DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant’s election without traverse of Group II (claim 2) in the reply filed on 4/10/2026 is acknowledged. Applicant has amended claims 3-8 and 11-33 to depend from claim 2. Therefore, claims 2-8 and 11-33 are pending and are examined on the merits herein.
Claims 1 and 34 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected inventions, there being no allowable generic or linking claim.
It is noted that claims 1 and 34 are not labeled with the proper claim identifier, as both claims state (Original) and not (Withdrawn). Applicant must amend these claims to reflect their change in status. See MPEP 714 II (C).
Information Disclosure Statement
The information disclosure statements (IDSs) submitted on 3/27/2023 and 7/18/2024 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner, except where noted.
The information disclosure statement filed 3/27/2023 fails to comply with 37 CFR 1.98(a)(2), which requires a legible copy of each cited foreign patent document; each non-patent literature publication or that portion which caused it to be listed; and all other information or that portion which caused it to be listed. No copy of non-patent literature documents 111 and 116 has been provided, and so these references have not been considered.
Claim Interpretation
Regarding the use of the term “activated” in the instant claims, it is noted that no specific definition is provided. Para. 111 of the instant specification notes that, “Activation of a Cas protein may include contacting one or more target sequences with a guide RNA sequence associated with the Cas protein. In some embodiments, the guide RNA of the Cas protein may help to activate the Cas protein's RNase activity by hybridizing to a complementary target RNA sequence.” Thus, this term will be interpreted as the ability of a claimed component to perform its claimed function.
It is noted that in para. 101 of the instant specification, “a,” “an,” and “the” in singular form also include plural references. Thus, in claim 2, “a reporter molecule” includes multiple reporter molecules. Therefore, there is no indefiniteness issue with regard to both the primary activator complex and signal amplifier complex both cleaving the reporter molecule.
Regarding the use of the terms “cage” and “caged,” the instant specification does not specifically define this term. Para. 16 states that caged molecules may have “caging structures or molecules,” and repeatedly throughout the specification, a caged molecule is described as comprising or creating a molecule with a stem-loop structure (e.g. paras. 36, 40, 43-44) or containing a modification or moiety (e.g. paras. 44 and 175). Figure 1G shows various cages that are simply additional sequences added to the 3’ or 5’ end of a gRNA or activator. Para. 64 notes that trans cage molecules may be “oligonucleotide sequences…that are not covalently linked to the amplifier or guide RNA which are able to interact with the amplifier or guide and act as a cage.” Para. 146 states, “A "trans caging molecule" can comprise any nucleic acid that binds to another nucleic acid such that a duplex structure is formed or created,” which is broader in scope than the function presented in para. 64. Though it appears that caged molecules can in some instances prevent or delay hybridization until removal of the cage (e.g. Figures 1C and 1H), this does not always appear to be the case (e.g. Figure 1G). Thus, a “trans cage” will generally be interpreted to be the use of two nucleic acid molecules as described in para. 147, though no particular function is required, and a cage generally can include the presence of a trans cage molecule, or when a trans cage is not specified, the presence of a stem-loop or some other sequence modification is required.
Claim Objections
Claims 4-7 are objected to because of the following identical informality: to properly capture the fact that one or more Cas-effector enzymes may be used, each claim should read “the first and/or second Cas-effector enzyme comprise(s).” Appropriate correction is required.
Claims 5-7 are objected to because of the following identical informality: the phrase “Cas effectors” should be amended to read “Cas-effector enzymes” to better match the language used in earlier claims. Appropriate correction is required.
Claim 14 is objected to because of the following informality: the phrase “the reporter molecule” should be removed from the claim, such that the body of the claim reads “wherein the detectable label comprises one or more fluorescent dyes.” Appropriate correction is required.
Claim 19 is objected to because of the following informality: in the final line of the claim, the word “or” should be removed between “locked nucleic acid (LNA)” and “moieties.” Appropriate correction is required.
Claim 20 is objected to because of the following informality: the claim should read “wherein one or more of the activator sequences comprise.” Appropriate correction is required.
Claim 22 is objected to because of the following informality: in line 1, the word “the” should be removed. Appropriate correction is required.
Claims 24-25 are objected to because of the following identical informality: in line 2, the word “the” should be inserted before “first and/or second guide RNAs.” Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 12, 18, 22, and 30 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 12 recites the limitation "the fluorophore" in line 2. There is insufficient antecedent basis for this limitation in the claim, as fluorophores are not discussed earlier in the claim or in claim 2, from which this claim depends. It will be interpreted as though “the fluorophore” is referring to “the detectable label” of claim 2.
Claim 18 is indefinite because in lines 1-2, the phrase “the guide RNA” is used, but it is unclear if this is referring to the first guide RNA, the second guide RNA, or both. For the purposes of claim interpretation, any of these options will be considered to meet the requirements of the claim.
Claim 22 is rejected due to the use of the phrase “modified to allow conditional interaction with the Cas-effector enzyme during the optimal time frame.” Specifically, it is unclear when “the optimal time frame” would be during the method of claim 2, and the instant specification does not define when such a time period would occur. Para. 172 of the instant specification states that in some embodiments, an optimal time frame may be after detection of a target nucleic acid, but at least one guide RNA would already have been used in the system of claim 2 to cleave the reporter molecule before any such detection could occur. Thus, the scope of the claim within the context of the system of claim 2 is indefinite. Prior art will be considered to read on the claim if it generally teaches modifications to guide RNAs or activator sequences that allow for any conditional interaction with Cas-effector enzymes.
Claim 30 recites the limitation "the individual" in its final line. There is insufficient antecedent basis for this limitation in the claim, as “an individual” is not recited earlier in the claim or in claim 2, from which this claim depends. As claim 2 already requires the presence of a sample, this limitation will be considered to be met by the prior art if any of the sample types listed in claim 30 are used with a system that reads on that of claim 2, as collection of said sample would naturally have already occurred in such an instance.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 2-5, 7-8, 11-15, 18-19, 23-27, 29-31, and 33 are rejected under 35 U.S.C. 103 as being unpatentable over Doudna et al. (WO 2017/218573 A1; cited in Applicant’s IDS) in view of Gootenberg et al. (Science, 20181).
Doudna teaches methods for detecting target RNA involving C2c2 proteins and guide RNA (para. 7). The reference states that C2c2 can cleave in both cis and trans fashion (paras. 187 and 347). Para. 381 and Figure 11 show that the C2c2 protein/guide RNA complex can bind to a target RNA while non-specifically cleaving a non-target labeled RNA. Figure 13A shows this where the non-target RNA is a fluorophore/quencher pair and the target RNA is described as an activator. Thus, the labeled cleaved non-target RNA acts as the claimed reporter molecule, the C2c2 protein/guide RNA complex acts as the primary activator complex, and the target RNA acts as the claimed activator to enable the non-specific non-target cleavage. Note that C2c2 is also known as Cas13a (para. 94).
Para. 162 of Doudna states that methods involving two target RNAs can be performed, where a first C2c2 protein binds with a first guide RNA that also hybridizes with a first RNA target, and a second C2c2 protein binds to a second guide RNA that also binds to a second RNA target. Both C2c2 proteins then perform RNA cleavage to produce a detectable signal. The detectable label can be a FRET or fluorophore/quencher pair (paras. 163-164). Thus, the reference shows that multiple C2c2 protein/guide RNA complexes can function similarly in a single method. Figures 13D and 27E of the reference also note that two C2c2 complexes can both perform trans cleavage in a reaction.
Aspect 46 on page 113, Aspect 52 on page 114, and claims 46 and 52 note that two target sequences can be distinct sequences on the same molecule. This teaching of Doudna would indicate to the ordinary artisan that two target sequences that are to be hybridized by two distinct C2c2 complexes may be close to each other on a given RNA molecule. In such an instance, simultaneous binding of both target sequences to both C2c2 complexes would likely not be efficient or even possible. Thus, if the ordinary artisan wanted to detect both target sequences, they would recognize that cleavage or other manipulation of the molecule would be needed.
In the context of mapping Doudna to the instant claims, the reference does not teach that the activation of a primary activation complex activates an activator sequence recognized by a second guide RNA.
Gootenberg teaches detection of nucleic acids with a SHERLOCK method (Abstract). Figure 1 shows the basics of this method with Cas13 enzymes, where trans cleavage of a reporter molecule occurs upon binding with a target sequence. In Figure S24, a positive feedback SHERLOCK reaction is shown, where a Cas13 cleaves a target, then that cleaved target is bound with a Csm6 protein, and the Csm6 protein cleaves a reporter molecule to produce a detectable signal.
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the guidance of Gootenberg to solve the problem posed by utilizing multiple target sequences close together on the same molecule in Doudna. Specifically, Gootenberg teaches that the product of a first Cas-cleavage reaction can be used to activate a second cleavage reaction. Thus, the ordinary artisan could use this concept to design the C2c2 systems of Doudna such that the first C2c2 protein complex hybridizes to the first target in the molecule, and cleaves the molecule so that the second target can then hybridize to the second C2c2 protein complex. The second C2c2 complex would thus act as the claimed signal amplifier, and the cleaved second target would act as the claimed activator sequence. Doudna specifically states in para. 187 that, “If the HEPNl and HEPN2 domains of the C2c2 protein are intact, once activated, the C2c2 protein cleaves the target RNA, but also cleaves non-target RNAs.” Thus, both the cleavage of the molecule and the cleavage of the fluorescent reporter would be possible for the C2c2 protein complexes. By designing the system in this way, it would ensure that both targets can be detected, as para. 162 of Doudna notes that the signal produced by each target molecule can be distinguishable from one another. Para. 186 of Doudna specifically notes that this type of detection, where different target sites are on the same RNA, can generally increase the sensitivity of detection for a particular RNA. Thus, such a system would be would be helpful in instances where it is desired to determine the presence of an RNA molecule generally in a sample, motivating the ordinary artisan, as the detectable signals generated by each C2c2 complex could be added together. Additionally, this detection method would be useful in instances where multiple closely packed mutations need to be distinctly detected, such as SNPs that are within the same RNA molecules, or to evaluate variants such as CNVs. There would be a reasonable expectation of success as Doudna teaches that the C2c2 system is “readily-programmable,” (para. 410), teaches that crRNA can be specifically designed (para. 411), and generally teaches that the target RNA can be any single-stranded RNA (para. 121), and so the design of the C2c2 complexes to perform the method of Doudna in view of Gootenberg (specifically, to target the desired sequences with the guide RNAs) would be possible.
Thus, claims 2-5, 7-8, 11, and 14 are prima facie obvious over Doudna in view of Gootenberg.
Regarding claim 12, Doudna teaches that a labeled detector RNA can contain various modifications (paras. 146-161). This labeled detector RNA can be the generator of detectable signal resulting from cleavage with the C2c2 complex, specifically resulting from non-target trans cleavage (e.g. paras. 8, 14, 17, and 124). The modifications to these detectors can include locked nucleic acids (LNAs; para. 156), which are specifically noted to be a type of caging in the instant specification (see para. 44). As the teachings of Doudna encompass using locked nucleic acids with the detectors in their methods, it would be prima facie obvious that such modified detectors could also be used in the method of Doudna in view of Gootenberg.
Thus, claim 12 is prima facie obvious over Doudna in view of Gootenberg.
Regarding claim 13, paras. 146 and 290 of Doudna state that labeled detectors can have self-complementarity and can produce fully or partially double stranded compounds (e.g. stem-loops). Thus, labeled detectors may have nucleic acids that hybridize to one another to form a duplex structure, which is encompassed by “trans cage” as described above in the “Claim Interpretation” section.
Thus, claim 13 is prima facie obvious over Doudna in view of Gootenberg.
Regarding claims 15 and 18-19, the guide RNAs of Doudna have stem-loop structures – see Figures 1, 4, 9-10, 13, 17, 19, and 27 for example.
Thus, claims 15 and 18-19 are prima facie obvious over Doudna in view of Gootenberg.
Regarding claim 23, Doudna teaches that when detecting two different target RNAs, the first C2c2 system can cleave A sequences and the second can cleave U sequences, where the first C2c2 system would not be capable of cleaving a polyU sequence and the second C2c2 system would not be capable of cleaving a polyA sequence. Thus, the ordinary artisan would recognize that the first C2c2 system would be capable of cleaving a polyA sequence, and the second C2c2 system would be capable of cleaving a polyU sequence. To ensure that these systems cleave the correct targets, as said targets would be on a single RNA molecule, as described above in the rejection of claim 2, it would be prima facie obvious to ensure that these target sequences included sequences that could not be cleaved by an undesired C2c2 system, to prevent unwanted cleavage, reduce background noise, and ensure the system works as intended to maximize accuracy. Thus, including a polyU sequence on the second target sequence (which is analogous to the claimed activator sequence) would be prima facie obvious. As such a design is already contemplated in the two C2c2 system of Doudna, there would be a reasonable expectation of success.
Thus, claim 23 is prima facie obvious over Doudna in view of Gootenberg.
Regarding claim 24, Doudna teaches that a given polynucleotide may be 100% complementary to the target region within the target nucleic acid sequence to which it hybridizes (para. 72). Thus, it would be prima facie obvious for the first and second guide RNAs of Doudna in view of Gootenberg to be 100% complementary to their respective target sequences.
Thus, claim 24 is prima facie obvious over Doudna in view of Gootenberg.
Regarding claim 25, Doudna teaches that a given polynucleotide need not be 100% complementary to its target sequence, and can be anywhere from 60-100% complementary (para. 72). Thus, it would be prima facie obvious that the first and second guide RNAs of Doudna in view of Gootenberg need not be 100% complementary to their respective target sequences.
Thus, claim 25 is prima facie obvious over Doudna in view of Gootenberg.
Regarding claims 26-27, Doudna teaches that the sample from which the target is taken can be from mammalian cells (para. 114) and bacteria and fungi (para. 119), and also notes that targets may be RNA from a virus (para. 121). It would be prima facie obvious that if the sample is from a particular type of organisms (mammal, bacteria, etc.), that the target nucleic acid can be specific to that organism, particularly in view of the fact that a target RNA can be “any single-stranded RNA” (para. 121).
Thus, claims 26-27 are prima facie obvious over Doudna in view of Gootenberg.
Regarding claims 29-30 and 33, Doudna teaches that samples can be a variety of bodily fluids and tissues, including many of those listed in instant claim 30, such as blood, serum, plasma, and urine (para. 117). This paragraph also specifically notes that biological samples can be used, and that cells in culture may be used.
Thus, claims 29-30 and 33 are prima facie obvious over Doudna in view of Gootenberg.
Regarding claim 31, para. 118 of Doudna teaches that samples can be acellular fluid.
Thus, claim 31 is prima facie obvious over Doudna in view of Gootenberg.
Regarding claim 32, it is noted that the instant specification does not define what constitutes an “environmental sample.” Doudna teaches that the sample may be plant cell lysate (para. 114), and thus would not contain cells, and would be a liquid environmental sample.
Thus, claim 32 is prima facie obvious over Doudna in view of Gootenberg.
Claim 6 is rejected under 35 U.S.C. 103 as being unpatentable over Doudna et al. (WO 2017/218573 A1; cited in Applicant’s IDS), in view of Gootenberg et al. (Science, 2018), and further in view of Zhang et al. (Cell, 2018).
Doudna in view of Gootenberg teaches the system of claims 2-5, 7-8, 11-15, 18-19, 23-27, 29-31, and 33, as noted above. However, Doudna teaches the uses of C2c2 (where the Cas-effector protein is Cas13a), and Gootenberg teaches the use of Cas13a, Cas13b, and Cas12a (see Figure 1 for example). Thus, neither reference teaches the use of Cas13d.
Zhang teaches the use of a Cas13d system. The reference teaches that Cas13 can generally cleave in both cis and trans fashion (page 212, column 2, para. 1). The reference notes potential uses for this enzyme – particularly highlighting its “robust activity for both target cleavage and binding,” (page 213, column 1, para. 2). The reference also notes that Cas13d and Cas13a have “overall similarity of their RNase activities,” (page 221, column 1, para. 1), and states that the enzyme can be effectively used for multiplexing applications and, “can be expected to enable improved properties for diverse biomolecular applications of RNA targeting,” (page 221, column 2, para. 1).”
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the guidance of Zhang to substitute the Cas13a of Doudna in view of Gootenberg with the Cas13d taught by Zhang. MPEP 2143 I (B) states, “The rationale to support a conclusion that the claim would have been obvious is that the substitution of one known element for another yields predictable results to one of ordinary skill in the art.” Zhang teaches that Cas13d can perform both cis and trans cleavage, has similar RNase capabilities compared to those of Cas13a, and can be used in RNA targeting and multiplexing reactions. These properties would provide a reasonable expectation of success that Cas13d could perform the same functions as Cas13a as described by Doudna in view of Gootenberg and result in the same trans cleavage of reporters/detection of fluorescent signals.
Thus, claim 6 is prima facie obvious over Doudna, in view of Gootenberg, and further in view of Zhang.
Claim 16 is rejected under 35 U.S.C. 103 as being unpatentable over Doudna et al. (WO 2017/218573 A1; cited in Applicant’s IDS), in view of Gootenberg et al. (Science, 2018), and further in view of Hendel et al. (Nature Biotechnology, 2015).
Doudna in view of Gootenberg teaches the system of claims 2-5, 7-8, 11-15, 18-19, 23-27, 29-31, and 33, as noted above. However, neither reference teaches that the guide RNAs can include modified nucleotide bases.
Hendel teaches an analysis of chemically modified guide RNAs, specifically 2′-O-methyl (M), 2′-O-methyl 3′phosphorothioate (MS), or 2′-O-methyl 3′thioPACE (MSP) modifications at both the 5′ and 3′ ends of sgRNAs (Abstract and page 985, column 2, para. 2). Hendel concludes that, “chemically synthesized sgRNAs can be used effectively for targeted genome editing, and we demonstrate that chemically modified sgRNAs significantly enhance genome editing efficiencies in human primary T cells and CD34+ HSPCs. Chemically synthesized and modified sgRNAs offer advantages over expressed or in vitro transcribed sgRNAs, including (i) increased efficacy, (ii) robust and scalable production of highly pure sgRNAs for biotechnological and therapeutic applications, (iii) greater flexibility in the sgRNA design in contrast to constraints on the first transcribed nucleotides imposed by the U6 or T7 promoters typically used for plasmid expression or in vitro transcription of sgRNAs, respectively, and (iv) enabling of a highly active RNA-only or RNP CRISPR platform with lower cytotoxicity in primary cells than DNA plasmid-based systems,” and that, “this study suggests that chemically modified sgRNAs such as those presented here have the potential to substantially improve a wide array of CRISPR/Cas bio technological and therapeutic applications,’ (page 989, column 1, paras. 2-3).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to incorporate one or more of the gRNA modifications described by Hendel into the gRNAs of Doudna in view of Gootenberg. Hendel teaches many reasons why the ordinary artisan would be motivated to incorporate these modifications, and clearly shows the chemical structure of said modifications and their place within the gRNA sequence (Figure 1a-b). This, combined with the fact that these modifications clearly are compatible for use with CRISPR systems, would provide a reasonable expectation of success.
Thus, claim 16 is prima facie obvious over Doudna, in view of Gootenberg, and further in view of Hendel.
Claim 17 is rejected under 35 U.S.C. 103 as being unpatentable over Doudna et al. (WO 2017/218573 A1; cited in Applicant’s IDS), in view of Gootenberg et al. (Science, 2018), and further in view of Yin et al. (Nature Chemical Biology, 2018).
Doudna in view of Gootenberg teaches the system of claims 2-5, 7-8, 11-15, 18-19, 23-27, 29-31, and 33, as noted above. However, neither reference teaches that the guide RNAs can include both DNA and RNA bases.
Yin teaches an examination of DNA-RNA chimeras as gRNA sequences (Abstract). The inclusion of up to 12 DNA bases in a 20 base guide sequence can still produce gene editing results (Figure 1), and the reference generally concludes that such modifications lead to a decrease in off-target activity while retaining similar levels of on-target activity (page 311, column 2, para. 2 and page 314, column 2, para. 2). Yin also shows that this hybrid gRNA is compatible with multiple types of Cas enzymes (page 314, column 2, para. 3). The reference also notes that the creation of such hybrid gRNAs can be done at reduced cost (page 316, column 1, para. 1).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the hybrid DNA-RNA guide oligonucleotide described by Yin in the method of Doudna in view of Gootenberg. Though trans cleavage activity (and the associated fluorescent detection) is important in the method of Doudna in view of Gootenberg, the amount of signal required for detection does not need to be high - see para. 132 of Doudna, which notes a signal increase of as little as 5% when the fluorophore is cleaved from the quencher, and also states that when the quencher is in proximity to the fluorophore, there may be no signal above background detected at all. This means that a large amount of fluorescent signal would not be required to effectively detect the targets of Doudna in view of Gootenberg. Therefore, the hybrid guide molecules of Yin could be used in Doudna in view of Gootenberg without fear of false negatives for a given sample, as only a small amount of fluorescent probe cleavage would be required for detection. Then, the guide molecule of Yin provides the additional benefit of less off-target cleavage of wholly undesired sequences, such as other RNA sequences in a sample that may generate background noise/signal, thus overall improving accuracy. As the guide molecule of Yin does not effect on-target hybridization/activity, the C2c2 systems would still be able to hybridize to their respective targets, thus providing a reasonable expectation of success.
Thus, claim 17 is prima facie obvious over Doudna, in view of Gootenberg, and further in view of Yin.
Claims 18-19, and 21-22 are rejected under 35 U.S.C. 103 as being unpatentable over Doudna et al. (WO 2017/218573 A1; cited in Applicant’s IDS), in view of Gootenberg et al. (Science, 2018), and further in view of Jain et al. (Angew. Chem. Int. Ed., 2018; cited in Applicant’s IDS).
Doudna in view of Gootenberg teaches the system of claims 2-5, 7-8, 11-15, 18-19, 23-27, 29-31, and 33, as noted above. However, neither reference teaches modifying gRNAs to allow for conditional interactions with Cas-effector enzymes.
Jain teaches a photocaging method for gRNAs that allows for remote photoactivation (Abstract). The basic outline of the method is shown in Figure 1a – a blocking oligonucleotide attaches to the gRNA, and this prevents use with the Cas-effector enzyme when it remains attached. When the blocking oligonucleotide is cleaved, than the gRNA can work with the Cas-effector enzyme to perform target cleavage. This blocking can work in a multiplex fashion (Figure 3). Jain concludes that their method “offers an attractive, simple approach to many researchers that they can adapt for their own applications by further modifying the protectors or by conjugating the protector to the sgRNA,” and “provides a rapid and simple approach for the light-mediated control of genome editing,” (page 12443, column 1, para. 2 and column 2, para. 1). This method also provides advantages over other approaches as no modification of the Cas enzyme itself is required, and the blocking oligonucleotides are “simple to design, chemically synthesize, modify, functionalize, purify, and characterize,” (page 12440, column 2, para. 2).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Jain to add the photocleavable blocking oligonucleotide described by the reference to the method of Doudna in view of Gootenberg. Jain provides a means by which Cas cleavage is essentially blocked until the appropriate photo-treatment is provided. This would be particularly useful in the method of Doudna in view of Gootenberg described above for the second C2c2 system, as this system is not desired to activate until after the first C2c2 system has cleaved the first target sequence. Thus, to prevent premature hybridization of the second gRNA to the second target, the blocking oligonucleotide of Jain could be used with the second gRNA. Then, after providing time for the first C2c2 system to operate, the photo-treatment could be performed to release the second gRNA on the second C2c2 for hybridization, and the method of Doudna in view of Gootenberg would continue as described above. Jain also provides additional motivation for using their particular method (no enzyme modification required, ease of designing the blocking oligonucleotide, multiplexed detection, and a simple approach), and there would be a reasonable expectation of success as this modification would not change how the C2c2 systems operate, it would only affect the timing of said operation.
It is noted that this blocking oligonucleotide can be considered a trans caging molecule, and so adds to the caging structures described above as they relate to claims 18-19.
Thus, claims 18-19, and 21-22 are prima facie obvious over Doudna, in view of Gootenberg, and further in view of Jain.
Claim 20 is rejected under 35 U.S.C. 103 as being unpatentable over Doudna et al. (WO 2017/218573 A1; cited in Applicant’s IDS), in view of Gootenberg et al. (Science, 2018), and further in view of Baughman et al. (WO 2020/0167597 A1).
Regarding claim 20, Doudna in view of Gootenberg teaches the system of claims 2-5, 7-8, 11-15, 18-19, 23-27, 29-31, and 33, as noted above. Doudna also states that the oligonucleotides, polynucleotides, and nucleic acids of their invention can include modified nucleic acids (paras. 67-68). As the activator sequences of Doudna in view of Gootenberg are the target sequences that hybridize to the C2c2 sequences, these targets would be nucleic acids/polynucleotides – this is further evidenced by the use of the phrasing “target nucleic acid” throughout Doudna (see paras. 69, 72, 74, and 121, for example). Thus, these target nucleic acids can include modified nucleic acids. Para. 121 of Doudna also notes that target RNA can be any single stranded RNA.
Baughman teaches the detection of single-stranded target nucleic acids with Cas protein systems, where the Cas protein performs off-target trans cleavage (Abstract). In a specific discussion of Cas13a systems and trans cleavage, Baughman mentions that single-stranded RNA sequences used with these systems can include structured assemblies, such as stem-loops and bubbles, and so target sequences can also include these structures (page 8, para. 4).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art, given the guidance provided by Baughman, that RNAs consisting of or containing stem loops could be used in the method of Doudna in view of Gootenberg. As these sequences naturally exist in RNA, and can be used with Cas13a systems, as evidenced by Baughman, the ordinary artisan would recognize that such targets could be used in the Cas13a system of Doudna in view of Gootenberg, particularly in view of the fact that Doudna teaches that any single stranded RNA can be used in their method. This would then amount to a simple substitution of the target RNA described by Baughman as the target(s) in the method of Doudna in view of Gootenberg. MPEP 2143 I (B) states, “The rationale to support a conclusion that the claim would have been obvious is that the substitution of one known element for another yields predictable results to one of ordinary skill in the art.” As any single stranded RNA can be used in the method of Doudna in view of Gootenberg, and the stem loop RNAs are simply single-stranded RNAs with self-complementarity that can form a duplex structure, they would fall under the scope of the targets of Doudna in view of Gootenberg. Thus, they could be used in Doudna in view of Gootenberg to the same result as described above in the rejection of claim 2.
Thus, claim 20 is prima facie obvious over Doudna, in view of Gootenberg, and further in view of Baughman.
Claim 28 is rejected under 35 U.S.C. 103 as being unpatentable over Doudna et al. (WO 2017/218573 A1; cited in Applicant’s IDS), in view of Gootenberg et al. (Science, 2018), and further in view of Broughton et al. (Nature Biotechnology, 20202; cited in Applicant’s IDS).
Doudna in view of Gootenberg teaches the system of claims 2-5, 7-8, 11-15, 18-19, 23-27, 29-31, and 33, as noted above. Although Doudna teaches that target nucleic acids can be viruses (para. 121), the reference does not specifically discuss targeting coronaviruses.
Broughton teaches the detection of SARS-CoV-2 with a CRISPR-Cas12 system (Abstract). Figure 1 states that guide RNAs were specifically designed to target SARS-CoV-2 and other coronavirus strains (see figure caption). The reference also notes the dangers associated with SARS-CoV-2 and the need for accurate testing (page 870, columns 1-2, joining para.).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the guidance provided by Broughton to examine SARS-CoV-2 in the method of Doudna in view of Gootenberg. Broughton provides motivation to examine this virus by highlighting its rapid spread, its potential for asymptomatic transmission, and its ability to cause pneumonia in patients (page 870, columns 1-2, joining para.), and provides a reasonable expectation of success in being able to target this virus with CRISPR systems, as Broughton was able to develop guide RNAs to target it. As Doudna teaches that viruses may be targeted in their invention, SARS-CoV-2 would be encompassed in such targets.
Thus, claim 28 is prima facie obvious over Doudna, in view of Gootenberg, and further in view of Broughton.
Conclusion
No claims are currently allowable.
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/FRANCESCA FILIPPA GIAMMONA/Examiner, Art Unit 1681
1 This reference was provided in Applicant’s IDS submitted 3/27/2023 in manuscript form. The final version, along with the supplemental information, is provided with this action.
2 Though this reference states that it is in a July 2020 issue, it was made publicly available on April 16, 2020 according to the webpage associated with the article.
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