Whither CRISPR? University of California/Berkeley Granted Another CRISPR Patent

By Kevin E. Noonan --

The U.S. Patent and Trademark Office granted U.S. Patent No. 10,113,167 today to the University of California/Berkeley, directed to an aspect of its CRISPR technology (where CRISPR is an acronym for Clustered Regularly lnterspaced Short Palindromic Repeats).  The interference between the Broad Institute and the University of California/Berkeley over patents directed to CRISPR technology has been in the spotlight over the past few years (see "CRISPR Interference Declared"; "PTAB Decides CRISPR Interference -- No interference-in-fact"; "PTAB Decides CRISPR Interference in Favor of Broad Institute -- Their Reasoning"; "University of California/Berkeley Appeals Adverse CRISPR Decision by PTAB"; and "Berkeley Files Opening Brief in CRISPR Appeal").  And while the Broad was successful in getting the Federal Circuit to affirm the PTAB's decision that there was no interference-in-fact between the parties' claims (see "Regents of the University of California v. Broad Institute, Inc. (Fed. Cir. 2018): Federal Circuit Affirms PTAB in Appeal of CRISPR Interference") and there have been reports of the outcomes of other skirmishes between the parties in the meantime (see "The CRISPR Chronicles -- Broad Institute Wins One and Loses One"), questions remain about how the rights to this technology will be apportioned between the parties and useful, reliable patent licenses will be granted to permit robust development and fulfillment of the many promises of CRISPR in a wide variety of genetic contexts.

The Broad's extensive patent portfolio survived the interference, particularly these patents directly at issue:

• U.S. Patent No. 8,697,359 -- claims 1-20
• U.S. Patent No. 8,771,945 -- claims 1-29
• U.S. Patent No. 8,795,965 -- claims 1-30
• U.S. Patent No. 8,865,406 -- claims 1-30
• U.S. Patent No. 8,871,445 -- claims 1-30
• U.S. Patent No. 8,889,356 -- claims 1-30
• U.S. Patent No. 8,895,308 -- claims 1-30
• U.S. Patent No. 8,906,616 -- claims 1-30
• U.S. Patent No. 8,932,814 -- claims 1-30
• U.S. Patent No. 8,945,839 -- claims 1-28
• U.S. Patent No. 8,993,233 -- claims 1-43
• U.S. Patent No. 8,999,641 -- claims 1-28

The Berkeley application-in-interference, U.S. Application No. 13/842,859, published as U.S. Patent Application Publication No. US 2014/0068797, remains in Patent Office limbo during the pendency of court proceedings; these claims include the following:

165.  A method of cleaving a nucleic acid comprising contacting a target DNA molecule having a target sequence with an engineered and/or non-naturally-occurring Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising
    a) a Cas9 protein; and
    b) a single molecule DNA-targeting RNA comprising
        i) a targeter-RNA that hybridizes with the target sequence, and
        ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment,
    wherein the activator-RNA and the targeter-RNA are covalently linked to one another with intervening nucleotides,
    wherein the single molecule DNA-targeting RNA forms a complex with the Cas9protein,
    whereby the single molecule DNA-targeting RNA targets the target sequence, and the Cas9 protein cleaves the target DNA molecule.

203.  An engineered and/or non-naturally occurring Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising
    a) a Cas9 protein, or a nucleic acid comprising a nucleotide sequence encoding said Cas9 protein; and
    b) a single molecule DNA-targeting RNA, or a nucleic acid comprising a nucleotide sequence encoding said single molecule DNA-targeting RNA;
    wherein the single molecule DNA-targeting RNA comprises:
        i) a targeter-RNA that is capable of hybridizing with a target sequence in a target DNA molecule, and
        ii) an activator-RNA that is capable of hybridizing with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment,
    wherein the activator-RNA and the targeter-RNA are covalently linked to one another with intervening nucleotides; and
    wherein the single molecule DNA-targeting RNA is capable of forming a complex with the Cas9 protein, thereby targeting the Cas9 protein to the target DNA molecule,
whereby said system is capable of cleaving or editing the target DNA molecule or modulating transcription of at least one gene encoded by the target DNA molecule.

224.  A Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising:
    a Cas9 protein; and
    a single molecule DNA-targeting RNA, or a nucleic acid comprising a nucleotide sequence encoding said single molecule DNA-targeting RNA,
    wherein the single molecule DNA-targeting RNA comprises:
        i) a targeter-RNA that is capable of hybridizing with a target sequence in a target DNA molecule, and
        ii) an activator-RNA that is capable of hybridizing with the targeter-RNA to form a double-stranded duplex of a protein-binding segment,
    wherein i) and ii) are arranged in a 5' to 3' orientation and are covalently linked to one another with intervening nucleotides;
    wherein the single molecule DNA-targeting RNA is capable of forming a complex with the Cas9 protein and hybridization of the targeter-RNA to the target sequence is capable of targeting the Cas9 protein to the target DNA molecule, and
    wherein the single molecule DNA-targeting RNA comprises one or more sequence modifications compared to a sequence of a corresponding wild type tracrRNA and/or crRNA.

The '167 patent granted today, includes the following claims:

1.  A non-naturally occurring DNA-targeting RNA, or a nucleic acid encoding the non-naturally occurring DNA-targeting RNA, wherein the non-naturally occurring DNA-targeting RNA comprises: (a) a targeter-RNA comprising: (i) a first nucleotide sequence that is complementary to a target sequence of a target DNA molecule, and (ii) a second nucleotide sequence that hybridizes with an activator-RNA, wherein the first and second nucleotide sequences are heterologous to one another; and (b) the activator-RNA, which hybridizes with the second nucleotide sequence of the targeter-RNA to form a double-stranded RNA (dsRNA) duplex of a protein-binding segment, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 8 to 15 base pairs, wherein the non-naturally occurring DNA-targeting RNA is capable of forming a complex with a Cas9 polypeptide and targeting the complex to the target sequence of the target DNA molecule.

12.  A non-naturally occurring DNA-targeting RNA that comprises: (a) a targeter-RNA comprising a nucleotide sequence that is complementary to a target sequence of a target DNA molecule, and (b) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA (dsRNA) duplex of a protein-binding segment, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 8 to 15 base pairs, wherein the non-naturally occurring DNA-targeting RNA comprises one or more of: a non-natural internucleoside linkage, a nucleic acid mimetic, a modified sugar moiety, and a modified nucleobase, and wherein the non-naturally occurring DNA-targeting RNA is capable of forming a complex with a Cas9 polypeptide and targeting the complex to the target sequence of the target DNA molecule.

19.  One or more nucleic acids encoding a non-naturally occurring DNA-targeting RNA that comprises: (a) a targeter-RNA comprising a nucleotide sequence that is complementary to a target sequence of a target DNA molecule, and (b) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA (dsRNA) duplex of a protein-binding segment, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 8 to 15 base pairs, wherein the non-naturally occurring DNA-targeting RNA is capable of forming a complex with a Cas9 polypeptide and targeting the complex to the target sequence of the target DNA molecule, and wherein the one or more nucleic acids comprises a first nucleotide sequence encoding the targeter-RNA and a second nucleotide sequence encoding the activator-RNA; wherein the first nucleotide sequence, the second nucleotide sequence, or both, is operably linked to a heterologous transcriptional control sequence and/or a heterologous translational control sequence.

36.  A composition comprising: (1) a non-naturally occurring DNA-targeting RNA, or a nucleic acid encoding the non-naturally occurring DNA-targeting RNA, wherein the non-naturally occurring DNA-targeting RNA comprises: (a) a targeter-RNA comprising a nucleotide sequence that is complementary to a target sequence of a target DNA molecule, and (b) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA (dsRNA) duplex of a protein-binding segment, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 8 to 15 base pairs, wherein the non-naturally occurring DNA-targeting RNA is capable of forming a complex with a Cas9 polypeptide and targeting the complex to the target sequence of the target DNA molecule; and (2) one or more of: a nuclease inhibitor, a buffering agent, a detergent, a polyamine, an adjuvant, a wetting agent, a stabilizing agent, an antioxidant, and a complexing agent.

54.  A composition comprising: (1) a Cas9 polypeptide, or a nucleic acid encoding the Cas9 polypeptide; and (2) a non-naturally occurring DNA-targeting RNA, or a nucleic acid encoding the non-naturally occurring DNA-targeting RNA, wherein the non-naturally occurring DNA-targeting RNA comprises: (a) a targeter-RNA comprising: (i) a first nucleotide sequence that is complementary to a target sequence of a target DNA molecule, and (ii) a second nucleotide sequence that hybridizes with an activator-RNA, wherein the first and second nucleotide sequences are heterologous to one another; and (b) the activator-RNA, which hybridizes with the second nucleotide sequence of the targeter-RNA to form a double-stranded RNA (dsRNA) duplex of a protein-binding segment, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 8 to 15 base pairs, wherein the non-naturally occurring DNA-targeting RNA is capable of forming a complex with the Cas9 polypeptide and targeting the complex to the target sequence of the target DNA molecule.

While the Broad was quicker off the mark in applying for and having granted patents on its flavor(s) of CRISPR, UC/Berkeley has a number of pending applications, including U.S. Serial No. 14/942,782, as well as the following eight other applications:

• U.S. Application No. 15/435,233, filed on 2-16-2017, which claims the benefit of U.S. Application No. 15/138,604;
• U.S. Application No. 15/925,544, filed on 3-19-2018, which claims the benefit of U.S. Application No. 15/138,604;
• U.S. Application No. 15/947,700, filed on 4-6-2018, which claims the benefit of U.S. Application No. 15/138,604;
• U.S. Application No. 15/947,718, filed on 4-6-2018, which claims the benefit of U.S. Application No. 15/138,604;
• U.S. Application No. 15/981,808, filed on 5-16-2018, which claims the benefit of U.S. Application No. 15/138,604;
• U.S. Application No. 15/981,809, filed on 5-16-2018, which claims the benefit of U.S. Application No. 15/138,604;
• U.S. Application No. 16/136,159, filed on 9-19-2018, which claims the benefit of U.S. Application No. 15/138,604; and
• U.S. Application No. 16/136,165, filed on 9-19-2018, which claims the benefit of U.S. Application No. 15/138,604.

Today's granted patent joins previously granted U.S. Patent No. 10,000,772, which contains claims directed to these embodiments of the invention:

1.  A method of modifying a target DNA molecule, the method comprising: contacting a target DNA molecule having a target sequence with a complex comprising: (a) a Cas9 protein; and (b) a DNA-targeting RNA comprising: (i) a targeter-RNA that hybridizes with the target sequence, and (ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA (dsRNA) duplex of a protein-binding segment, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 10 to 15 base pairs, wherein said contacting takes place outside of a bacterial cell and outside of an archaeal cell, thereby resulting in modification of the target DNA molecule.

This patent has eight pending related applications:

• U.S. Application No. 15/138,604, filed on 4-26-2016, which claims the benefit of U.S. Application No. 14/685,502;
• U.S. Application No. 15/435,233, filed on 2-16-2017, which claims the benefit of U.S. Application No. 14/685,502;
• U.S. Application No. 15/947,700, filed on 4-6-2018, which claims the benefit of U.S. Application No. 14/685,502;
• U.S. Application No. 16/136,159, filed on 9-19-2018, which claims the benefit of U.S. Application No. 14/685,502;
• U.S. Application No. 15/925,544, filed on 3-19-2018, which claims the benefit of U.S. Application No. 14/685,502;
• U.S. Application No. 15/947,718, filed on 4-6-2018, which claims the benefit of U.S. Application No. 14/685,502;
• U.S. Application No. 15/981,808, filed on 5-16-2018, which claims the benefit of U.S. Application No. 14/685,502;
• U.S. Application No. 15/981,809, filed on 5-16-2018, which claims the benefit of U.S. Application No. 14/685,502;
• U.S. Application No. 16/136,165, filed on 9-19-2018, which claims the benefit of U.S. Application No. 14/685,502; and
• U.S. Application No. 15/343,156, filed on 11-3-2016, which claims the benefit of U.S. Application No. 14/685,502.

With regard to today's issued claims, they are not limited to the type of cell in which the CRISPR reaction occurs (nor, indeed, are limited to any cell at all).  The subject matter eligibility of the claimed RNA molecules is presumably supported by limitations to "non-naturally occurring" (reminiscent of limiting transgenic animals to "non-human" embodiments) as well as specific embodiments of non-naturally occurring nucleic acid forms (comprising a "non-natural internucleoside linkage, a nucleic acid mimetic, a modified sugar moiety, and a modified nucleobase"); indeed, the prosecution file history shows applicants overcame subject matter eligibility rejections under 35 U.S.C. § 101.  While there are dependent claims directed to more specific embodiments of the claimed RNAs, the breadth of scope, necessarily limited extent of disclosure of specific embodiments, and functional characterization of the claimed RNAs (e.g., "the activator-RNA, which hybridizes with the second nucleotide sequence of the targeter-RNA to form a double-stranded RNA (dsRNA) duplex of a protein-binding segment") raise legitimate questions regarding whether these claims will stand up to inevitable challenge on 35 U.S.C. § 112 grounds.

All this is to say that the patent situation remains somewhat murky, at least with regard to which entity, or both, or another, will ultimately have sufficiently strong or comprehensive patent protection to give licensees confidence in their rights to develop CRISPR technology to fulfill its great promise.

Comments

[MaxDrei]

Meanwhile, at the EPO there has already been a reasoned Decision by the Opposition Division, with major repercussions in this struggle for priority between the rival East Coast and West Coast teams. As I understand it, the earlier patents are pre-AIA. It will be interesting to see how the later patents make out i) under the AIA vs ii) the EPC.

[Kevin E Noonan]

As I understand things, Max, the EPO decision was based on the Broad running afoul of the priority claiming requirements of the EPC. These won't affect the US patents/applications so we could get different outcomes.

Thanks for the comment

[MaxDrei]

Indeed! What will be interesting is how the respective laws arrive at different outcomes, given a common fact base of filing events by the rival filers, when both jurisdictions claim to grant to the party that was "First to File".