CRISPR Interference Declared

By Kevin E. Noonan --

CRISPR (an acronym for Clustered Regularly lnterspaced Short Palindromic Repeats), which is part of a system for altering chromosomal sequences in situ in a cell in combination with a bacterially derived protein called Cas9, has been hailed as the "Breakthrough of the Year" for 2015, and rightfully so.  Just as the discovery of bacterial restriction enzymes by Cohen and Boyer in 1972 provided the ability to dissect DNA at specific sites in the DNA sequence, CRISPR provides a mechanism for inserting or deleting specific DNA sequences using CRISPR-associate targeting RNAs and the Cas9 RNA-guided DNA endonuclease enzyme.  It provides for the first time the type of specificity for altering DNA that the polymerase chain reaction (PCR) provided a generation ago for amplifying specific DNA, as illustrated by this schematic:

Given the commercial potential of this method, patenting is an obvious concern and, as it turns out, more than one group of inventors has filed patent applications on the reagents, methods, and cells produced or used to produce CRISPR modifications.  Because these applications were filed prior to March 16, 2013, the inventor(s) who first invented this invention have the right to patent it, and disputes regarding who was the first to invent are resolved using a procedure called an interference.

In this case, on January 11, 2016, the U.S. Patent and Trademark declared Interference No. 106,048 under the provisions of 37 C.F.R. § 41.203(b), naming Feng Zhang and his colleagues, the named inventor of the Broad Institute/MIT's patents, as the Junior Party, and Jennifer Doudna and her colleagues at UC/Berkeley as Senior Party (see Declaration).  These designations stem from which party was the first to file a patent application and are important, because (without evidence to the contrary) the PTO considers the Senior Party to be the presumptive inventor (and thus the burden to establish prior right based on earlier invention is borne by the Junior Party).

Paradoxically, the Broad Institute/MIT inventors have obtained a number of patents even though the Berkeley inventors have the earliest filing date and no issued patents (which is important because an interference cannot resolve the dispute between patents granted on the same invention).  This is because the Broad availed itself of priority examination ("fast track") provisions of the law.  In fact, the Broad attempted to provoke an interference with the Berkeley patents, albeit involving a fewer number of patents and claims than are involved in this interference.  The Broad patents (and their involved claims, which constitute all of the granted claims of all of the Broad patents) are:

• Patent 8,697,359 – claims 1-20
• Patent 8,771,945 – claims 1-29
• Patent 8,795,965 – claims 1-30
• Patent 8,865,406 – claims 1-30
• Patent 8,871,445 – claims 1-30
• Patent 8,889,356 – claims 1-30
• Patent 8,895,308 – claims 1-30
• Patent 8,906,616 – claims 1-30
• Patent 8,932,814 – claims 1-30
• Patent 8,945,839 – claims 1-28
• Patent 8,993,233 – claims 1-43
• Patent 8,999,641 – claims 1-28*,

while only one pending application from the Berkeley group is involved in the interference:

• US 2014-0068797 A1 - claims 165, 200, 202-218, 220-222 and 224-247.

Representative claims of each party are as follows:

The Broad/MIT:

1.    An engineered, non-naturally occurring CRISPR-Cas system comprising one or more vectors comprising:
    a) a first regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with a target sequence of a DNA molecule in a eukaryotic cell that contains the DNA molecule, wherein the DNA molecule encodes and the eukaryotic cell expresses at least one gene product, and
    b) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding a Type-II Cas9 protein,
    wherein components (a) and (b) are located on same or different vectors of the system, whereby the guide RNA targets and hybridizes with the target sequence and the Cas9 protein cleaves the DNA molecule, whereby expression of the at least one gene product is altered; and, wherein the Cas9 protein and the guide RNA do not naturally occur together.

5.    A method of altering expression of at least one gene product comprising introducing into a eukaryotic cell containing and expressing a DNA molecule having a target sequence and encoding the gene product an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)--CRISPR associated (Cas) (CRISPR-Cas) system comprising one or more vectors comprising:
    a) a first regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with the target sequence, and
    b) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding a Type-II Cas9 protein,
    wherein components (a) and (b) are located on same or different vectors of the system, whereby the guide RNA targets the target sequence and the Cas9 protein cleaves the DNA molecule, whereby expression of the at least one gene product is altered; and, wherein the Cas9 protein and the guide RNA do not naturally occur together.

6.    A CRISPR-Cas system-mediated genome editing method comprising introducing into a eukaryotic cell containing and expressing a DNA molecule having a target sequence and encoding at least one gene product an engineered, non-naturally occurring CRISPR-Cas system comprising one or more vectors comprising:
    a) a first regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with the target sequence, and
    b) a second regulatory element operable in a eukaryotic cell operably linked to a nucleotide sequence encoding a Type-II Cas9 protein,
    wherein components (a) and (b) are located on same or different vectors of the system, whereby expression of the at least one gene product is altered through the CRISPR-Cas system acting as to the DNA molecule comprising the guide RNA directing sequence-specific binding of the CRISPR-Cas system, whereby there is genome editing; and, wherein the Cas9 protein and the guide RNA do not naturally occur together.

Berkeley:

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.

In an interference, the PTO establishes one or more "counts," which is a phantom claim that encompasses the interfering subject matter.  The parties provide evidence of conception of at least one embodiment falling within the scope of the count and reduction to practice (actual or constructive, i.e., by filing a patent application having an enabling disclosure of said one embodiment).  While there are several complicated scenarios that can arise in an interference, generally a party that conceived first and reduced to practice first, and did not abandon, suppress or conceal the invention, will prevail.  The sole Count in the CRISPR interference reads as follows:

Count 1

A method, in a eukaryotic cell, of cleaving or editing a target DNA molecule or modulating transcription of at least one gene encoded thereon, the method comprising:
    contacting, in a eukaryotic cell, a target DNA molecule having a target sequence with an engineered and/or non-naturally-occurring Type II Clustered Regularly lnterspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising:
    a) a DNA-targeting RNA comprising
        i) a targeter-RNA or guide sequence that hybridizes with the target sequence, and
        ii) an activator-RNA or tracr sequence that hybridizes with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment, and
    b) a Cas9 protein,
    wherein the DNA-targeting RNA forms a complex with the Cas9 protein, thereby targeting the Cas9 protein to the target DNA molecule, whereby said target DNA molecule is cleaved or edited or transcription of at least one gene encoded by the target DNA molecule is modulated.

The interference will proceed in two stages.  The first stage involves the parties presenting motions that can modify the count, have certain claims declared outside the scope of the count (or vice versa), and ask for a finding that the claims are invalid under any of the provisions of the patent statute.  If these motions are not decided in a way that would disqualify one or both parties, then the interference will move to a second stage, where the Junior Party (The Broad) will present its proofs of conception and reduction to practice and the Senior Party will be permitted to oppose.  The Senior Party is under no obligation to present proofs earlier than its earliest filing date unless the Junior Party evinces evidence of (at least) earlier conception.  In practice, the parties can both be expected to submit their evidence.

The parties can also settle the inference privately, with the losing party filing a Concession of Priority and the prevailing party (usually) granting a license to the loser; such settlement agreements are kept confidential but must be filed.  Otherwise, everything else in an interference is public information and can be found under the Patent Trial and Appeal Board (PTAB) on the Office's PAIR website.  Office rules mandate the times for these two stages and an interference is usually concluded with a decision (if not earlier settled) within 36 months of the Declaration.

* The Broad/MIT also have several pending applications, including US 2014-0186919 A1; US 2014-0179770 A1; US 2014-0242664 A1; US 2014-0310830 A1; US 2014-0189896 A1; US 2014-0242699 A1; US 2014-0357530 A1; US 2015-0020223 A1; and US 2015-0079681 A1.  Should allowable claims be presented in any of these applications such claims may become involved in this interference.

Comments

[Disbelievin' Doug]

Thanks for the update. This technology is indeed a big deal, but in view of the Sequenom decision, I wager that the PTO will wash its hand of all of this by ruling that all the claims at issue are directed to ineligible subject matter under 101.

[D]

Which Office rule mandates the 36 month time frame?

[skeptical]

My brother "Cynical" would harumph and say that all of this is "directed to" a law of nature, add in a snippet of leaf-cutting, a magic microscope, and call it a day as he locks up the patent office doors on his way out.

;-)

[Kevin E. Noonan]

Dear Doug:

It might be tough for that to stick, seeing how the reaction occurs in bacteria in nature and its application to mammals requires human intervention.

[Barry Buchbinder]

Historical note/correction:
Cohen and Boyer did not discover restriction enzymes. See .
Cohen and Boyer got the first patent for recombinant DNA, which was invalid because, among other reasons, the conception was by Peter Lobban and, independently, Paul Berg. See . Albert Halluin wrote an article on it. (Why the patent was never challenged ....)

[Shahid Hasan]

good job! Kevin

[A Rational Person]

"It might be tough for that to stick, seeing how the reaction occurs in bacteria in nature and its application to mammals requires human intervention."

Kevin,

You just call that human intervention activity "conventional" so the claim could still be determined to be patent ineligible under Alice-Mayo.

[Kevin E. Noonan]

Thanks, Barry. As usual, things are not as simple as they seem.

Bacterial restriction was observed as early as the 1950's as a way that bacteria protected themselves from lambda virus infection. These enzymes, later termed Type I enzymes, were not useful for cloning or gene mapping because they cleaved at a relatively random position some distance from the recognition sites.

Ham Smith and Dan Nathan were the first to identify the Type II enzymes, including EcoR1, and to use them to prepare a physical map of SV40 viral DNA. Paul Berg indeed was the first person to make a recombinant plasmid (and to trigger the need to the Asilomar conference to discuss the potential risks of recombinant DNA).

But Cohen and Boyer were the first to realize that restriction digestion + ligase treatment could provide an effective way to prepare recombinant DNA:

Cohen, S. N., Chang, A. C. Y., Boyer, H. W. & Helling, R. B. (1973) Proc. Natl. Acad. Sci. USA 70, 3240-3244.

All this was summarized nicely by Richard Roberts in PNAS:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1087929/

Thanks for giving me the opportunity to clarify.

[Kevin E. Noonan]

Dear Rational:

I don't see producing the type of guide RNA in a non-bacterial cell combined with a bacterial nuclease as being conventional in any way.

I am pessimistic about overcoming Alice/Mayo but not despondent about it.

Thanks for the comment

[A Rational Person]

"I don't see producing the type of guide RNA in a non-bacterial cell combined with a bacterial nuclease as being conventional in any way."

Kevin,

Based on the reasoning of Mayo, the court can, if it wishes, just keep subdividing the parts of the claim until the sub-parts are each either conventional or directed to laws of nature. For example in Alice, the step of determining whether the level of 6-thiouguanine was less than or below a particular amount was subdivided so that the act of determining could be classified as separate "conventional step" and the particular level could be held to be a separate "step" directed a "law of nature."

Of course, the court in Mayo improperly held that the two wherein clauses were two separate "wherein steps" and not part of the determination step (b).

Also, under the holding of Myriad, DNA, despite all evidence to the contrary, is just "information" and not a physical molecule. Also, based on the holding of Myriad, despite all evidence to the contrary and despite what has been taught in chemistry classes around world for decades, breaking two covalent bonds in a first molecule to produce a second molecule DOES NOT produce a second molecule that is different from the first molecule.

From Myriad:

"Also, based on Myriad, any step that involved breaking a covalent bond at a particular place in a molecule involved the "conventional step" of breaking the covalent bond.

Nor are Myriad’s claims saved by the fact that isolating DNA from the human genome severs chemical bonds and thereby creates a nonnaturally occurring molecule. Myriad’s claims are simply not expressed in terms of chemical composition, nor do they rely in any way on the chemical changes that result from the isolation of a particular section of DNA."

The scientific ignorance of the Supreme Court is breathtaking.

[A Rational Person]

The last part of my previous post should have read:

Also, based on Myriad, any step that involves breaking a covalent bond at a particular place in a molecule involves merely involves "conventional step" of breaking the covalent bond and therefore cannot be used as a basis for patent eligibility.

From Myriad:

"Nor are Myriad’s claims saved by the fact that isolating DNA from the human genome severs chemical bonds and thereby creates a nonnaturally occurring molecule. Myriad’s claims are simply not expressed in terms of chemical composition, nor do they rely in any way on the chemical changes that result from the isolation of a particular section of DNA."

The scientific ignorance of the Supreme Court is breathtaking.

[Paul Cole]

@ A Rational Person

If you want to understand Myriad, it really pays to read Chakrabarty, American Fruit Growers and Hartranft.

By WHAT reasoning and jurisprudence does gBACA1 fall into the category "composition of matter" or "manufacture"? gBRCA1 may be a newly created molecule, but does that mean that it falls into either category?

[skeptical]

To Mr. Cole's point, the act of "creation by man" is not enough.

Take purification of salt water into potable water as a loose example.

Pure water may be created (and for argument's sake, let's say that the process takes a vat of salt water, breaks that vat down to all of its elements, and then efficiently removes all elements except hydrogen and oxygen, which are then recombined into pure water).

Clearly, the final product is something made by man.

Also as clearly, that final product itself is not eligible for patenting.

Note that an argument to deny a patent that might be attempted to be uses might be 102, since pure water is known.

But that is a different "test" and in truth but an additional block (patentability as opposed to eligibility).

Now one may still (possibly) obtain a patent on the process, but the end item itself, although "technically" a manufacture according to any legal definition of that term, remains outside the realm of eligibility precisely because it is a manufacture that nature itself can make.

There is no difference in kind from what is in "the warehouse of nature."

[A Rational Person]

Skeptical,

I understand that merely being "man-made" is not enough to make a product eligible if the product, such as pure water, exists in nature in the same form as the man-made product. But Mr. Cole is arguing that a new molecule that is formed by breaking covalent bonds and that does not exist in nature in its man-made form is not patent eligible. When has that ever been the rule before Myriad?

In your example of purifying water, even though you may be breaking down water in your purification process you are still ending up with water and salt at the end. The water and salt may now be separated, but they are still the same compounds with which you started, i.e., there are no "new" molecules being formed in your proposed process.

Also, if pure water did not exist in nature, and your process produced water of a purity that did not exist in nature and it was unobvious how to produce water of a particular purity, then it seems reasonable that you should be able to claim as a product water of particular purity. For example, see the following from MPEP 2144.04:

"Pure materials are novel vis-à-vis less pure or impure materials because there is a difference between pure and impure materials. Therefore, the issue is whether claims to a pure material are unobvious over the prior art. In re Bergstrom, 427F.2d 1394, 166 USPQ 256 (CCPA 1970). Purer forms of known products may be patentable, but the mere purity of a product, by itself, does not render the product unobvious."

[A Rational Person]

@Paul

"By WHAT reasoning and jurisprudence does gBACA1 fall into the category "composition of matter" or "manufacture"? gBRCA1 may be a newly created molecule, but does that mean that it falls into either category??"

Claim 1 of U.S. Patent No. 5,747,282 at issue in the Myriad case reads as follows:

1. An isolated DNA coding for a BRCA1 polypeptide, said polypeptide having the amino acid sequence set forth in SEQ ID NO:2.

The DNA molecule in Claim 1 was admitted by the court to be nonnaturally occurring. Therefore, the DNA molecule should have been found by the Supreme Court to be both a new composition of matter and a manufacture.

[skeptical]

A Rational Person,

Thank you for thinking through the loose example.

Where the example becomes tighter (and why Myriad was decided as it was), are the spaces where you "effectively" have something already in nature's warehouse (note: this is not a time dependent 102 argument).

While Myriad's claim was to an isolated coding, that mere isolation itself was deemed to effectively be the same as the un-isolated coding because (to the Court's eye), the "Gist" (to borrow the term) of the isolated portion was EFFECTIVELY the same as the non-isolated portion. While in fact different, they were not different "enough."

That's my attempt at translating what the Court said anyway.

[A Rational Person]

@Paul

The problem is for me is that there is no "isolation of DNA" exception to 35 USC 101 specifically spelled out in the statute. In the absence of such an explicit exception, a DNA molecule should have been treated as any other chemical.

When you break the covalent chemical bonds of a first DNA to form a second DNA molecule, the second DNA molecule is a different molecule than the first DNA molecule and has different properties than the first DNA.

This no different than breaking a covalent bond in ethanol, a drinkable alcohol and generally considered safe in limited quantities, to form methane (a gas) and a methanol (a liquid that is poisonous when drunk). If methane and methanol did not exist naturally, no chemist would consider them part of "nature's warehouse" because ethanol can be broken down to form them.

Also, the idea that the difference between the second and first DNA molecule is not "significant" or "different enough" is rebutted by the fact that that the party challenging the patents in Myriad was willing to spend the money to invalidate the patents. If the isolated DNA molecule was not significantly different than the full naturally occurring DNA molecule the people challenging parties could have simply used the full naturally occurring DNA molecule.

I think you give the Court too much credit with respect to their scientific knowledge and logical reasoning.

[A Rational Person]

@Paul,

My apologies, my last post should have been

@Skeptical

[skeptical]

A Rational Person,

I think that you have overstated the situation with your response.

Your counter example has a logical error, in that the example you have chosen does in fact make a difference in kind. Not all breaking of bonds has that same result, so you are attempting to make a logical leap that does not fit.

Further, your attempt to use the actual battle (and the "expense" or even "futility") of letting the challenger of Myriad's patent to use the "effective" other item - the so called item already in the warehouse of nature INSTEAD of the Myriad CLAIMED item is also a use of "false" logic.

Yes there is a difference, and yes, that difference does have real world effects. But those are just not the effects that the Court is looking at from a differentiating patent perspective.

(Again, do not "shoot the messenger" - this is just my take on what the Court stated)

What the Court did was to look at the (in)famous "Gist," and decide - based on that "Gist" whether "using the full molecule" or the claimed portion made ENOUGH of a difference for the aim of what that use was going to be.

Here, the answer was "no," as (again, as I take the Court's writing) EVEN using the full molecule, one would need to engage in (conventional) processes that would get to the same EFFECTIVE place as the claim in order to have the utility in question. Effectively, the "allowance" of the claim would mean that the full molecule you offer as an option would NOT be a viable option.

Personally, I think that your position has legs BECAUSE it shows that a specific utility - NOT found in nature - accompanies man's doing something with something found in nature. To me this is indeed "different enough."

Was this argument made? I do seem to recall that THAT answer is yes (even on this very blog).

Was that argument LISTENED to? Or, was there an Ends already in mind?

[A Rational Person]

@Skeptical,

True, not all breaking of covalent bonds would produce a different material. For example in the case of a polymer or any compound with repeating units such as cellulose (remember the baseball bat "isolated" from a tree example during oral argument?) and some type of DNA and RNA molecules, when you separate out a portion of the molecule the new molecule formed is essentially the same material but a smaller piece.

But that wasn't the case with the isolated
DNA molecule in Myriad. And my concern is that no one on the Court understood the difference as well (see the baseball bat example during oral argument).

Your point about the theoretical false logic of the expense of the suit is well-taken. I am also no expert on the specific field of the patent and its economics. There was also a strong political component to this suit with amici (wrongly) concerned about patenting human beings and their DNA. But I would still guess that from a practical point, people in this field would believe there was a significant different in working with the isolated DNA molecule instead of the full DNA molecule.

I also don't think it's just a case of arguments not being listened to by the Court. I think it's also a case that they just do not understand basic chemistry.

For example, I would not be surprised that if an attorney ever based an argument on the analogy that iron loses weight as it rusts that at least one member of the Supreme Court would buy this erroneous analogy (in fact, as has been known for centuries now, iron gains weight as it rusts by reacting with oxygen to form iron oxide).

[skeptical]

Comment noting the allowance to play with human embryos by UK scientists using CRISPR...

Stuck in moderation...?

Too "sensitive" to post...?

[Donald Zuhn]

Skeptical:

All of the comments we have received in the past week have been published. You will have to re-send your earlier comment.

Don

[skeptical]

Sorry Don, did not save the comment.

If I recall correctly, the comment was noting a certain lack of ethical concern tangent to this article in that recently, the UK has approved "playing" with human embryos using the CRISPR technology.

The article I read did not detail any limits as to the grant to use CRISPR on the human embryos, and visions of the Island of Doctor Moreau danced in my head.