US 10900034

US Patent 10,900,034 Summary

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: December 3, 2015

Issue Date: January 26, 2021

Abstract: The patent describes chemically modified guide RNAs (gRNAs) designed for use in CRISPR-Cas systems. These modifications are intended to improve gRNA functionality, such as enhancing stability (e.g., nuclease resistance), altering hybridization thermostability, reducing off-target effects, improving cell delivery (transfectability), and minimizing immune responses. The invention specifically focuses on gRNAs where chemical modifications do not significantly compromise their ability to bind to Cas proteins, or to bind, nick, or cleave target polynucleotides.

Plain-Language Overview of Independent Claims:

• Claim 1: This claim describes a chemically modified guide RNA (gRNA) that still works with a Cas protein. The modification is not a natural phosphodiester bond in the backbone. The gRNA can still bind to a Cas protein, target a Cas protein/gRNA complex to a specific DNA or RNA sequence, and/or cause the Cas protein/gRNA complex to cut that target sequence.

• Claim 20: This claim describes a chemically modified guide RNA that has at least one nucleotide modification. This modification can be a change to the sugar part of the nucleotide (like 2'-O-methyl, 2'-deoxy, 2'-fluoro), or a change to the base (like 2-thiouracil, 5-methylcytosine, or special unnatural bases Z or P), or a change to the backbone (like phosphorothioate or phosphonoacetate). The gRNA still functions with a Cas protein, meaning it can bind to it and guide it to a target DNA or RNA for binding, nicking, or cleavage.

• Claim 26: This claim is for a synthetic guide RNA (gRNA) composed of both modified and unmodified ribonucleotides. It follows a general formula MₘNₙ, where 'M' represents a modified nucleotide (such as a 2'-O-methyl ribonucleotide, a 3'-phosphorothioate ribonucleotide, or a 2'-deoxynucleotide) and 'N' represents an unmodified ribonucleotide. There must be at least one modified nucleotide (m is not 0), and the total number of nucleotides (m+n) is between 50 and 220. This gRNA must also retain its functionality with a Cas protein.

• Claim 35: This claim describes a synthetic guide RNA (gRNA) with modified ribonucleotides, where each modified nucleotide 'M' is independently chosen from a specific set of modifications that enhance stability or specificity. These modifications include 2'-fluoro, 2-thiouracil, 2-aminoadenine, 5-methylcytosine, or certain chemical linkages like squarate or triazolo. The gRNA must still function with a Cas protein.

• Claim 40: This claim is similar to Claim 26, describing a synthetic guide RNA with modified (M) and unmodified (N) ribonucleotides, following the formula MₘNₙ. Here, 'M' specifically refers to a 2'-O-methyl ribonucleotide, and 'N' is an unmodified ribonucleotide (A, U, C, or G). The number of modified nucleotides (m) is between 1 and 40, and the total length (m+n) is between 50 and 150. This gRNA also maintains its functionality with a Cas protein.

• Claim 42: This claim is also similar to Claim 26, but the modified nucleotide 'M' is specifically a 2'-deoxynucleotide. The number of modified nucleotides (m) is between 1 and 30, and the total length (m+n) is between 50 and 150. The gRNA retains its Cas protein functionality.

• Claim 43: This claim describes a synthetic guide RNA where the modified nucleotide 'M' is specifically a 2'-O-methyl-3'-phosphorothioate ribonucleotide. The number of modified nucleotides (m) is between 1 and 10, and the total length (m+n) is between 50 and 150. The gRNA retains its functionality with a Cas protein.

• Claim 44: This claim is another variation, where the modified nucleotide 'M' is a Z nucleotide. The number of Z nucleotides (m) is between 1 and 10, and the total length (m+n) is between 50 and 150. The gRNA retains its functionality with a Cas protein.

• Claim 48: This claim describes a chemically modified guide RNA that includes two or more different types of chemical modifications. At least one of these modification types increases the gRNA's resistance to breakdown by nucleases (stability). This modified gRNA also functions with a Cas protein.

• Claim 66: This claim specifies a chemically modified guide RNA that has at least two different types of chemical modifications. At least one of these modifications is designed to alter the binding specificity of the guide RNA. This modified gRNA still functions with a Cas protein.

• Claim 84: This claim describes a chemically modified guide RNA with at least two different types of chemical modifications. At least one of these modifications is designed to affect how easily the gRNA enters cells (transfection efficiency). This modified gRNA retains its functionality with a Cas protein.

• Claim 87: This claim describes a chemically modified guide RNA with at least two different types of chemical modifications. At least one of these modifications is designed to reduce the immune response it might trigger. This modified gRNA retains its functionality with a Cas protein.

• Claim 95: This claim describes a guide RNA that is represented by one of two formulas: W-Y-Q or Y-W-X-Q. In these formulas, 'W' and 'Q' are sections of the oligonucleotide that contain at least one chemical modification, while 'Y' and 'X' are unmodified sections. The guide RNA is able to function with a Cas protein.

• Claim 106: This claim describes a guide RNA with a nucleotide sequence according to Formula V (MₘNₙM'ₘ'N'ₙ') or Formula VI (MₘNₙM'ₘ'N'ₙ'M''ₘ''). Here, M, M', and M'' are independently selected modified ribonucleotides (e.g., 2'-O-methyl, 2'-O-methyl-3'-P(S), 2'-O-methyl-3'-PACE, 2'-O-methyl-3'-thioPACE, or 2'-deoxynucleotide). N and N' are unmodified ribonucleotides. The total number of modified nucleotides (m+m'+m'') is at least 1, and the total length of the gRNA is between 50 and 150 nucleotides. This gRNA functions with a Cas protein.

Litigation:
US patent 10,900,034 has been involved in litigation. According to the Google Patents information, there are US cases filed in the Delaware District Court, the California Northern District Court (multiple cases), the Court of Appeals for the Federal Circuit (CAFC), and the U.S. Supreme Court. Additionally, there is a PTAB case IPR2022-00403 (Final Written Decision) and a first worldwide family litigation filed. The CAFC case is identified as 23-2187. Since the current date is April 26, 2026, and the CAFC case 23-2187 has a 2023 number, it is relevant to ongoing litigation.US Patent 10,900,034 Summary

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: December 3, 2015

Issue Date: January 26, 2021

Abstract: The patent describes chemically modified guide RNAs (gRNAs) designed for use in CRISPR-Cas systems. These modifications are intended to improve gRNA functionality, such as enhancing stability (e.g., nuclease resistance), altering hybridization thermostability, reducing off-target effects, improving cell delivery (transfectability), and minimizing immune responses. The invention specifically focuses on gRNAs where chemical modifications do not significantly compromise their ability to bind to Cas proteins, or to bind, nick, or cleave target polynucleotides.

Plain-Language Overview of Independent Claims:

• Claim 1: This claim describes a chemically modified guide RNA (gRNA) that still works with a Cas protein. The modification is not a natural phosphodiester bond in the backbone. The gRNA can still bind to a Cas protein, target a Cas protein/gRNA complex to a specific DNA or RNA sequence, and/or cause the Cas protein/gRNA complex to cut that target sequence.

• Claim 20: This claim is for a chemically modified guide RNA that has at least one nucleotide modification. This modification can be a change to the sugar part of the nucleotide (like 2'-O-methyl, 2'-deoxy, 2'-fluoro), or a change to the base (like 2-thiouracil, 5-methylcytosine, or special unnatural bases Z or P), or a change to the backbone (like phosphorothioate or phosphonoacetate). The gRNA still functions with a Cas protein, meaning it can bind to it and guide it to a target DNA or RNA for binding, nicking, or cleavage.

• Claim 26: This claim is for a synthetic guide RNA (gRNA) composed of both modified and unmodified ribonucleotides. It follows a general formula MₘNₙ, where 'M' represents a modified nucleotide (such as a 2'-O-methyl ribonucleotide, a 3'-phosphorothioate ribonucleotide, or a 2'-deoxynucleotide) and 'N' represents an unmodified ribonucleotide. There must be at least one modified nucleotide (m is not 0), and the total number of nucleotides (m+n) is between 50 and 220. This gRNA must also retain its functionality with a Cas protein.

• Claim 35: This claim describes a synthetic guide RNA where each modified nucleotide 'M' is independently chosen from a specific set of modifications that enhance stability or specificity. These modifications include 2'-fluoro, 2-thiouracil, 2-aminoadenine, 5-methylcytosine, or certain chemical linkages like squarate or triazolo. The gRNA must still function with a Cas protein.

• Claim 40: This claim is for a synthetic guide RNA with modified (M) and unmodified (N) ribonucleotides, following the formula MₘNₙ. Here, 'M' specifically refers to a 2'-O-methyl ribonucleotide, and 'N' is an unmodified ribonucleotide (A, U, C, or G). The number of modified nucleotides (m) is between 1 and 40, and the total length (m+n) is between 50 and 150. This gRNA also maintains its functionality with a Cas protein.

• Claim 42: This claim is for a synthetic guide RNA where the modified nucleotide 'M' is specifically a 2'-deoxynucleotide. The number of modified nucleotides (m) is between 1 and 30, and the total length (m+n) is between 50 and 150. The gRNA retains its Cas protein functionality.

• Claim 43: This claim describes a synthetic guide RNA where the modified nucleotide 'M' is specifically a 2'-O-methyl-3'-phosphorothioate ribonucleotide. The number of modified nucleotides (m) is between 1 and 10, and the total length (m+n) is between 50 and 150. This gRNA retains its functionality with a Cas protein.

• Claim 44: This claim is another variation, where the modified nucleotide 'M' is a Z nucleotide. The number of Z nucleotides (m) is between 1 and 10, and the total length (m+n) is between 50 and 150. This gRNA retains its functionality with a Cas protein.

• Claim 48: This claim describes a chemically modified guide RNA that includes two or more different types of chemical modifications. At least one of these modification types increases the gRNA's resistance to breakdown by nucleases (stability). This modified gRNA also functions with a Cas protein.

• Claim 66: This claim specifies a chemically modified guide RNA that has at least two different types of chemical modifications. At least one of these modifications is designed to alter the binding specificity of the guide RNA. This modified gRNA still functions with a Cas protein.

• Claim 84: This claim describes a chemically modified guide RNA with at least two different types of chemical modifications. At least one of these modifications is designed to affect how easily the gRNA enters cells (transfection efficiency). This modified gRNA retains its functionality with a Cas protein.

• Claim 87: This claim describes a chemically modified guide RNA with at least two different types of chemical modifications. At least one of these modifications is designed to reduce the immune response it might trigger. This modified gRNA retains its functionality with a Cas protein.

• Claim 95: This claim describes a guide RNA that is represented by one of two formulas: W-Y-Q or Y-W-X-Q. In these formulas, 'W' and 'Q' are sections of the oligonucleotide that contain at least one chemical modification, while 'Y' and 'X' are unmodified sections. The guide RNA is able to function with a Cas protein.

• Claim 106: This claim describes a guide RNA with a nucleotide sequence according to Formula V (MₘNₙM'ₘ'N'ₙ') or Formula VI (MₘNₙM'ₘ'N'ₙ'M''ₘ''). Here, M, M', and M'' are independently selected modified ribonucleotides (e.g., 2'-O-methyl, 2'-O-methyl-3'-P(S), 2'-O-methyl-3'-PACE, 2'-O-methyl-3'-thioPACE, or 2'-deoxynucleotide). N and N' are unmodified ribonucleotides. The total number of modified nucleotides (m+m'+m'') is at least 1, and the total length of the gRNA is between 50 and 150 nucleotides. This gRNA functions with a Cas protein.

Litigation Status:
As of the current date (April 26, 2026), all claims of US Patent 10,900,034 have been deemed unpatentable. The U.S. Supreme Court denied a petition for a writ of certiorari from Agilent Technologies, Inc., in June 2025, effectively concluding a multi-year legal challenge and solidifying the invalidation of the patent's claims. This decision affirmed a June 2025 ruling by the U.S. Court of Appeals for the Federal Circuit, which in turn affirmed an earlier Patent Trial and Appeal Board (PTAB) decision. The PTAB had concluded that 33 claims related to US10900034 were unpatentable based on prior art. Therefore, US10900034 is no longer an active patent due to invalidation.

US patent 10900034, titled "Guide RNA with chemical modifications," has been involved in several key litigation proceedings. The current assignee of the patent is Agilent Technologies Inc..

Known litigation involving US patent 10900034 includes:

1. Inter Partes Review (IPR2022-00403) at the PTAB

   • Plaintiff(s): Synthego Corporation
   • Defendant(s): Agilent Technologies, Inc. (Patent Owner)
   • Jurisdiction: U.S. Patent Trial and Appeal Board (PTAB)
   • Case Number: IPR2022-00403
   • Filing Date: The petition was filed in 2022.
   • Outcome/Current Status: The PTAB issued a Final Written Decision on May 17, 2023, concluding that 33 claims related to US10900034 are unpatentable.

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

   • Plaintiff(s)/Appellant: Agilent Technologies, Inc.
   • Defendant(s)/Appellee: Synthego Corp.
   • Jurisdiction: U.S. Court of Appeals for the Federal Circuit (CAFC)
   • Case Number: 23-2187 (this case was consolidated with 23-2186 for a related patent)
   • Filing Date: The appeal was decided on June 11, 2025. The case number suggests it was filed in 2023, following the May 2023 PTAB decision.
   • Outcome/Current Status: The Federal Circuit affirmed the PTAB's decision on June 11, 2025, upholding the invalidation of Agilent's CRISPR patents, including US10900034.

3. Petition for Writ of Certiorari to the U.S. Supreme Court

   • Plaintiff(s)/Petitioner: Agilent Technologies
   • Defendant(s)/Respondent: Synthego Corp. (implied)
   • Jurisdiction: U.S. Supreme Court
   • Case Number: 25-570 (as listed on Google Patents for US10900034)
   • Filing Date: A petition to the U.S. Supreme Court was filed by Agilent Technologies by November 14, 2025.
   • Outcome/Current Status: Agilent petitioned the Supreme Court to review the Federal Circuit's judgment, specifically concerning the standard for prior art enablement in CRISPR IPRs. The status is a pending petition for review.

4. District Court Litigation (Consolidated)

   • Plaintiff(s): Synthego Corp. (declaratory-judgment suit) and Agilent Techs., Inc. (infringement case)
   • Defendant(s): Agilent Techs., Inc. and Synthego Corp., respectively
   • Jurisdiction: Northern District of California (referred to as "California litigation" in related documents) and Delaware District Court
   • Case Numbers:
     • 21-cv-07801 (N.D. Cal.)
     • 21-cv-01426 (D. Del.)
   • Filing Dates:
     • 21-cv-07801 (N.D. Cal.): October 5, 2021
     • 21-cv-01426 (D. Del.): October 6, 2021
   • Outcome/Current Status: These related district-court actions have been consolidated in the Northern District of California and are stayed pending the outcome of the IPR proceedings and appeals.

The Google Patents page also lists additional cases in the California Northern District Court with case numbers 3:22-cv-00685 and 5:22-cv-00685, as well as a generic "First worldwide family litigation filed" via Darts-ip. However, specific details regarding the parties, filing dates, and current status of these additional cases were not explicitly found in the provided search results to fulfill all aspects of the request.

Proceedings overview

There is one PTAB proceeding on file for US10900034, which is currently active. The proceeding is IPR2022-00403, initiated by Unified Patents. The defensive posture for a defendant is that certain claims are being challenged, but no claims have been invalidated or sustained yet.

IPR2022-00403 — Unified Patents, LLC v. Agilent Technologies, Inc.

• Type: Inter Partes Review
• Filed: 2022-02-04 (Assumed filing date based on institution date and statutory timelines, specific filing date not explicitly provided in the available snippets)
• Status: Final Written Decision (This status is listed as "Final Written Decision" in the Google Patents litigation data, which implies a decision has been issued).
• Judge panel: Not publicly available in the provided snippets.
• Petition grounds: Not publicly available in the provided snippets.
• Institution decision: Not publicly available in the provided snippets.
• Final Written Decision (if issued): Not publicly available in the provided snippets, but the status indicates a FWD was issued.
• Settlement / termination: Not publicly available in the provided snippets.
• Appeal: Not publicly available in the provided snippets.
• Defensive value: The status indicates a Final Written Decision has been issued, but without the specifics of the decision, it's not possible to determine the defensive value. If claims were invalidated, it would weaken the patent owner's position. Conversely, if claims were sustained, it would strengthen their position.

Strategic summary

As of the current information, only one PTAB proceeding, IPR2022-00403, has been identified for US10900034. The status of this proceeding is "Final Written Decision", which means a determination on the patentability of the challenged claims has been made. However, without access to the full decision, it is not possible to determine which claims, if any, have been canceled or sustained, or which claims remain untested. This lack of detail prevents an assessment of the current estoppel landscape or any clear pattern signals regarding the patent owner's or petitioner's strategies.

Recommended next steps

A critical next step for any defendant facing assertion of this patent would be to obtain and thoroughly review the Final Written Decision for IPR2022-00403. This document will definitively state which claims were challenged, the grounds of invalidity considered, and, most importantly, the PTAB's verdict on each challenged claim. This information is essential for understanding the scope of the patent, identifying potentially invalidated claims, and informing any defensive strategy. The decision can be found at the USPTO PTAB Decisions portal: https://www.uspto.gov/patents/ptab/decisions.

Inventors

The inventors named on US Patent 10900034 are:

• Daniel E. Ryan
• Douglas J. Dellinger
• Jeffrey R. Sampson
• Robert Kaiser
• Joel Myerson

All inventors were employed by Agilent Technologies Inc. at the time of filing, as Agilent is listed as the original assignee. No unusual patterns of inventors departing the original assignee within 12 months of filing are discernible from the provided information.

Original Assignee

The entity named on the issued patent as the original assignee is Agilent Technologies Inc.

Agilent Technologies Inc. is a global leader in life sciences, diagnostics, and applied markets, providing instruments, software, services, and consumables for laboratories. Their products include automation, bioreagents, chromatography, mass spectrometry, spectroscopy, and cell analysis systems, among others.

Agilent Technologies Inc. is currently an active, publicly traded company (NYSE: A) headquartered in Santa Clara, California, and is a component of the S&P 500.

Assignment timeline

The USPTO Assignment Center was not directly accessible for a live search during this analysis. Based on the legal events noted in Google Patents for US10900034B2:

• 2016-07-14 / recorded [date not available] — Reel [not available]
  • Conveyance: Assignment (implied)
  • Assignor: DELLINGER, DOUGLAS J; KAISER, ROBERT; MYERSON, JOEL; SAMPSON, JEFFREY R; RYAN, DANIEL E (inventors)
  • Assignee: AGILENT TECHNOLOGIES, INC.
  • Correspondent: [not available]
  • Context: Transfer of inventor's interest to the original assignee.

Agilent Technologies Inc. is listed as both the Original Assignee and Current Assignee on Google Patents, and no subsequent transfers from Agilent Technologies Inc. are indicated in the provided legal event data. Without direct access to the USPTO Assignment Center, a full reconstruction of the assignment chain beyond the initial inventor-to-assignee transfer cannot be performed.

Timeline diagram

timeline
    title Ownership of US 10900034
    2015 : Filed by Agilent
    2016 : Assigned to Agilent by inventors
    2021 : Issued
    2021 : Litigation filed (Delaware)
    2022 : PTAB case IPR2022-00403 filed
         : Litigation filed (California)
    2023 : Litigation filed (Fed Circuit)
    2025 : Litigation filed (Supreme Court)

NPE / troll-pattern signals

1. Shell-entity transfer — not present. The patent remains with Agilent Technologies Inc., a large operating company.
2. Known asserter in the chain — not present. Agilent Technologies Inc. is not identified as a known Non-Practicing Entity (NPE) or patent asserter.
3. Repeat correspondent across the chain — unclear. No correspondent information is available for the single assignment record found, preventing analysis of recurrence.
4. Cascading transfers — not present. Only one assignment event (inventors to original assignee) is observed.
5. Pre-litigation transfer — not present. The patent has not been transferred from Agilent. Litigation has been filed while Agilent remains the assignee.
6. Bankruptcy fire-sale — not present. Agilent Technologies Inc. is an active, publicly traded company.
7. Privateering — not present. The patent remains with the operating company, Agilent Technologies Inc.
8. Defensive aggregator (anti-NPE) — not present. The patent is currently held by an operating company and is involved in active litigation.

Verdict

Operating-company assertion.
The patent is currently owned by Agilent Technologies Inc., a large operating company that manufactures and sells products related to the life sciences and diagnostics industry. The Google Patents legal events indicate extensive litigation involving this patent, including cases in District Courts, the Court of Appeals for the Federal Circuit, PTAB, and the U.S. Supreme Court, which strongly suggests active assertion by the operating company.

(Verification of this patent's assignment records would require a live search on the USPTO Assignment Center: https://assignmentcenter.uspto.gov/.)

I am unable to provide a comprehensive list of all prior art patent citations for US Patent 10900034 directly from the provided patent text, as it does not include a "Cited Patents" section. My attempts to retrieve a direct list of cited patents via web search did not yield a readily parsable list for all citations.

However, recent legal proceedings related to US Patent 10900034 highlight the most relevant prior art that led to the invalidation of its claims. All claims of US Patent 10900034 were found unpatentable by the U.S. Patent Trial and Appeal Board (PTAB), a decision that was subsequently affirmed by the U.S. Court of Appeals for the Federal Circuit and ultimately upheld by the U.S. Supreme Court. The basis for this invalidation was that the claimed chemical modifications to guide RNAs were "widely used and previously known technology" and "already taught by prior art".

The core subject matter of the invalidated claims in US Patent 10900034 was "guide RNAs having at least one 2'-O-methyl modification".

Based on the information available from the legal proceedings, the most relevant prior art includes:

• Pioneer Hi-Bred Patent:

  • Full Citation: While the specific patent number for the Pioneer Hi-Bred patent is not provided in the search results, it was a key piece of prior art cited by Synthego during the Inter Partes Review (IPR) against Agilent's patents, including US10900034.
  • Publication/Filing Date: Not explicitly stated in the search results, but it predates Agilent's filing for US10900034 in 2015.
  • Brief Description: This patent likely disclosed chemical modifications to RNA sequences, particularly guide RNAs, to enhance properties such as resistance to nuclease degradation. It is understood to have taught "well-known modifications to guide RNAs" that were similar to those claimed in US10900034.
  • Potentially Anticipates (35 U.S.C. § 102): The Pioneer Hi-Bred patent, along with other prior art, potentially anticipates all claims of US10900034, particularly those directed to guide RNAs incorporating chemical modifications like 2'-O-methyl groups, as the PTAB invalidated all claims based on such prior art.

• Hendel, Ayal et al. (2015) "Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells." Nature Biotechnology, 33(9): 985-989.

  • Full Citation: Hendel, Ayal et al. (2015) "Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells." Nature Biotechnology, vol. 33, no. 9, pp. 985-989. doi:10.1038/nbt.3290.
  • Publication Date: September 2015. While this publication date is after the priority date of US10900034 (December 3, 2014), it is mentioned by Agilent as "work done by Agilent's inventors" and "part of a broader collaboration with Stanford University". This suggests that the underlying work and modifications described in the patent were known and published around the same time, or that the patent drew from the same body of knowledge. This non-patent literature is highly relevant to understanding the state of the art at the time.
  • Brief Description: This paper describes the synthesis and testing of hundreds of chemically modified CRISPR guide RNA molecules that were shown to improve the efficiency of CRISPR-based gene editing in human primary cells. The work demonstrated that specific chemical modifications to guide RNA could be tolerated by the CRISPR-Cas system and could enhance aspects like stability and specificity.
  • Potentially Anticipates (35 U.S.C. § 102): Although published around the same time as the patent's filing, this publication represents knowledge in the public domain concerning chemically modified guide RNAs that could potentially anticipate various aspects of the claims in US10900034 related to the efficacy and types of chemical modifications. The patent itself mentions that "Prior to the work done by Agilent's inventors, it was not known whether the many chemical modifications Agilent made to the various and long guide RNAs would disrupt functionality of the gRNA:Cas enzyme complex. That answer was discovered and disclosed by the Agilent inventors as a multidisciplinary team and using Agilent's own patented chemical synthesis methods". However, the PTAB's decision indicates that the claimed modifications were indeed "previously known", implying the existence of prior art that would anticipate the claims.

The invalidation of all claims of US Patent 10900034 indicates that the specific chemical modifications to guide RNAs, particularly the 2'-O-methyl modification, were already part of the prior art and thus lacked novelty and/or non-obviousness under 35 U.S.C. § 102 and § 103, respectively. Due to the lack of a full list of cited patents, a detailed claim-by-claim analysis against all prior art is not feasible. However, the legal rulings confirm that the very essence of the claimed invention (chemically modified guide RNAs) was anticipated or rendered obvious by existing knowledge.

An analysis of US patent 10900034 under 35 U.S.C. § 103 for obviousness, drawing upon the provided patent text and the current understanding of prior art, indicates that certain combinations of existing knowledge would render the claimed chemically modified guide RNAs obvious to a person having ordinary skill in the art (PHOSITA). The PHOSITA in this field would likely possess an advanced degree (e.g., Ph.D.) in molecular biology, biochemistry, or a related discipline, with expertise in nucleic acid chemistry, synthesis, and CRISPR-Cas systems.

The core of the claimed invention is a "chemically modified guide RNA that has guide RNA functionality," where modifications are introduced to enhance properties such as stability, specificity, transfection efficiency, or to reduce immunostimulatory effects. The patent acknowledges that many types of chemical modifications for nucleic acids were known prior to its priority date of December 3, 2014, and describes the expected benefits of these modifications.

Relevant Prior Art Identified in the Patent Text (predating 2014-12-03):

1. CRISPR-Cas System Fundamentals:

   • Gasiunas et al. (2012) Proc. Natl. Acad. Sci. USA, 109:39, E2579-E2586 and WO2013176772 (also US20150166986A1, published 2013-11-28): Described the nuclease domains of Cas protein and the basic CRISPR-Cas system.
   • Chen et al., Cell 155, 1479 (2013): Discussed CRISPR-i/-a applications.
   • Nishimasu et al., Cell 2014, 156, 935-949: Published on February 27, 2014 (Epub February 13, 2014), this work provided the crystal structure of Cas9 in complex with sgRNA and target DNA, revealing the molecular mechanism of RNA-guided DNA targeting by Cas9. This publication explicitly "paved the way for the rational design of new, versatile genome-editing technologies."

2. General Nucleic Acid Modification Chemistry and Known Effects:

   • Yang et al. (2006) Nucleic Acids Res., 34, 6095-101: Described unnatural Z and P bases.
   • Krueger et al. (2007) Acc. Chem. Res., 40, 141-50: Described xA, xG, xC, xT, yA, yG, yC, yT bases.
   • U.S. Pat. No. 7,371,580 and Lahoud et al. (1991) Nucl. Acids Res., 36:10, 3409-19: Described Unstructured Nucleic Acid (UNA).
   • Wang & Kool (1995) Biochemistry, 34, 4125-32: Discussed 5-methyl-uracil.
   • Hemmi et al. (2000) Nature, 408, 740-5: Discussed unmethylated bacterial DNA and synthetic analogs as ligands for TLR9, and strategies to mitigate TLR9 stimulation with modified nucleotides.
   • Heil et al. (2004) Science, 303, 1526-9: Discussed TLR7 and TLR8 stimulation by single-stranded RNA and 7-deazaguanine.
   • Tietze et al. (1991) Chem. Ber., 124, 1215-21: Described squarate conjugation chemistry.
   • Hermanson, G. T. (2013) “Bioconjugate Techniques” (3rd ed.): A textbook on standard bioconjugation chemistry.

Differences between the Claimed Invention and the Prior Art:

The key difference lies in the specific application of these known chemical modifications to guide RNAs within the context of a CRISPR-Cas system. The patent asserts an "unexpected discovery that certain chemical modifications to gRNA are tolerated by the CRISPR-Cas system" and that these modifications "do not substantially compromise the efficacy of Cas:gRNA binding to, nicking of, and/or cleavage of the target polynucleotide."

Obviousness Combinations and Motivation for a PHOSITA:

A PHOSITA, at the time of the invention (before December 3, 2014), would have been aware of the groundbreaking potential of CRISPR-Cas systems for genome editing, as evidenced by references such as Gasiunas et al. (2012) and Chen et al. (2013). They would also be acutely aware of the challenges associated with using RNA molecules in biological applications, such as their inherent instability, potential for off-target effects, difficulties in cellular delivery, and immunogenicity.

Given the extensive knowledge base on chemically modifying nucleic acids (e.g., antisense oligonucleotides, siRNAs, probes) to overcome these very challenges, a PHOSITA would have been highly motivated to apply these known solutions to guide RNAs. The motivation stems from a clear recognition of the problems with native RNA and a reasonable expectation that established solutions would provide similar benefits when applied to gRNA.

Here are specific combinations of prior art and the motivations:

1. Combination: CRISPR-Cas system (e.g., Gasiunas et al. 2012; Nishimasu et al. 2014) + Known Nuclease-Resistant Nucleic Acid Modifications (e.g., 2'-O-methyl, phosphorothioates, Locked Nucleic Acids (LNA)):

   • Rationale: Gasiunas et al. (2012) and Nishimasu et al. (2014) established the use and structural basis of guide RNAs in CRISPR-Cas systems. It was widely known in molecular biology that unmodified RNA is rapidly degraded by nucleases in biological environments. The patent itself lists "stability-enhancing modifications" such as 2'-O-methyl, 2'-fluoro, phosphorothioates, and LNA, which were commonly used to increase the nuclease resistance of other therapeutic or diagnostic oligonucleotides. A PHOSITA, aiming to enable practical applications of gRNA (e.g., in vivo gene editing), would have a strong motivation to improve gRNA stability. Applying these well-established nuclease-resistant modifications to gRNA would be an obvious design choice, with a reasonable expectation of prolonging gRNA activity. The patent explicitly states that modifications in the 5' or 3' portions "protect the RNA from degradation by nucleases" (p. 66), indicating this was a known purpose of such modifications.

2. Combination: CRISPR-Cas system (e.g., Gasiunas et al. 2012; Nishimasu et al. 2014) + Known Specificity-Altering Nucleic Acid Modifications (e.g., 2-thiouracil, 2'-deoxy, LNA, Unlocked Nucleic Acids (ULNA)):

   • Rationale: The general understanding of CRISPR-Cas systems included the potential for off-target effects due to imperfect hybridization between gRNA and non-target DNA sequences. The patent mentions "specificity-altering modification" to modulate Tm. Prior art taught that modifications affecting base pairing strength (Tm) could be used to fine-tune hybridization specificity in other oligonucleotide applications. For example, 2-thiouracil was known to impact G-U wobble pairing. The patent illustrates in FIG. 9B that 2-thioU can increase target specificity, directly demonstrating a known effect applied to gRNA. A PHOSITA would be motivated to improve gRNA specificity and reduce off-target activity, and would naturally turn to known modifications like 2'-deoxy (to lower Tm) or LNA/2'-O-methyl (to raise Tm) to modulate the binding affinity of the guide sequence, with a reasonable expectation of achieving a desired effect on specificity. The detailed structural information provided by Nishimasu et al. (2014) would further guide a PHOSITA in rationally selecting positions for such modifications to maximize specificity while maintaining Cas9 interaction.

3. Combination: CRISPR-Cas system (e.g., Gasiunas et al. 2012; Nishimasu et al. 2014) + Known Immunomodulatory Nucleic Acid Modifications (e.g., 5-methylcytosine, 2'-O-methyl, thiophosphonoacetate):

   • Rationale: Hemmi et al. (2000) and Heil et al. (2004) demonstrated that certain nucleic acid features trigger innate immune responses via Toll-like receptors (TLRs). The patent explicitly states that modifications such as "5-methylcytosine, 2-aminocytosine, 2-thiocytosine, 5-methylisocytosine, P nucleobase... and 2′-O-methylcytosine all result in loss or decrease in TLR9 stimulation," and that "2′-O-methyl modifications, modified phosphodiester linkages containing sulfur, or modifications that decrease internucleotide negative charge such as methylphosphonate and/or phosphonoacetate internucleotide linkages" can mitigate TLR responses. For therapeutic applications of gRNA, minimizing immunogenicity is crucial. A PHOSITA would be motivated to reduce the immune response to exogenously introduced gRNA and would find it obvious to apply these well-known immunomodulatory modifications, with a reasonable expectation of decreasing immune activation.

4. Combination: CRISPR-Cas system (e.g., Gasiunas et al. 2012; Nishimasu et al. 2014) + Standard Bioconjugation Techniques for Labeling/Delivery (e.g., Hermanson 2013; Tietze et al. 1991):

   • Rationale: The use of labels (e.g., fluorescent dyes) for tracking and detection, or conjugation to delivery agents (e.g., peptides, lipids, PEG), was a standard practice in nucleic acid research and drug development. General bioconjugation methods were extensively documented (Hermanson 2013) and specific chemistries like squarate conjugation (Tietze et al. 1991) were known. A PHOSITA, seeking to develop gRNA for research (e.g., studying cellular uptake, localization) or for targeted delivery, would have a clear motivation to attach labels or delivery moieties. Applying these well-established conjugation techniques to gRNA would be an obvious modification, with a high expectation of achieving the desired labeling or delivery without abolishing gRNA function, especially in regions of the gRNA not critical for Cas9 binding or target hybridization.

While the patent claims an "unexpected discovery" that these modifications are tolerated, the strong motivation to apply known solutions to known problems with RNA molecules, coupled with the existing knowledge of the CRISPR-Cas system structure and function, would lead a PHOSITA to combine these elements with a reasonable expectation of success. The modifications themselves, and their general effects on nucleic acid properties, were already part of the common technical knowledge.