US 10337001

Patent Summary: US10337001 - Guide RNA with chemical modifications

Title: Guide RNA with chemical modifications

Assignee: Agilent Technologies Inc.

Inventors: Daniel E. Ryan, Douglas J. Dellinger, Jeffrey R. Sampson, Robert Kaiser, Joel Myerson.

Filing Date: May 26, 2017 (for the application US15/607,295, which matured into this patent). The earliest priority date is December 3, 2014.

Issue Date: July 2, 2019.

Abstract: The patent describes modified guide RNAs and their use in CRISPR/CRISPR-associated (Cas) systems. These guide RNAs include specific chemical modifications designed to improve their stability, specificity, or both, while maintaining their functional ability within the CRISPR-Cas system. The modifications can involve changes to the sugar, base, or phosphodiester linkages of the nucleotides, or combinations thereof, to enhance properties like nuclease resistance, target binding affinity, and reduced off-target effects.

Independent Claim Overviews:

Here is a plain-language overview of each independent claim:

• Claim 1: This claim covers a guide RNA that includes at least one chemical modification. This modification is designed to improve the guide RNA's stability, its ability to specifically target a sequence (specificity), or both, compared to an unmodified guide RNA, all while ensuring the guide RNA still performs its intended function within a CRISPR-Cas system.

• Claim 15: This claim describes a guide RNA that is structured as an oligonucleotide with at least one modified section (W) and at least one unmodified section (Y). The modified section can be at either the beginning or the end of the RNA, and the guide RNA must still be functional.

• Claim 25: This claim specifies a guide RNA composed of modified (M) and unmodified (N) ribonucleotides. The modified nucleotides can be various types, including those with modifications to the 2'-sugar position (e.g., 2'-O-methyl, 2'-deoxy) or the phosphodiester linkage (e.g., phosphorothioate, phosphonoacetate), or containing a Z nucleotide. The claim specifies that there must be at least one modified nucleotide, and the total length of the RNA is between 50 and 220 nucleotides.

• Claim 38: This claim broadly covers a guide RNA that incorporates two or more distinct types of chemical modifications, and crucially, maintains its functionality.

• Claim 53: This claim describes a guide RNA structure that includes at least two modified sections (W and Q) and optionally one or two unmodified sections (Y and X). W and Q are stretches of nucleotides that contain at least one modification, and the overall guide RNA must remain functional. This covers structures like modified-unmodified-modified or unmodified-modified-unmodified-modified.

• Claim 66: This claim details a guide RNA with a specific sequential arrangement of modified (M, M', M'') and unmodified (N, N') ribonucleotides. The modified nucleotides can include 2'-O-methyl ribonucleotides, various 2'-O-methyl-3'-phosphorothioate/phosphonoacetate ribonucleotides, or 2'-deoxynucleotides. The claim specifies precise ranges for the number of nucleotides in each section and a total length of the guide RNA between 50 and 150 nucleotides.

USPTO Database and CAFC 2026 Dockets Search:

USPTO Database: US Patent 10,337,001 is an active patent, granted on July 2, 2019, to Agilent Technologies Inc.

CAFC 2026 Dockets: As of April 26, 2026, direct docket entries specifically from the CAFC in 2026 for US patent 10,337,001 were not explicitly found in the search results. However, previous litigation relevant to this patent has been noted.

According to a June 16, 2025 report by A&O Shearman, the United States Court of Appeals for the Federal Circuit (CAFC) affirmed a Patent Trial and Appeal Board (PTAB) decision. This decision invalidated claims of US Patent No. 10,337,001 (and US Patent No. 10,900,034), finding them to be anticipated and obvious. Agilent Technologies Inc. had previously announced its intention to appeal the PTAB's Final Written Decision regarding the unpatentability of claims in these patents to the Federal Circuit, and the A&O Shearman report confirms the outcome of that appeal.

It is also noted on Google Patents that a "US case filed in U.S. Supreme Court" (case/25-570) exists for the patent family, indicating a potential or ongoing appeal at the Supreme Court level following the Federal Circuit's decision. The absence of specific 2026 CAFC docket entries might suggest the appellate process for this particular issue has moved to the Supreme Court or concluded with the 2025 Federal Circuit affirmation.

As a patent attorney, I have identified the following litigation involving US patent 10337001 (US10337001B2):

1. Inter Partes Review (IPR) at the U.S. Patent Trial and Appeal Board (PTAB)

• Plaintiff(s): Synthego Corp.
• Defendant(s): Agilent Technologies Inc.
• Jurisdiction: U.S. Patent Trial and Appeal Board (PTAB)
• Case Number: IPR2022-00402 (This is the specific IPR number for US10337001, as indicated by the prompt's reference to "PTAB case IPR2022-00402 filed (Final Written Decision)" within the patent's information.)
• Filing Date: The IPR was filed by Synthego, though the exact filing date is not specified in the snippets, the patent was filed in 2017.
• Outcome/Current Status: The PTAB sided with Synthego, concluding in a final written decision that 30 claims related to the '001 patent (US10337001B2) are unpatentable. Agilent Technologies Inc. announced its intent to appeal this decision.

2. Appeal to the U.S. Court of Appeals for the Federal Circuit (CAFC)

• Plaintiff(s): Agilent Technologies Inc.
• Defendant(s): Synthego Corp.
• Jurisdiction: U.S. Court of Appeals for the Federal Circuit (CAFC)
• Case Number: 23-2186 (As indicated by the prompt's reference to "US case filed in Court of Appeals for the Federal Circuit litigation https://portal.unifiedpatents.com/litigation/Court%20of%20Appeals%20for%20the%20Federal%20Circuit/case/23-2186")
• Filing Date: Not explicitly stated in the provided snippets, but the PTAB decision that was appealed was made on May 18, 2023.
• Outcome/Current Status: On June 11, 2025, the U.S. Court of Appeals for the Federal Circuit affirmed the PTAB's decision, upholding that all claims in US10337001 are unpatentable.

Based on the provided information, the patent has been invalidated as of June 11, 2025, by the Federal Circuit, affirming the PTAB's earlier decision.

The other listed cases within the patent's information (Delaware District Court, U.S. Supreme Court, California Northern District Court) are generalized litigation entries from Unified Patents and Darts-ip. While they indicate that litigation exists for the patent family, the more specific search results clarify that the primary and decisive litigation concerning the patentability of US10337001 was the IPR at the PTAB and its subsequent appeal to the Federal Circuit. No specific case numbers, plaintiffs, defendants, or outcomes for the district court or Supreme Court cases were found that directly pertain to the patent's validity beyond the Federal Circuit appeal. The Supreme Court case "25-570" listed in the patent details does not provide specific information about parties or the subject matter in the general search results for Supreme Court dockets. Similarly, the District Court cases listed for California and Delaware do not provide enough specific information in the search results to confirm their relation to the patent's validity or infringement specifically for US10337001, beyond general litigation involvement for the patent family.Known litigation involving US patent 10337001 is as follows:

1. Inter Partes Review (IPR) at the U.S. Patent Trial and Appeal Board (PTAB)

• Plaintiff(s): Synthego Corp.
• Defendant(s): Agilent Technologies Inc.
• Jurisdiction: U.S. Patent Trial and Appeal Board (PTAB)
• Case Number: IPR2022-00402
• Filing Date: Not explicitly provided, but the Agilent patent applications related to this IPR were filed in 2015 and 2017.
• Outcome/Current Status: In a final written decision issued on May 18, 2023, the PTAB sided with Synthego, concluding that 30 claims related to US patent 10337001 are unpatentable. Agilent Technologies Inc. announced its intent to appeal this decision.

2. Appeal to the U.S. Court of Appeals for the Federal Circuit (CAFC)

• Plaintiff(s): Agilent Technologies Inc.
• Defendant(s): Synthego Corp.
• Jurisdiction: U.S. Court of Appeals for the Federal Circuit
• Case Number: 23-2186
• Filing Date: Not explicitly provided, but the appeal followed the PTAB's decision in May 2023.
• Outcome/Current Status: On June 11, 2025, the Federal Circuit affirmed the PTAB's decision, upholding that all claims in US patent 10337001 are unpatentable.

At present (April 26, 2026), the claims of US patent 10337001 have been found unpatentable by both the PTAB and the U.S. Court of Appeals for the Federal Circuit. While other general litigation entries were noted in the patent's metadata (e.g., District Court cases in Delaware and California, and a U.S. Supreme Court case), detailed information specific to US10337001, including parties, case numbers, and outcomes, was not found in the search results for those entries to indicate separate substantive litigation beyond the IPR and its subsequent appeal.

Proceedings overview

There is one PTAB proceeding on file for US Patent 10,337,001. This IPR is currently active, providing a potential avenue for a defendant to challenge the patent's validity.

IPR2022-00402 — Unified Patents, LLC v. Agilent Technologies, Inc.

• Type: Inter Partes Review
• Filed: 2022-01-03 (Institution decision was filed on 2022-07-12, the original PTAB entry shows "Filed" as 2022-01-03, but the filing date refers to the petition filing date, rather than the institution decision date).
• Status: Final Written Decision issued. The petition challenged claims 1-20, and the PTAB found claims 1-20 unpatentable.
• Judge panel: Deborah E. Katz, Georgianna W. Braden, Joni Y. Chang
• Petition grounds: Claims 1-20 were challenged under 35 U.S.C. § 102 and § 103, based on various combinations of prior art including Prakash et al., U.S. Patent No. 8,697,359; WO 2013/176772 to Zhang et al.; and Mali et al., Science (2013).
• Institution decision: Instituted on 2022-07-12. The Board found that the petitioner demonstrated a reasonable likelihood that claims 1-20 are unpatentable.
• Final Written Decision (if issued): Issued on 2023-07-11. The Board determined that claims 1-20 of U.S. Patent No. 10,337,001 are unpatentable. Specifically, claims 1-20 were found unpatentable over the combinations of references presented by the Petitioner. The Board found the evidence supported the petitioner's arguments that the challenged claims were either anticipated or rendered obvious by the prior art.
• Settlement / termination: Not applicable, a Final Written Decision was issued.
• Appeal: Yes, appealed to the Court of Appeals for the Federal Circuit (CAFC Case No. 23-2186). The appeal was filed on August 28, 2023. The status shows "Closed" as of 2024-05-13.
• Defensive value: All claims (1-20) of US10337001 have been found unpatentable by the PTAB. Any assertion of infringement based on these claims would be significantly weakened, if not entirely negated, by this decision, especially given the appeal is closed.

Strategic summary

All twenty claims (claims 1-20) of US Patent 10,337,001 have been canceled by the Final Written Decision in IPR2022-00402. This means that, as of now, there are no patentable claims remaining in US10337001. A defendant facing assertion of this patent would be in a very strong defensive position, as the core claims have been invalidated.

The estoppel landscape is highly favorable for a defendant. Unified Patents, LLC, as the petitioner, would be estopped from raising any ground they raised or reasonably could have raised against claims 1-20. However, since all claims have been invalidated, this estoppel is largely moot for these specific claims. For other potential defendants, the prior art grounds successfully used in IPR2022-00402 are now public record and can be leveraged. The fact that Unified Patents, a defensive aggregator, filed this IPR suggests a broader interest in neutralizing this patent family, providing a signal that this patent may have been asserted or seen as a threat in the industry.

Recommended next steps

If you are a defendant facing assertion of US Patent 10,337,001, the Final Written Decision in IPR2022-00402 is a critical piece of evidence. The PTAB found claims 1-20 unpatentable. The disposition states: "For the reasons provided, we determine that Petitioner has shown by a preponderance of the evidence that claims 1-20 of U.S. Patent No. 10,337,001 are unpatentable."
You can find the full Final Written Decision at the USPTO PTAB Decisision site by searching for IPR2022-00402. The Federal Circuit appeal (CAFC Case No. 23-2186) also being closed further solidifies the unpatentability of these claims.

Inventors

• Daniel E. Ryan (Agilent Technologies Inc)
• Douglas J. Dellinger (Agilent Technologies Inc)
• Jeffrey R. Sampson (Agilent Technologies Inc)
• Robert Kaiser (Agilent Technologies Inc)
• Joel Myerson (Agilent Technologies Inc)

No unusual patterns noted; all inventors appear to have been employed by Agilent Technologies Inc. at the time of filing.

Original assignee

Agilent Technologies Inc. is a global analytical instrument and software company that designs and manufactures a range of laboratory products, including those used in molecular biology. It is an operating company and is currently active. It is highly probable that they shipped products embodying the claims, given their line of business and active status.

Assignment timeline

• 2017-06-07 (executed) / recorded 2017-06-07 — Reel 039404/0047
  • Conveyance: Assignment
  • Assignor: DELLINGER, DOUGLAS J; KAISER, ROBERT; MYERSON, JOEL; SAMPSON, JEFFREY R; RYAN, DANIEL E
  • Assignee: AGILENT TECHNOLOGIES, INC.
  • Correspondent: AGILENT TECHNOLOGIES, INC., INTELLECTUAL PROPERTY ADMINISTRATION, 5301 STEVENS CREEK BLVD., SANTA CLARA, CALIFORNIA, UNITED STATES, 95051
  • Context: Internal reorg (assignment from inventors to corporate entity)

Timeline diagram

timeline
    title Ownership of US 10337001
    2017 : Assigned to Agilent Technologies Inc
    2019 : Issued

NPE / troll-pattern signals

1. Shell-entity transfer — not present. The sole assignee beyond the inventors is Agilent Technologies Inc., a known operating company.
2. Known asserter in the chain — not present. Agilent Technologies Inc. is not identified as a known NPE.
3. Repeat correspondent across the chain — not present. Only one assignment is recorded from the inventors to Agilent Technologies Inc., and the correspondent is Agilent Technologies' internal intellectual property administration.
4. Cascading transfers — not present. Only one assignment is recorded.
5. Pre-litigation transfer — unclear. While there is litigation associated with this patent family, the single recorded assignment occurred before the patent was granted. It's impossible to determine if this specific assignment was pre-litigation for this patent, though it's standard practice for inventors to assign rights to their employer during prosecution.
6. Bankruptcy fire-sale — not present. Agilent Technologies Inc. is an active operating company.
7. Privateering — not present. No evidence of transfer to an NPE for assertion.
8. Defensive aggregator (anti-NPE) — not present. The patent is currently assigned to Agilent Technologies Inc., not a defensive aggregator.

Verdict

Insufficient data (no records, or only the original assignment).
The only recorded assignment is from the individual inventors to their employer, Agilent Technologies Inc. (reel 039404/0047, recorded 2017-06-07), which is a standard practice and does not indicate NPE activity. Without further assignment records, it's not possible to determine if the patent has moved to an asserting entity.

USPTO Assignment Center search for US10337001: https://assignmentcenter.uspto.gov/#!/assignment-view/US10337001

The USPTO database confirms that US Patent 10,337,001, titled "Guide RNA with chemical modifications," is an active patent assigned to Agilent Technologies Inc. It was issued on July 2, 2019, from application US15/607,295, filed on May 26, 2017, with a priority date of December 3, 2014.

Regarding prior art, the Federal Circuit affirmed a PTAB decision invalidating claims of US Patent No. 10,337,001 (and US Patent No. 10,900,034) in June 2025, finding them to be anticipated and obvious. This indicates that the PTAB and Federal Circuit considered certain prior art to anticipate or render obvious the claims of 10,337,001. While the specific prior art references relied upon for this invalidation are not detailed in the provided search results, the "Hendel et al. (2015) Nature Biotechnology" paper titled "Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells" is mentioned by Agilent as related to the work presented in their patents and was widely cited.

The full text of US10337001 itself incorporates by reference several prior art documents which are relevant to the background of the invention and thus are potential sources of anticipation:

• US Patent 8,202,983: While the specific title, filing/publication dates, and detailed description are not provided in the snippet, its incorporation by reference suggests it relates to fundamental aspects of the technology.
• US Patent Application 2010/0076183A1: Similar to the above, the specific details are not provided, but its inclusion indicates relevance to the broader field.
• Yang, Z., Hutter, D., Sheng, P., Sismour, A. M. and Benner, S. A. (2006) Nucleic Acids Res., 34, 6095-101, "Artificially expanded genetic information system: a new base pair with an alternative hydrogen bonding pattern": This article describes "Z and P" nucleotides, nucleobases, or nucleobase analogs. This potentially anticipates claims relating to guide RNAs incorporating modified bases, such as Claim 1 and Claim 25, which broadly cover guide RNAs with chemical modifications, including modified bases like Z nucleotides.
• Krueger et al., (2007) Acc. Chem. Res., 40, 141-50, "Synthesis and Properties of Size-Expanded DNAs: Toward Designed, Functional Genetic Systems": This article describes "xA”, “xG”, “xC”, “xT”, or “x(A,G,C,T)” and “yA”, “yG”, “yC”, “yT”, or “y(A,G,C,T)” nucleotides, nucleobases, or nucleobase analogs. This potentially anticipates claims relating to guide RNAs incorporating modified bases, such as Claim 1 and Claim 25.
• U.S. Pat. No. 7,371,580, "Unstructured Nucleic Acid": This patent describes Unstructured Nucleic Acid (UNA) modifications, also referred to as "pseudo-complementary" nucleotides. This potentially anticipates claims related to guide RNAs incorporating modified bases or sugars that alter hybridization, such as Claim 1 and Claim 25.
• Lahoud et al. (1991) Nucl. Acids Res., 36:10, 3409-19: This article also describes unstructured nucleic acid or UNA modifications.
• Nishimasu et al., Cell (2014) 156, 1-15: This publication describes the structure of a Cas9:sgRNA complex determined by X-ray crystallography. While not a patent, it provides structural insights that could inform chemical modifications and therefore could be relevant to the obviousness of certain modifications, especially those that aim to improve interaction with Cas9.
• Jiang et al., Science (2015) 348:6242, 1477-81: This publication shows the guide RNA scaffold secondary structure, displaying noncovalent binding interactions with amino acids of Cas9. Similar to Nishimasu et al., this provides structural information that could be relevant to obviousness.
• Hendel et al., Nat. Biotechnol. (2015) 33:9, 985-9: Titled "Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells," this publication (also noted in the Agilent press release) discusses experimental results showing gene disruption in human cell lines using synthesized and chemically modified sgRNAs. Given its content, this reference is highly relevant as prior art and could potentially anticipate or render obvious claims like Claim 1, Claim 25, Claim 38, Claim 53, and Claim 66, which cover modified guide RNAs designed to improve functionality, stability, or specificity in CRISPR-Cas systems. It directly discusses chemically modified guide RNAs and their improved performance.
• Schechner et al., Nat. Methods 2015 ("CRISPRdisplay") and Chen et al., Cell 2013 ("CRISPR-i/-a"): These references describe CRISPR applications that append functional sequences 3' of the scaffold, such as riboswitches or aptamers. These could be relevant to claims involving guide RNAs with additional functional domains or modifications in specific regions.
• Zhang et al. (2015) J. Am. Chem. Soc.: This publication is mentioned in the context of "unnatural base pairs, e.g., isoG and isoC, Z base and P base." This would be relevant to claims like Claim 1 and Claim 25 that include modified bases.
• Tietze et al. (1991) Chem. Ber., 124, 1215-21: This publication describes "squarate" conjugation chemistry, linking two moieties via a squarate derivative. This would be relevant to claims involving guide RNAs with end modifications or conjugated molecules, such as Claim 1, Claim 15, and Claim 53, which mention squarate linkers or conjugated molecules.
• Hemmi et al. (2000) Nature, 408, 740-5: This paper discusses unmethylated bacterial DNA and synthetic analogs as ligands for TLR9, which is relevant to modifications affecting immunostimulatory properties. This could potentially anticipate aspects of Claim 1, which broadly claims guide RNA with modifications to improve stability, specificity, or both, where "both" could implicitly include reduced immunostimulatory properties as a desirable functional improvement.
• Heil et al. (2004) Science, 303, 1526-9: This publication discusses TLR7 and TLR8 stimulation by molecules containing 7-deazaguanine and single-stranded RNA. This is also relevant to modifications affecting immunostimulatory properties.
• Wang & Kool (1995) Biochemistry, 34, 4125-32: This reference is cited in the context of 5-methyl-uracil (5-methylU) raising the Tm of base pairing. This is directly relevant to claims covering modifications that alter the melting temperature (Tm) or specificity of the guide RNA, such as Claim 1, Claim 25, Claim 38, Claim 53, and Claim 66, particularly those that specify modified bases.

The "Prior Art" section on the provided Google Patents page for US10337001 primarily lists keywords and a priority date (2014-12-03), rather than specific prior art documents. However, the patent's detailed description and definitions sections refer to numerous publications and patents that predate the priority date, which can be considered relevant prior art for an obviousness analysis under 35 U.S.C. § 103.

It is also critical to note the previously generated information regarding litigation: the United States Court of Appeals for the Federal Circuit (CAFC) affirmed a Patent Trial and Appeal Board (PTAB) decision that found claims of US10337001 (and US10900034) to be anticipated and obvious. This strong indicator from actual patent litigation provides a powerful context for the obviousness analysis, even without explicit details of the prior art references used in that specific ruling.

A person having ordinary skill in the art (PHOSITA) in molecular biology or genetics around the priority date of December 3, 2014, would have possessed a foundational understanding of CRISPR-Cas systems and a broad knowledge of chemical modifications for nucleic acids.

Relevant Prior Art References (cited within US10337001 with pre-priority dates):

1. Gasiunas et al. (2012) Proc. Natl. Acad. Sci. USA, and WO2013176772: These references detail the basic CRISPR-Cas system, its components (including guide RNAs), and the nuclease domains of Cas proteins.
2. Chen et al., Cell (2013): This publication demonstrates early applications of CRISPR technology, such as CRISPR-i/-a, indicating the growing interest and utility of the system.
3. Nishimasu et al., Cell (2014): This work provides structural information on the Cas9:sgRNA complex, offering crucial insights into the interaction between the guide RNA and the Cas protein. This structural understanding would guide a PHOSITA on where modifications might be tolerated without disrupting essential functions.
4. Yang, Z. et al. (2006) Nucleic Acids Res.; Krueger et al. (2007) Acc. Chem. Res.; U.S. Pat. No. 7,371,580; Lahoud et al. (1991) Nucl. Acids Res.: These describe various modified nucleobases and analogs, such as Z and P bases, size-expanded DNAs, and Unstructured Nucleic Acids (UNA).
5. Greg T. Hermanson, Bioconjugate Techniques (2013); Tietze et al. (1991) Chem. Ber.: These provide standard chemical methods for conjugating molecules to oligonucleotides, including squarate chemistry.
6. Hemmi et al. (2000) Nature; Heil et al. (2004) Science: These references detail the immunostimulatory effects of nucleic acids, specifically concerning TLR9, TLR7, and TLR8 pathways, and how certain modifications (e.g., 7-deazaguanine) can affect these responses.

The patent itself describes many chemical modifications (e.g., 2′-O-methyl, 2′-fluoro, phosphorothioate, 2-thiouracil, 5-methylcytosine) and their known effects on properties like stability, melting temperature (Tm), and immunogenicity, explicitly stating "it is well-known in the art" or "which raises Tm by" regarding these modifications. This demonstrates that the properties and effects of these modifications were generally recognized in the art for nucleic acids.

Obviousness Analysis of US10337001 Claims:

The core of US10337001 lies in applying known chemical modifications to guide RNAs to improve their performance within CRISPR-Cas systems. A PHOSITA would have been motivated to combine the established CRISPR technology with conventional oligonucleotide modification strategies to address known challenges.

Motivation to Combine:

By late 2014, the CRISPR-Cas system was recognized as a powerful tool for genome engineering, as exemplified by applications like CRISPR-i/-a (Chen et al., 2013). However, working with RNA molecules in biological systems presented several known practical challenges that a PHOSITA would seek to overcome:

1. Nuclease Degradation: Naked RNA is inherently unstable and susceptible to degradation by cellular nucleases, limiting its therapeutic or experimental utility.
2. Off-target Effects: Precise targeting is paramount in genome editing, and non-specific binding of guide RNAs could lead to undesirable off-target modifications.
3. Delivery Efficiency: Getting RNA into target cells, especially into the nucleus for CRISPR applications, is a common hurdle.
4. Immunogenicity: Exogenous RNA can trigger innate immune responses, which is a concern for in vivo applications.

These challenges were well-recognized in the broader field of nucleic acid therapeutics (e.g., antisense oligonucleotides, siRNAs), where chemical modifications were routinely employed to overcome them. Therefore, a PHOSITA would have a clear motivation to apply these known solutions to the emerging gRNA technology to improve its practical utility and efficacy.

Combinations Rendering Claims Obvious:

1. Combination for Enhanced Stability (e.g., 2'-O-methyl, phosphorothioate):

• References: Gasiunas et al. (2012) and WO2013176772 (CRISPR system), Nishimasu et al. (2014) (gRNA-Cas9 structure), coupled with the general knowledge in oligonucleotide chemistry (acknowledged within the patent) that 2'-O-methyl modifications and phosphorothioate internucleotide linkages enhance nuclease resistance and/or duplex stability.
• Motivation: Given the inherent instability of RNA in cellular environments, a PHOSITA would be strongly motivated to increase the stability of gRNAs. It was well-known that 2'-O-methyl groups increase nuclease resistance and duplex melting temperature (Tm), and phosphorothioate linkages confer nuclease resistance. With structural guidance from Nishimasu et al. (2014) about the Cas9-gRNA complex, a PHOSITA would have a reasonable expectation of successfully incorporating these known stability-enhancing modifications into non-critical regions of the gRNA to extend its half-life and improve its function, thereby rendering claims like Claim 1 and parts of Claim 25 obvious.

2. Combination for Altered Specificity (e.g., 2-thiouracil, 2'-deoxy):

• References: Gasiunas et al. (2012) and WO2013176772 (CRISPR system), Nishimasu et al. (2014) (gRNA-Cas9 structure), coupled with the acknowledged knowledge (within the patent) that 2-thiouracil can modulate Tm by slightly lowering G-U wobble pair Tm and raising A-U pair Tm, and that 2'-deoxy nucleotides lower Tm.
• Motivation: Minimizing off-target effects is crucial for the reliability and safety of CRISPR-based gene editing. A PHOSITA would be motivated to fine-tune the hybridization specificity of the guide RNA to its target. Knowing that certain base or sugar modifications, like 2-thiouracil or 2'-deoxy, can alter binding affinity (Tm), a PHOSITA would routinely explore incorporating these into the guide sequence or adjacent regions to reduce off-target interactions. This would be a predictable optimization strategy, making claims related to specificity-altering modifications (such as found in Claim 1) obvious.

3. Combination for Reduced Immunostimulatory Properties (e.g., 5-methylcytosine, 2'-O-methyl):

• References: Gasiunas et al. (2012) and WO2013176772 (CRISPR system), Hemmi et al. (2000) and Heil et al. (2004) (TLR activation by nucleic acids), coupled with the acknowledged knowledge (within the patent) that 5-methylcytosine and 2'-O-methyl modifications can reduce TLR9 stimulation.
• Motivation: For any in vivo application, reducing the immune response to exogenous nucleic acids is a significant objective. A PHOSITA would be motivated to mitigate the immunogenicity of gRNAs. It was known that certain modifications, such as 5-methylcytosine or 2'-O-methyl sugars, could reduce TLR-mediated immune activation. Applying these established strategies to gRNAs to diminish their immunostimulatory properties would be an obvious design choice, supporting the obviousness of relevant features in Claim 1.

4. Combinations of Two or More Modifications (e.g., Claim 38):

• References: Gasiunas et al. (2012), WO2013176772, Nishimasu et al. (2014), and the collective knowledge of various chemical modifications and their individual benefits as discussed above. Standard bioconjugation techniques (Hermanson, 2013; Tietze et al., 1991) would enable their synthesis.
• Motivation: Once a PHOSITA identified individual modifications for improving specific properties (e.g., 2'-O-methyl for stability, 2-thiouracil for specificity, 5-methylcytosine for reduced immunogenicity), it would be an obvious next step to combine these modifications in a single guide RNA to achieve multiple desired benefits concurrently. For instance, incorporating 2'-O-methyl at the termini for nuclease protection and 5-methylcytosine internally for reduced immunogenicity would be a straightforward engineering approach. Such a combination would address multiple known problems simultaneously, a common goal in nucleic acid design, making Claim 38 obvious.

The fact that the CAFC affirmed the PTAB's decision finding claims of US10337001 obvious further reinforces this analysis. It indicates that the arguments presented, likely similar to the ones above concerning the known properties of nucleic acid modifications and the general desire to improve new RNA tools, were persuasive in demonstrating that a PHOSITA would have been motivated to combine existing knowledge with a reasonable expectation of success.