaction conditions, or compatibility considerations for these reagents in the context of immobilized Cas9-sgRNA complexes.
Applying the Wands factors further demonstrates that undue experimentation would be required:
Quantity of experimentation: The claim encompasses treatment with multiple chemically distinct reagents, alone or in combination, without guidance as to which reagent(s) are operative, under what conditions, or for what purpose. A person of ordinary skill in the art would be required to screen numerous reagents, concentrations, and treatment conditions to determine whether any embodiment is functional.
Amount of guidance: The specification provides no guidance explain how treatment with any of the recited reagents affects Cas9 activity, sgRNA binding, DNA fragmentation, or immobilization. In particular, the claim no longer includes any asserted functional effect (such as reduced binding specificity) that could provide a basis (even if flawed) for understanding why these reagents are included.
Working Examples: The specification also lacks working examples demonstrating treatment of CRISPR/Cas9 enzymes with betaine, dimethyl sulfoxide, ethanol, ethylene glycol, dimethylacetamide, dimethylformamide, and/or sulphalane or any combination thereof in the claimed method.
Nature of the Invention: The claimed method involves protein-RNA-DNA interactions in an immobilized enzymatic system. Such systems are sensitive to solvent composition, protein conformation, and RNA stability, and are not tolerant of arbitrary reagent inclusion without careful optimization.
State of the prior art: The prior art does not establish that the listed reagents are routinely or predictably used to treat CRISPR/Cas9 enzymes, particularly immobilized Cas9 complexes, to facilitate DNA fragmentation. Organic solvents such as DMFA, DMSO, ethanol For example, DMFA has been shown to cause substrate inhibition and changes in protein conformation (see Miroliaei et al., IJBCB, 2002, Abstract and throughout), while Ulbrich-hofmann et al., measured the efficiency of both soluble and immobilized enzymes in organic solvents and found that kinetics were impacted by the “reversible and irreversible denaturation of the enzyme protein” and argued that “irreversible inactivation is especially strong in dimethylformamide” solutions (see Ulbrich-hofmann et al., Enzyme and Microbial Technology,1993, Abstract). Furthermore, organic solvents like DMA has been shown to disrupt hydration shell necessary for proper folding and function (see Panuszko et al., J. of Mol. Liquids, 2019). Several of the recited reagents are more commonly associated with protein denaturation or organic chemistry applications rather than enzymatic reactions. Mattos and Ringe demonstrate that “polar solvents that can easily strip water from the surface of the protein and compete strongly for hydrogen bonds between protein atoms (e.g. dimethyl sulfoxide [DMSO], dimethylformamide [DMF], formamide) usually denature the structure to a largely unfolded state” (see Mattos et al., Current Opinion in Structural Biology, 2001, pg. 761 ¶3) and would not be predicted to be compatible with the CRISPR/Cas9 mediated DNA fragmentation of the claimed method.
Relative skill of those in the art: A person of ordinary skill in the art would likely hold a PhD or equivalent experience in molecular biology or related field, with a familiarity in CRISPR/Cas9 guide design. Although a person of ordinary skill in the art would possess substantial expertise in molecular biology and CRISPR systems, such skill does not compensate for the absence of disclosure where the claim encompasses numerous speculative embodiments without guidance.
The predictability of the art: The effect of treating CRISPR/Cas9 enzymes with organic solvents or chaotropic agents is highly unpredictable, particularly when the enzymes are immobilized on a solid support. Some reagents would reasonably be expected to inactivate Cas9 or disrupt nucleic acid structure and binding.
The breadth of the claims: Claims 17 and 40 broadly encompass treatment with any of the listed reagents, individually or in combination, without limitation as to conditions or functional outcome. This breadth is not commensurate with the disclosure in the specification.
Considering all of the above, the specification fails to enable the full scope of claims 17 and 40 without undue experimentation. A person of ordinary skill in the art would not be able to practice the claimed method across the full scope based on the disclosure provided. Accordingly, claims 17 and 40 remain rejected under 35 USC 112(a) for lack of enablement.
In regards to the rejected claims 1-16, 19-39, and 42-50 under 35 U.S.C. § 103, the applicant's arguments filed 01/29/2026 have been fully considered but they are not persuasive. Applicant contends that the combination of Gormley, Zhou, and Jian-Kui fails to teach or suggest the limitation in amended independent claims 1 and 37 requiring that “the sgRNA comprise (AT)n, where n is 5-20.” Applicant asserts that this sequence motif is neither explicitly disclosed nor suggested by any of the cited references.
However, this argument misapplies the legal standard for obviousness. The reference Jian-Kui et al. (CN108103586A) teaches a random sgRNA library in which they “set the first of the 20 nucleotides of the sgRNA recognition sequence to G, and set the 2nd to 20th nucleotides to random nucleotides, where N is randomly A, T, C, or G.( see Fig. 2, [0039]). A person of ordinary skill in the art would understand that a randomized 19-mer (positions 2-20) drawn from A, T, G, and C bases will include all 419 possible permutations, including low-complexity motifs such as (AT)n, where n is 5-20.
It is well established that a claim limitation may be inherently disclosed in the prior art even if not expressly states. See In re Peterson, 315 F.3d 1325, 1330, 65 USPQ2d 1379, 1382-83 (Fed. Cir. 2003); In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655, 1658 (Fed. Cir. 1990). The randomer sgRNA pool described in Jian-Kui necessarily includes sequences matching the claimed (AT)n motif as part of the guide population. The applicant has provided no evidence that such sequence are excluded from the disclosed libraries, nor has any unexpected result been demonstrated for the (AT)-n motif versus other random sequences.
Applicant also argues that Gormley and Zhou do not disclose sgRNA or Cas9 targeting via this (AT)n motif or randomers. This is acknowledged in the previous rejection. However, Zhou supports the immobilization of nucleases (including Cas9) on a solid support, and Gormley teaches immobilized enzyme-based fragmentation of nucleic acids with retention of resulting fragments on the support, which the applicant does not argue. The rationale for combining these teaching with Jian-Kui’s randomer Cas9 system is based on well-known techniques for enzyme immobilization and DNA fragmentation, with the predictable results of generating an immobilized library of DNA fragments.
Finally, the applicants’ argument that no specific motivation exist to combine the references is unpersuasive. The motivation to use CRISPR/Cas9 with randomer sgRNA (as per Jian-Kui) in an immobilized solid-phase system (as per Gormley and Zhou) would have arisen from the desire to perform high-throughput non-sequence specific DNA fragmentation and immobilization suitable for sequencing library preparation, goals that are clearly addressed by the cited references. Accordingly, the rejection of claims 1, 37, and their dependent claims is maintained.
New Rejection
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-15, 17, 19-40, and 42-50 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
Claims 1 and 37 recite methods in which CRISPR/Cas9 enzymes are associated with single guide RNAs (sgRNA) “wherein the sgRNA comprise (AT)n, wherein n is 5-20.” The specification fails to enable this limitation across its full scope of the claimed sgRNA sequences.
The specification fails to enable sgRNAs comprising such repetitive motifs across the claimed range of n=5-20. CRISPR/Cas9 systems, including the most widely used Streptococcus pyogenes Cas9 (SpCas9), rely on a guide RNA containing a target-recognition (protospacer) sequence of approximately 20 nucleotides (see Ann Ran et al., Nature Protocols, (2013), Abstract, Fig. 1, and throughout). Fu et al., demonstrate that guides 15 nt and below are non-functional (see Fu et al., Nat Biotechnol., 2014, Fig. 1 and throughout). Furthermore, the structural and biochemical properties of SpCas9 do not allow for extended guide recognition beyond 20 nt (see Jiang et al., Science, 2016, Figs. 1-4 and throughout; Zhang et al., Genome Biol., 2017. Fig. 1). The crystal structure from Jiang shows the 5′ end of the guide lies inside the cavity formed between the HNH and RuvC nuclease domains, placing a limit on the guide length relative to the PAM (see Jiang et al., Fig. 4). This protospacer is flanked by a short PAM sequence observed to bind the metal ions associated with the Cas9 protein in crystal structure, limiting the distance between the 5’ end of the sgRNA and the PAM sequence (see Jiang et al., Fig. 2). Furthermore, Ann Ran et al., demonstrated that in vivo attempts to use sgRNA longer than 20 nt, and observed that extra 5’ nucleotides were trimmed, suggesting that only the canonical 20 nt region is protected/function by the Cas9 complex (see Ann Ran et al., Cell, 2013, Fig. 1). Therefore, an sgRNA comprising (AT)n where n=20 results in a homopolymeric 40-nucleotide motif, far exceeding the maximum recognized length of known Cas9 systems. There are no Cas9 variants known in the art that use a 40-nt protospacer region, and extending the guide in this way would not be expected to result in functional DNA binding or cleavage.
The specification does not address these concerns or provide any experimental evidence showing that such sgRNAs are functional within the claimed range.
Applying the Wands factors:
Quantity of experimentation: Determining whether sgRNAs comprising of (AT)n- motifs function effectively across the entire range of n = 5 to 20 would require extensive empirical screening across multiple target sequences and structural context. As the length of repetitive sequences increases, particularly, where n exceeds 10, such sgRNA are increasingly prone to undesirable secondary structure formation, reduced stability, impaired Cas9 targeting efficiency and loss of cleavage activity. The specification does not provide any guidance as to which values of n are operable, thereby requiring substantial trail-and-error experimentation.
Amount of guidance: The specification does not describe how to design, evaluate, or optimize sgRNAs comprising repetitive (AT)n motifs, nor does it address known challenges associated with low-complexity guide sequences. The absence of any design principles or functional criteria leaves a person of ordinary skill in the art without meaningful direction for practicing the full scope of the claim.
Working Examples: The specification also lacks working examples demonstrating the successful use of sgRNAs comprising (AT)n motifs, particularly for higher values of n. No experimental data or prophetic examples are provided showing that sgRNA containing (AT)n sequences in the claimed range effectively direct Cas9-mediated cleavage.
Nature of the Invention: The invention resides in the field of library preparation for high-throughput nucleic acid sequencing and uses CRISPR/Cas9, a technology that is highly sensitive to guide RNA sequence composition and structure. Minor sequence changes can significantly affect function, making enablement particularly demanding.
State of the prior art: At the time of filing, CRISPR/Cas9 was well known in the art, though the prior art did not teach or suggest that sgRNA comprising long repetitive motifs were functional or routinely usable. To the contrary many papers report that 20 nt guide length for Cas9, while Wang et al. report that Cas9 cleavage is reduced using guides with either very high or very low GC content. Specifically showing that SpCas9 is ineffective at cleaving target DNA when the guides contain less than 25% GC content, which indicate that guides with (AT)n where n is 8 or more (80%) would be ineffective, given the 20 nt protospacer length (see Wang et al., Science. 2014 January 3; 343(6166): Fig. 3, pg. 4 para 5).
Relative skill of those in the art: A person of ordinary skill in the art would likely hold a PhD or equivalent experience in molecular biology or related field, with a familiarity in CRISPR/Cas9 guide design. However, even with such skill, without specific guidance or examples, the POSITA would face trial-and-error screening across many potential (AT)-n lengths and sequence context to determine functional guides.
The predictability of the art: CRISPR/Cas9 guide activity is moderately to highly unpredictable, especially when using noncanonical or repetitive sequences. Guide structure, target DNA context, and sequence composition all affect function. The effect of long (AT) tracts on Cas9 targeting is not reliably predictable from general knowledge.
The breadth of the claims: Claims 1 and 37 broadly encompass any sgRNA comprising (AT)-n, for n ranging from 5 to 20, including embodiments that are chemically impossible, and others that exceed known functional limits of Cas9 systems. The breadth of the claims is not commensurate with the disclosure.
Considering all of the above, the specification fails to enable the full scope of Claims 1 and 37 without undue experimentation. The claims encompass embodiments that cannot be made as written and others that would not be expected to function in CRISPR/Cas9 systems Accordingly, claims 1 and 37 are rejected under 35 USC 112(a) for lack of enablement. Claims 2-15, 17, 19-36, 38-40, and 42-50 do not correct these deficiencies and are likewise rejected under 35 USC 112(a).
Conclusion
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Matthew H Raymonda whose telephone number is (703)756-5807. The examiner can normally be reached Monday - Friday 10:00 am - 4:00 pm.
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/MATTHEW HAROLD RAYMONDA/Examiner, Art Unit 1684 /AARON A PRIEST/Primary Examiner, Art Unit 1681