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 .
Claim Status
Claims 1, 3, 5, 9-11, 14-16, 19, 24, 26, 29-31, 34-35, 37, 41-43, 47, 49-51, 54, 56, 63-64, 67, and 70 are under examination. No other claims are currently pending in the present application.
Effective Filing Date
The present application, filed on December 9, 2022 is a 371 or PCT/US2021/036966, filed on June 11, 2021, which claims priority to U.S. Provisional application no: 63/038,507, filed on June 12, 2020. Therefore, the effective filing date of the present application is determined to be June 12, 2020.
Drawings
The drawings filed on December 9, 2022 are acceptable.
Information Disclosure Statement
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
It is noted that no information disclosure statement has been filed in this application.
Claim Objections
Claim 5 is objected to because of the following informalities: Claim 5 appears to have a duplicate line “…; the first Cas protein/gRNA complex comprises an inactive Cas protein and the second Cas protein/gRNA complex comprises an inactive Cas protein, or wherein the first Cas protein/gRNA complex comprises an inactive Cas protein and the second Cas protein/gRNA complex comprises an inactive Cas protein”. Appropriate correction is required.
Claim Rejections - 35 USC § 112
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.
Claims 11, 14, 15, 16, 19, 31, 34, 42, 43, 67, and 70 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
Claims 11, 15, 31, 42, and 67 recite that one or more of: the adapter oligonucleotide, the Cas protein, the gRNA, the target nucleic acid molecules, a transposon end sequence tag, or a target endonuclease is “attached to an affinity label”. It is unclear whether the limitation “attached” is meant to require that the molecule recited by the claim comprises an affinity label (for example, a biotinylated adapter oligonucleotide), or whether the claim simply requires that the molecule is in some way associated (i.e. attached) to the affinity label (for example, an adapter oligonucleotide ligated to a target sequence that is bound to a biotinylated dCas9 protein). In other words, it is unclear whether the claim requires that the “attachment” is: (A) a direct/covalent attachment as in the example wherein a biotinylated adapter is attached to an affinity label because the adapter is directly interacting/covalently linked to the affinity label or encompasses (B) “indirect” part of the same intermolecular complex as in the example where an adapter is “attached” to an affinity label because the adapter is ligated to a target sequence that is bound to a biotinylated Cas9-gRNA complex.
Claims 14, 16, 19, 34, 43, and 70 are rejected as indefinite because they depend on, and thus include the indefinite limitations of, claims 11, 15, 31, 42, and 67.
For the purposes of compact prosecution, claims reciting that a molecule is “attached” to an affinity label have been given their broadest reasonable interpretation that encompasses (B) indirect attachment.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 24, 26, 29-31, 34, 35, 47, 49, 51, 54, 56, and 63 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Peter et al., US 20170107560 A1, published April 20, 2017.
Regarding claim 24, Peter et al. teaches methods for enriching target nucleic acid molecules comprising binding target nucleic acid molecules with Cas9-gRNA(s) and isolating the complexes (i.e. separating the target molecules from nontarget molecules) (Peter et al., abstract and figure 1).
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Peter et al. further teaches that a single DNA sequence (i.e. target molecule) can be bound by several Cas9-gRNA complexes (i.e. at a first and second locus) to allow for capture of long target fragments (Peter et al., paragraph 0049).
Therefore, Peter et al. teaches all of the recited method steps of the present claim.
Regarding claim 26, Peter et al. teaches that a mixture of wild type and cleavage-deficient Cas9-gRNA complexes may be used such that the wild type Cas9 proteins cleave the target segment from adjacent regions, and cleavage deficient Cas9 proteins are used to bind to one or more regions within the target segment, allowing for efficient capture of the target segments (i.e. the first locus is bound by an active/inactive Cas9 and the second locus is bound by an inactive/active Cas9, respectively) (Peter et al., paragraph 0049 and figures 2-3).
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Regarding claim 29, Peter et al. teaches that the active Cas protein/gRNA complex cuts the target nucleic molecules (Peter et al., paragraph 0049).
Regarding claim 30, Peter et al. teaches ligating adaptors to the Cas9-gRNA cut end(s) of the target molecules (Peter et al., paragraph 0060).
Regarding claim 31, Peter et al. teaches that the adapter oligonucleotide, the Cas protein, the gRNA, or the target nucleic acid are attached to an affinity label (Peter et al., paragraphs 0054-0062)
Regarding claim 34, Peter et al. teaches the separation is performed by binding target molecules bound to the affinity label to a label partner and eluting the bound target nucleic acid molecules (Peter et al., paragraph 0056).
Regarding claim 35, Peter et al. teach the method may be used to isolate several different fragments from the sample, where each fragment is bound by a plurality of different Cas9-gRNA complexes (i.e. a set of first and a set of second Cas/gRNA complexes specific to first and second loci of a plurality of target regions) (Peter et al., paragraph 0059).
Regarding claim 47, Peter et al. teach that the method (described above for the rejection of claim 24 in this section) can be practiced using other enzymes including rare-cutting restriction enzymes (i.e. endonucleases), engineered nucleases such a ZFN (zinc finger nuclease), a TALEN (Transcription Activator Like effector nuclease), or a transposable element.
Regarding claim 49, Peter et al. teach separating Cas9-fragmented target sequences from nontarget sequences after binding the second Cas9-gRNA complex to the target sequences (Peter et al., figure 3 and paragraph 0056).
Regarding claim 51, Peter et al. teach the first, second, and third Cas9-gRNA complexes (i.e. endonucleases) bind to different loci of the target region (Peter et al., figure 1).
Regarding claim 54, Peter et al. teach releasing target nucleic acids from the Cas9-gRNA complexes (Peter et al., paragraph 0056).
Regarding claim 56, Peter et al. teach the target endonucleases comprise Cas9 (Peter et al., figure 1).
Regarding claim 63, Peter et al. teach ligating adaptor(s) to one or both ends after Cas9 fragmentation (Peter et al., paragraph 0060)
Claim Rejections - 35 USC § 103
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.
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 1, 3, 5, 9-11, and 14 are rejected under 35 U.S.C. 103 as being unpatentable over Lee et al., “CRISPR-Cap: multiplexed double-stranded DNA enrichment based on the CRISPR system”, Nucleic Acids Research, 2019, Vol. 47, No.1 e1, published September 12, 2018 in view of Nachmanson et al., “Targeted genome fragmentation with CRISPR/Cas9 enables fast and efficient enrichment of small genomic regions and ultra-accurate sequencing with low DNA input (CRISPR-DS)”, Genome Res. 2018 28: 1589-1599, published September 19, 2018 and Aalipour et al., “Deactivated CRISPR Associated Protein 9 for Minor-Allele Enrichment in Cell-Free DNA”, Clinical Chemistry 64:2 307-316, published February 1, 2018.
Regarding claim 1, Lee et al. teach a method of enriching for target nucleic acid molecules comprising binding target molecules to a first (active) Cas protein/gRNA complex and separating the fragmented target molecules from nontarget molecules (Lee et al., Figure 1)
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Similarly, Nachmanson et al. teach a method of enriching for target molecules comprising targeted Cas9 digestion of target region(s), separation of cut target fragments from nontarget molecules by size selection, and a second enrichment step comprising targeted hybridization capture to further enrich for target molecules (Nachmanson et al., Abstract and Figure 1-2)
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Neither Lee et al. nor Nachmanson et al. teach a second enrichment step using a second CRISPR-Cas9/gRNA complex. However, Aalipour et al. teach a method of enriching for rare alleles in clinical samples comprising binding fragmented sample DNA with an enzymatically inactive “dCas9” gRNA complex (Aalipour et al., figure 1).
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Aalipour et al. further teach methods for selectively enriching for target sequences using active Cas9 proteins (page 307, column 2 -page 308, column 1) require subsequent multiplex PCR or hybridization capture to further enrich rare alleles to sufficiently high abundance in a purified sample for reliable detection. Aalipour et al. teach that Cas9-based enrichment of target sequences exhibits robust single-base-pair discrimination compared to traditional hybrid capture (Aalipour et al. page 311, column 1).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the methods taught by Lee et al. and Nachmanson et al. with the teachings of Aalipour et al. to arrive at the presently claimed invention with a reasonable expectation of success. Specifically, the ordinary artisan would have been motivated to modify the methods of Lee et al. and Nachmanson et al. comprising active Cas9 selective cleavage of target loci followed by purification of target sequences by immunoprecipitation of the Cas9-gRNA-target complex (Lee et al.) or size selection and hybrid capture (Nachmanson et al.) with the methods taught by Aalipour et al. comprising using an enzymatically inactive “dCas9” to enrich for rare alleles in a clinical sample. The ordinary artisan would have been motivated to use a dCas9, taught by Aalipour et al. in place of the hybridization capture step, or in addition to the first immunoprecipitation step following the targeted fragmentation steps taught by Lee et al. and Nachmanson et al. because of the teaching of Aalipour et al. that Cas9-based enrichment of target sequences exhibits robust single-base-pair discrimination compared to traditional hybrid capture (Aalipour et al. page 311, column 1).
Regarding claim 3, Aalipour et al. teaches separating fragmented target nucleic acid molecules from nontarget molecules (Aalipour et al., figure 1)
Regarding claim 5, Lee et al. in view of Nachmanson et al. and Aalipour et al. teach sequentially binding an active Cas9-gRNA complex to target molecules, separating the targets from the sample, binding a second (inactive) Cas9-gRNA complex to the target molecules, and separating the targets from the sample.
This combination of references does not explicitly teach all other possible permutations of active/inactive first complexes followed by active/inactive second complexes. However, these embodiments of the claimed invention are simple rearrangements of steps of the claimed method. Absent unexpected results, it would have been prima facie obvious to perform the binding steps with any combination of active/inactive first and second complexes.
Regarding claim 9, Lee et al. and Nachmanson et al. each teach active Cas protein(s) cut the target nucleic acid molecules(s) (Lee et al., figure 1 ; Nachmanson et al., figure 1).
Regarding claim 10, Nachmanson et al. teach ligating an adapter to the cut ends of the target molecules (Nachmanson et al., figure 2).
Regarding claim 11, Lee et al. teach the gRNA is biotinylated (i.e. attached to an affinity label) (Lee et al., figure 1). Similarly, Aalipour et al. teach the dCas9 is conjugated to a 6x-His tag (i.e. an affinity label) (Aalipour et al., figure 1).
Regarding claim 14, Lee et al. and Aalipour et al each teach separating the target molecules from the sample comprises binding the affinity label to an affinity label partner (Streptavidin-coupled magnetic beads, Lee et al., figure 1 ; anti-6xHis antibodies-coupled to magnetic beads, Aalipour et al., figure 1).
Claims 15-16 are rejected under 35 U.S.C. 103 as being unpatentable over Lee et al., Nachmanson et al., and Aalipour et al. as applied to claims 1, 3, 5, 9-11, and 14 above, and further in view of Slesarev et al., “CRISPR/Cas9 targeted capture of mammalian genomic regions for characterization by NGS” Scientific Reports (2019) 9:3587, published March 5, 2019.
Regarding claims 15 and 16, as described in the 103 rejections above, Lee et al. in view of Nachmanson et al. and Aalipour et al. teach methods comprising binding target nucleic acid molecules with a (set of) Cas protein/gRNA complexes, separating the bound targets from the sample, and binding the targets with a (set of) second Cas protein/gRNA complexes, wherein the first and second complexes comprise any combination of active and/or inactive Cas protein. Lee et al. in view of Nachmanson et al. and Aalipour et al. further teach ligating adapter oligonucleotides to the cut ends of the target molecules.
Lee et al. in view of Nachmanson et al. and Aalipour et al. do not explicitly teach that the adapter oligonucleotides are attached to an affinity label.
However, Slesarev et al. teach methods wherein an RNA-Guided Endonuclease (RGEN) cuts fragmented genomic DNA at gRNA-directed target sites (Slesarev et al., Figure 1, step 2). Slesarev et al. further teaches ligating a biotinylated (i.e. affinity-labeled) adapter to the cut ends, capturing the biotinylated adapters with streptavidin-coupled beads, and eluting the captured target sequences from the beads by cleaving the adapter with an endonuclease or denaturing the streptavidin by heating in the presence of detergent and proteinase (Slesarev et al., Figure 1, step 3/4).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the method taught by Lee et al. in view of Nachmanson et al. and Aalipour et al. comprising: gRNA-targeted cleavage of a target site, ligation of a sequencing adapter, separation of the adapter-ligated targets, elution from the capture substrate, and capture of target sequences with a second active or inactive Cas-gRNA complex with the teachings of Slesarev et al. that Cas-cleavage sites can be ligated to biotinylated adapters, captured on streptavidin-coupled beads, separated from a sample mixture, and eluted from the beads prior to further purification (library construction, size selection, etc.). The ordinary artisan would have been motivated to use biotinylated adapters, taught by Slesarev et al. as the adapters in the method taught by Lee et al. in view of Nachmanson et al. and Aalipour et al. because of the teaching of Slesarev et al. that an affinity label (biotin) can be incorporated into: the cut target molecule 3’ ends (RGEN-TdT), an adapter (RGEN-R), a gRNA (RGEN-D) with reasonable expectation of success in enriching the target sequences by capture on streptavidin beads (Slesarev, Table 1 and page 2).
Regarding claim 16, Slesarev et al. and Lee et al. teach eluting affinity-captured target nucleic acid molecules prior to further processing (i.e. in the method taught by Lee et al. in view of Nachmanson et al. and Aalipour et al., prior to the second enrichment step (the binding of the second Cas-gRNA complex)).
Claim 19 is rejected under 35 U.S.C. 103 as being unpatentable over Lee et al., Nachmanson et al., Aalipour et al., and Slesarev et al., as applied to claims 15-16 above, and further in view of Peter et al., US 2017/0107560 A1, published April 20, 2017.
Regarding claim 19, Lee et al. in view of Nachmanson et al., Aalipour et al., and Slesarev et al. teach methods of enriching target nucleic acid molecules binding target nucleic acid molecules with a (set of) Cas protein/gRNA complexes, separating the bound targets from the sample, and binding the targets with a (set of) second Cas protein/gRNA complexes, wherein the first and second complexes comprise any combination of active and/or inactive Cas protein. Lee et al. in view of Nachmanson et al., Aalipour et al., and Slesarev et al. further teach ligating adapter oligonucleotides to the cut ends of the target molecules, wherein any of the gRNA, the target molecule, the Cas protein, or the adapter can comprise an affinity label. Lee et al. in view of Nachmanson et al., Aalipour et al., and Slesarev et al. specifically teach that the complex comprising the gRNA, an affinity-tagged Cas protein, target nucleic acids, and adapters can be enriched by capturing the labeled Cas protein with an anti-6x His antibody linked to a bead (an antibody directed against the affinity label) (Aalipour, Figure 1). Therefore, Lee et al. in view of Nachmanson et al., Aalipour et al., and Slesarev et al. teach that the adapter oligonucleotide is attached to an affinity label (the labeled Cas protein) through the intermolecular complex (see the interpretation (b) in the 112(b) rejection in section 7).
Lee et al. in view of Nachmanson et al., Aalipour et al., and Slesarev et al. do not teach that the antibody is an anti-Cas antibody linked to a bead.
However, Peter et al. teach methods of enriching for target nucleic acids wherein a Cas-gRNA (comprising either a functional or enzymatically inactive Cas “dCas”) complex bound to a target nucleic acid molecule are captured using an antibody against the Cas protein that may expressly be linked to an affinity support, a bead (Peter et al., paragraph 0049 and 0054).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the method taught by Lee et al., in view of Nachmanson et al., Aalipour et al., and Slesarev et al. comprising capturing intermolecular complexes of adapter-ligated target nucleic acid and 6xHis-tagged Cas-gRNA by binding the 6xHis-Cas with anti-6xHis antibodies coupled to beads (i.e. wherein the adapter is indirectly attached to an affinity label (the anti-6xHis antibody)) with the teachings of Peter et al. that such intermolecular complexes are captured with an antibody against the Cas protein itself rather than an antibody against an additional affinity tag (6x-His) attached to the Cas protein. Peter et al. additionally teach that the Cas protein may alternately be fused with affinity tags such as poly-histidine (i.e. 6x-His). The ordinary artisan would have been motivated to substitute the affinity/capture pair (6x-His:Cas and anti-6x-His:bead) taught by the method of Lee et al., in view of Nachmanson et al., Aalipour et al., and Slesarev et al. with the affinity/capture pair (Cas and anti-Cas:bead) taught by Peter et al. with a reasonable expectation of success because Peter et al. teaches that these methods of capture (directly capturing Cas protein with an antibody against Cas or capturing His-tagged Cas with an antibody against the His tag) are interchangeable, convenient methods for isolating complexes comprising Cas protein (Peter et al., paragraph 0054).
Claim 37 is rejected under 35 U.S.C. 103 as being unpatentable over Lee et al., Nachmanson et al., and Aalipour et al. as applied to claims 1, 3, 5, 9-11, and 14 above, and further in view of Gourguechon et al., WO 2017/031360 A1, published February 23, 2017.
Regarding claim 37, as described in the 103 rejections above, Lee et al. in view of Nachmanson et al. and Aalipour et al. teach methods comprising binding target nucleic acid molecules with a (set of) Cas protein/gRNA complexes, separating the bound targets from the sample, and binding the targets with a (set of) second Cas protein/gRNA complexes, wherein the first and second complexes comprise any combination of active and/or inactive Cas protein. Lee et al. in view of Nachmanson et al. and Aalipour et al. further teach ligating adapter oligonucleotides to the cut ends of the target molecules.
Lee et al. in view of Nachmanson et al. and Aalipour et al. do not teach that the adapter is attached to the target molecules through a transposase tethered to the Cas protein.
However, Gourguechon et al. teach methods for the enrichment of target nucleic acids using a nucleic acid-guided nuclease system (Gourguechon et al., abstract), wherein a Cas protein fused to (i.e. tethered to) a transposase inserts an adapter (i.e. a transposon end sequence) into a target nucleic acid molecule (Gourguechon et al., paragraph 0007). Gourguechon et al. teach that methods comprising capture of and simultaneous adapter attachment to target sequences are less time-consuming, expensive, and require fewer hands-on steps relative to hybridization-based sequence capture methods and allow for the removal of “off-target” sequences that can remain after hybridization-based capture (Gourguechon et al., paragraph 0002).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the method taught by Lee et al. in view of Nachmanson et al. and Aalipour et al. comprising distinct binding and adapter ligation steps with the teachings of Gourguechon et al. that transposase-tethered to a Cas protein allows for Cas binding and adapter attachment to a target nucleic acid to occur at the same time. The ordinary artisan would have been motivated to make this modification with a reasonable expectation of success because it advantageously combines the capture and adapter attachment step into a single process step and Gourguechon et al. teach using the Cas-transposase fusion in a substantially similar method of enriching for specific target sequences from a complex population of input nucleic acids.
Claims 41-43 are rejected under 35 U.S.C. 103 as being unpatentable over Lee et al., Nachmanson et al., Aalipour et al., and Gourguechon et al. as applied to claim 37 above, and further in view of Agilent, US 2017/0044592 A1, Published February 16, 2017 .
Regarding claim 41, Lee et al., Nachmanson et al., Aalipour et al., and Gourguechon et al. do not explicitly teach that the transposase protein in the dCas9-transposase fusion protein (Gourguechon et al., paragraph 0212) is Tn5.
However, Agilent teaches methods comprising fragmenting a genome comprising using Cas9-transposase fusion proteins that bind to specific target sequences and insert adapter sequences near the target site (Agilent, paragraphs 0096-0102 and 0113). Agilent further teaches that the transposase fused to the Cas9 can be Tn5 (Agilent, paragraph 0121).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have selected Tn5 as the Cas9-transposase fusion protein taught by Lee et al., Nachmanson et al., Aalipour et al., and Gourguechon et al. because Agilent teaches that such fusion proteins can be loaded with adaptors primed to generate NGS libraries to allow for one-step integration of NGS adaptors at specific genomic sites and advantageously produce an NGS library rapidly and without hybrid-selection approaches (Agilent, paragraph 0113). The ordinary artisan would have been motivated to choose Tn5 with a reasonable expectation of success because of the teaching of Agilent that Cas9-Tn5 fusion proteins allow for rapid production of targeted NGS libraries without relying on hybrid capture methods (Agilent, paragraph 0113).
Regarding claim 42, Agilent teaches that the adaptor molecules loaded into the Cas9-Tn5 fusion proteins can be biotinylated (i.e. may be attached to an affinity label) (Agilent, paragraph 0113).
Regarding claim 43, Agilent teaches that fragments containing the biotinylated adaptor molecules (i.e. target nucleic acids to which the Cas9 bound and the Tn5 integrated the biotinylated adaptor (i.e. an affinity tag)) can be separated from the DNA that was not “tagmented” (i.e. pulled down with an affinity label partner) (Agilent, paragraph 0113).
Claims 47 and 50 are rejected under 35 U.S.C. 103 as being unpatentable over Agilent in view of Peter et al.
Claim 47 is rejected under 102(a)(1) above, but is included here for clarity.
Regarding claim 50, as discussed in the 102(a)(1) rejection of claims 47 and 63 above, Peter et al. teaches teach methods for enriching target nucleic acid molecules comprising binding target nucleic acid molecules with Cas9-gRNA(s) and isolating the complexes (i.e. separating the target molecules from nontarget molecules) (Peter et al., abstract and figure 1). Peter et al. further teaches that a single DNA sequence (i.e. target molecule) can be bound by several Cas9-gRNA complexes (i.e. at a first and second locus) to allow for capture of long target fragments (Peter et al., paragraph 0049). Peter et al. teach ligating adaptor(s) to one or both ends after Cas9 fragmentation (Peter et al., paragraph 0060).
Briefly, Agilent teaches binding two Cas9-gRNA complexes to sequences flanking a target nucleic acid sequence and separating the cleaved products from nontarget nucleic acids by size selection (Agilent, figure 1, see below) or pull-down of biotinylated Cas9 or adapters (Agilent, paragraph 0113).
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Agilent does not teach binding a third Cas9-gRNA complex (i.e. a third nuclease) to the target nucleic acids to effect a subsequent enrichment for the target nucleic acids.
However, as described in the 102(a)(1) rejection of claim 47 above, Peter et al. teach a method wherein repetitive sequences are targeted and cut by a first active Cas9-gRNA complex (Peter et al., figure 2). Peter et al. further teach that the target sequences, released from larger DNA fragments by the cleaving of the first Cas9 enzyme, can then be bound at a different locus by a Cas9-gRNA complex that may be catalytically inactive (Peter, figure 3).
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Peter et al. teaches that the capture step (after cleaving/tagging) allows for further purification of the target sequences onto a support such as a bead and washing to remove unbound nucleic acids from the desired target nucleic acids (Peter et al., paragraph 0056).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have combined the teachings of Agilent and Peter et al. by performing the targeted Cas cleavage of two distinct loci flanking a target nucleic acid sequence, separating the nucleic acids from the Cas proteins, taught by Agilent, and subsequently using the dCas9 capture step taught by Peter et al. to specifically enrich for nucleic acid fragments containing a specific sequence complementary to the dCas9-gRNA.
The ordinary artisan would have been motivated to combine the methods of Agilent and Peter et al. with a reasonable expectation of success because of the teaching of Peter et al. that the subsequent capture step in a very similar method comprising Cas9-directed fragmentation at sites flanking target nucleic acid sequences allows for further purification of the target sequences onto a support such as a bead and washing to remove unbound nucleic acids from the desired target nucleic acids.
Claims 47 and 64 are rejected under 35 U.S.C. 103 as being unpatentable over Peter et al. in view of Gourguechon et al.
Claim 47 is rejected under 102(a)(1) above, but is included here for clarity.
Regarding claim 64, as discussed in the 102(a)(1) rejection of claims 47 and 63 above, Peter et al. teaches teach methods for enriching target nucleic acid molecules comprising binding target nucleic acid molecules with Cas9-gRNA(s) and isolating the complexes (i.e. separating the target molecules from nontarget molecules) (Peter et al., abstract and figure 1). Peter et al. further teaches that a single DNA sequence (i.e. target molecule) can be bound by several Cas9-gRNA complexes (i.e. at a first and second locus) to allow for capture of long target fragments (Peter et al., paragraph 0049). Peter et al. teach ligating adaptor(s) to one or both ends after Cas9 fragmentation (Peter et al., paragraph 0060).
Peter et al. do not teach that an adapter is attached to the target molecules through a transposase tethered to the Cas protein.
However, Gourguechon et al. teach methods for the enrichment of target nucleic acids using a nucleic acid-guided nuclease system (Gourguechon et al., abstract), wherein a Cas protein fused to (i.e. tethered to) a transposase inserts an adapter (i.e. a transposon end sequence) into a target nucleic acid molecule (Gourguechon et al., paragraph 0007). Gourguechon et al. teach that methods comprising capture of and simultaneous adapter attachment to target sequences are less time-consuming, expensive, and require fewer hands-on steps relative to hybridization-based sequence capture methods and allow for the removal of “off-target” sequences that can remain after hybridization-based capture (Gourguechon et al., paragraph 0002).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the method taught by Peter et al. comprising distinct binding and adapter ligation steps with the teachings of Gourguechon et al. that transposase-tethered to a Cas protein allows for Cas binding and adapter attachment to a target nucleic acid to occur at the same time. The ordinary artisan would have been motivated to make this modification with a reasonable expectation of success because it advantageously combines the capture and adapter attachment step into a single process step and Gourguechon et al. teach using the Cas-transposase fusion in a substantially similar method of enriching for specific target sequences from a complex population of input nucleic acids.
Claims 47, 63, 67 and 70 are rejected under 35 U.S.C. 103 as being unpatentable over Peter et al. in view of Slesarev et al.
Claims 47 and 63 are rejected under 102(a)(1) above, but are included here for clarity.
Regarding claim 67, as discussed in the 102(a)(1) rejection of claims 47 and 63 above, Peter et al. teach methods for enriching target nucleic acid molecules comprising binding target nucleic acid molecules with Cas9-gRNA(s) and isolating the complexes (i.e. separating the target molecules from nontarget molecules) (Peter et al., abstract and figure 1).
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Peter et al. further teaches that a single DNA sequence (i.e. target molecule) can be bound by several Cas9-gRNA complexes (i.e. at a first and second locus) to allow for capture of long target fragments (Peter et al., paragraph 0049). Peter et al. teach ligating adaptor(s) to one or both ends after Cas9 fragmentation (Peter et al., paragraph 0060).
Peter et al. do not teach that the adapter oligonucleotides are attached to an affinity label.
However, Slesarev et al. teach methods wherein an RNA-Guided Endonuclease (RGEN) cuts fragmented genomic DNA at gRNA-directed target sites (Slesarev et al., Figure 1, step 2). Slesarev et al. further teaches ligating a biotinylated (i.e. affinity-labeled) adapter to the cut ends, capturing the biotinylated adapters with streptavidin-coupled beads, and eluting the captured target sequences from the beads by cleaving the adapter with an endonuclease or denaturing the streptavidin by heating in the presence of detergent and proteinase (Slesarev et al., Figure 1, step 3/4).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the method taught by Peter et al. comprising: gRNA-targeted cleavage of a target site, ligation of a sequencing adapter to the cut ends, separation of the adapter-ligated targets, elution from the capture substrate, and capture of target sequences with a second active or inactive Cas-gRNA complex with the teachings of Slesarev et al. that Cas-cleavage sites can be ligated to biotinylated adapters, captured on streptavidin-coupled beads, separated from a sample mixture, and eluted from the beads prior to further purification (library construction, size selection, etc.). The ordinary artisan would have been motivated to use biotinylated adapters, taught by Slesarev et al. as the adapters in the method taught by Peter et al. because of the teaching of Slesarev et al. that an affinity label (biotin) can be incorporated into: the cut target molecule 3’ ends (RGEN-TdT), an adapter (RGEN-R), a gRNA (RGEN-D) with reasonable expectation of success in enriching the target sequences by capture on streptavidin beads (Slesarev et al., Table 1 and page 2).
Regarding claim 70, Slesarev et al. teach capturing biotinylated adapters ligated to the target nucleic acid molecules on streptavidin coupled beads (i.e. an affinity label partner).
Conclusion
No claim is allowed.
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/Z.M.T./Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682