DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. 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
2. Applicant’s election without traverse of Group I, claims 1-22 and 27-30, in the reply filed on 17 April 2026 is acknowledged.
Claim Rejections - 35 USC § 112
3. 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.
4. Claim 4 is 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 4 recites the phrase “wherein gRNA is targeted to the target sequence and comprises a protospacer adjacent motif compatible with the CRISPR-Cas12a-based endonuclease.” Applicant’s specification ([0030] and [0039]) indicates that the protospacer adjacent motif (PAM) is present on the target DNA, and not the gRNA as indicated by the claim. It is unclear if this claim is attempting to require the gRNA itself to comprise the PAM sequence for Cas12a (i.e., TTTN) or how this limitation is intended to limit the metes and bounds of this claim, and therefore this claim is indefinite. For the purpose of prosecution, this claim is being interpreted in line with the applicant’s specification and the prior art in that the PAM region is comprised within the target DNA and compatible with the CRISPR-Cas12a-based endonuclease (e.g., the claim is being read as “wherein gRNA is targeted to the target sequence and the target sequence comprises a protospacer adjacent motif compatible with the CRISPR-Cas12a-based endonuclease”).
5. The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
6. Claim 14 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 14 recites the phrase “Wherein fragmenting further the double-stranded nucleic acid…. is accomplished in a single step or in two or more steps.” This phrase seeks to add additional limitations to the steps provided in claim 1 lines 8 and 9, however, performing the method steps of lines 8 and 9 in claim one in “a single step or in two or more steps” encompasses all of the possible number of steps in which these method steps could be performed. Therefore claim 14 does not further limit the method of claim 1 and fails to satisfy the requirements on U.S.C. § 112(d).
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 103
7. 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.
8. 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.
9. Claims 1-8, 13, 14, 16-21, 29 and 30 are rejected under 35 U.S.C. 103 as being unpatentable over Kennedy et al (International Patent Application No. WO 2018175997, published 27 September 2018) in view of Crawford et al (United Sates Patent Application No. US 20190300935, effectively filed 07 January 2019) and Stella et al (Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity, Cell, 175, 1856-1871, published 13 December 2018).
Regarding claim 1, Kennedy teaches a method for the target sequencing of double-stranded nucleic acids (abstract) comprising cleaving double-stranded nucleic acids with a plurality of endonuclease-guide ribonucleic acid (gRNA) complexes (cutting with a targeted endonuclease (e.g., CRISPR-associated (Cas) enzyme/guideRNA complex, for example Cas9 or Cpf1 [0015]; A total of 12 gRNAs were designed, which excised TP53 into 7 different fragments [00290]), ligating a first adapter to the targeted sites of the double-stranded nucleic acid fragments (ligation of DS adapters [00165]) and that adapters comprises regions complementary to PCR primers (i.e., priming sites; [00122]), further fragmenting the double-stranded nucleic acids fragments at random sites (multiple targeted endonucleases are used… in combination with random or semi-random methods of nucleic acid fragmentation [00174]), and adding a second adapter at the random sites (adapter sequences at both ends [0013] and [00130]). Kennedy teaches that the library is selectively amplified using primers complementary to the adapters at each end ([0035]).
Kennedy does not specifically teach that the double-stranded nucleic acids are dephosphorylated, and while they teach Cpf1 as a targeted endonuclease useful for their method they do not specifically teach that Cpf1 generates nucleic acid fragments having phosphorylated 5' end overhangs at target sites.
However, Crawford teaches a similar method of CRISPR mediated targeted cleavage (abstract and FIG 1), and specifically teaches blocking (i.e., dephosphorylating) the DNA source with a phosphatase ([0004] and [0131]).
Stella teaches that Cpf1 (i.e., CRISPR-Cas12a) cuts yield a 5'-phosphorylated product (pg. 1863 column 2 ¶ 3) and a DNA overhang (pg. 1856 column 2 ¶ 2).
It would have been obvious to one having ordinary skill in the art to have modified the method taught by Kennedy to have included the dephosphorylated step taught by Crawford and to have further used Cpf1 (a specific endonuclease taught by Kennedy) to generate 5'-phosphorylated overhangs as taught by Stella to arrive at the instantly claimed invention with a reasonable expectation of success. The ordinary artisan would have been motivated to make these modifications because Crawford teaches that blocking the source DNA blocks the accessible DNA ends prior to targeted cleavage ([0004]) and Kennedy specifically teaches Cpf1 as an endonuclease alternate for their method (having the cleavage pattern generating a 5'-phosphorylated end and an overhang as taught by Stella. In addition, the ordinary artisan would have recognized that the known techniques in the cited references could have been combined with predictable results because the known techniques in the cited references predictably result in the targeted cleavage of DNA molecules for sequencing.
Regarding claims 2 and 3, Kennedy teaches that the plurality of endonuclease-gRNA complexes are ribonucleoproteins ([0031]) such as Cpf1 (i.e., Cas12a; [0031] and [0081]).
Regarding claim 4, Kennedy in view of Stella teaches that the gRNA binds to a target-specific motif (i.e., a target sequence; Kennedy [0081]) and the target sequence comprises a protospacer adjacent motif compatible with CRISPR-Cas12a (Stella; 1856 column 2 ¶ 1).
Regarding claim 5, Kennedy in view of Crawford and Stella does not specifically teach the limitations of claim 5.
However, Crawford teaches designing a template oligonucleotide for the transcription of the guide RNAs ([0128]). It is noted here that the DNAs were purchased from IDT, but custom oligonucleotides purchased from IDT are synthesized by IDT, therefore this is considered to meet the limitation “synthesizing double-stranded nucleic acids.” Crawford teaches transcribing the DNA into the gRNAs ([0128]) and complexing the gRNAs with CRISPR-Cas9 ([0131]).
One having ordinary skill in the art would have recognized that the method taught by Kennedy in view of Crawford and, including the synthesis of double-stranded nucleic acids encoding gRNA, transcribing the double-stranded nucleic acids in vitro into the gRNAs, and complexing the gRNAs with CRISPR-Cas9 could have been modified to produce gRNAs compatible with CRISPR-Cas12a using the gRNA characteristics taught by Stella (pg. 1861 column 2 ¶ 1, FIG S5, and the RNA oligonucleotides in the Key Resource Table) to arrive at the instantly claimed invention with a reasonable expectation of success. The ordinary artisan would have been motivated to make this modification because Kennedy specifically teaches that Cpf1 is a suitable targeted endonuclease for this method ([0081]). In addition, one having ordinary skill in the art would have recognized that the known techniques in the cited references could have been combined with predictable results because the known techniques in the cited references predictably result in the formation and use of CRISPR-gRNA complexes.
Regarding claim 6, Stella teaches that gRNAs were purchased from IDT (Key Resources Table – oligonucleotides).
Regarding claim 7, Kennedy teaches that the double-stranded nucleic acid is genomic DNA ([00248]).
Regarding claim 8, Crawford teaches dephosphorylating double-stranded nucleic acids to provide dephosphorylated double-stranded nucleic acids ([0004] and [0131]).
Regarding claim 13, Kennedy teaches enriching the CRISPR cut targets by size selection after fragmentation ([0026], [00260] and [00283]).
Regarding claims 14 and 16, Kennedy teaches random fragmentation by mechanical or acoustic shearing ([00174]), end-repair, and ligation steps (i.e., one or more steps; [00104]).
Regarding claims 17 and 29, Kennedy in view of Crawford and Stella does not specifically teach a pulldown reaction targeted to the first adapter to generate pulldown products.
However, Kennedy teaches capturing the adapter ligated DNA by hybridization to a biotinylated probe ([00285]) and streptavidin bead pulldown ([00151]). Kennedy additionally teaches a biotinylated adapter ([0033]).
It would have been obvious to one having ordinary skill in the art to have modified the method taught by Kennedy to have performed the biotin pulldown step using the biotinylated adapter taught by Kennedy rather that a biotinylated hybridization probe to arrive at the instantly claimed invention with a reasonable expectation of success. The ordinary artisan would have been motivated to make this modification because Kennedy teaches the steps of biotin pulldown as a purification method, and using the biotinylated adapter as the pulldown target would reduce reagent usage, time, and the risks of off target pulldown due to improper hybridization by eliminating the hybridization pulldown entirely.
Regarding claim 18, Kennedy teaches size selection of the library after amplification ([00170]), library quantification and sequencing on the Illumina MiSeq platform (i.e., massively parallel sequencing; [00297]).
Regarding claim 19, Kennedy teaches that targeted endonucleases have the ability to specifically and selectively excise precise sequence regions of interest ([00160]).
Regarding claim 20, Kennedy teaches methods to improve the PCR amplification of region comprising high GC content ([00144]).
Regarding claim 21, Kennedy teaches that size selection improves the performance of degraded DNA in CRISPR-DS, however it is noted that the MPEP states that a reference can be relied upon for all that it discloses, even non-preferred embodiments (i.e., omitting the size selection step prior to adapter ligation; [00304] and MPEP 2123(I)).
Regarding claim 30, Kennedy teaches performing post-capture PCR on adapter-ligated DNA pulled down by streptavidin beads ([00296]).
10. Claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over Kennedy et al (International Patent Application No. WO 2018175997, published 27 September 2018) in view of Crawford et al (United Sates Patent Application No. US 20190300935, effectively filed 07 January 2019) and Stella et al (Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity, Cell, 175, 1856-1871, published 13 December 2018) as applied to claim 8 above, and further in view of Ruan (United States Patent Application No. US 20180135120, published 17 May 2018).
Regarding claim 9, the method of claim 8 is discussed fully above and incorporated here.
Neither Kennedy, Crawford nor Stella teach that existing 5' overhangs are removed from the double-stranded nucleic acids prior to dephosphorylation.
However, Ruan teaches the removal of 5' overhangs by filling in or removing the overhangs ([0105]).
It would have been obvious to one having ordinary skill in the art to have modified the method taught by Kennedy in view of Crawford and Stella with the 5' overhang removal taught by Ruan to arrive at the instantly claimed invention with a reasonable expectation of success. The ordinary artisan would have been motivated to make this modification because Crawford teaches that blocking the ends of the source DNA to make it unavailable for ligation ([0004] and [0060]), and the blunting method taught by Ruan would further reduce unwanted ligation of adapters comprising overhangs as needed after CRISPR-Cas12a digestion. In addition, the ordinary artisan would have recognized that the known techniques in the cited references could have been combined with predictable results because the known techniques in the cited references predictably result in the preparation of oligonucleotides for adapter ligation and sequencing.
11. Claims 10-12 are rejected under 35 U.S.C. 103 as being unpatentable over Kennedy et al (International Patent Application No. WO 2018175997, published 27 September 2018) in view of Crawford et al (United Sates Patent Application No. US 20190300935, effectively filed 07 January 2019) and Stella et al (Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity, Cell, 175, 1856-1871, published 13 December 2018) as applied to claim 1 above, and further in view of Lok (United States Patent Application US 20090156431, published 18 June 2009).
Regarding claim 10, Kennedy teaches that adapters are suited to ligate a multiple nucleotide overhang ([00123]) but neither Kennedy, Crawford nor Stella teach that the adapters comprise degenerate overhang bases compatible with the phosphorylated 5' end overhangs of the double-stranded nucleic acid fragments.
However, Lok teaches adapters comprising degenerate 5' overhangs ([0057]).
It would have been obvious to one having ordinary skill in the art to have modified the generic adapters taught by Kennedy in view of Crawford and Stella with the degenerate overhangs taught by Lok to arrive at the instantly claimed invention with a reasonable expectation of success. The ordinary artisan would have been motivated to make this modification because Kennedy specifically teaches that adapters are suited to ligate to multiple nucleotide overhangs ([00123]) that result from cleavage with the plurality of Cpf1 endonucleases ([0015]). In addition, one having ordinary skill in the art would have recognized that the known techniques in the cited references could have been combined with predictable results because the known techniques in the cited references predictably result in adapters for ligation and preparation of sequencing libraries.
Regarding claim 11, Kennedy teaches that the adapters comprise a single molecule identifier (i.e., a unique molecular identifier; [00108]).
Regarding claim 12, Kennedy teaches that the first adapter comprises a 5' biotin modification (a biotinylated oligonucleotide; [0033]).
12. Claim 15 is rejected under 35 U.S.C. 103 as being unpatentable over Kennedy et al (International Patent Application No. WO 2018175997, published 27 September 2018) in view of Crawford et al (United Sates Patent Application No. US 20190300935, effectively filed 07 January 2019) and Stella et al (Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity, Cell, 175, 1856-1871, published 13 December 2018) as applied to claim 1 above, and further in view of Chen et al (International Patent Application No. WO 2021034974, with valid priority to 19 August 2019).
Regarding claim 15, Kennedy in view of Crawford and Stella teach the method of claim 1 as discussed fully above and incorporated here.
Neither Kennedy, Crawford nor Stella teach that the further fragmenting at random sites and adding the second adapter comprises using a transposase.
However, Chen teaches the addition of an adapter to a barcode tagged fragment (e.g., the fragments comprising the first adapter taught by Kennedy) using a transposome ([0035] and FIG 12).
It would have been obvious to one having ordinary skill in the art to have modified the method taught by Kennedy in view of Crawford and Stella with the transposase-based adapter tagging taught by Chen to arrive at the instantly claimed invention with a reasonable expectation of success. The ordinary artisan would have been motivated to make this modification because Chen teaches transposase-based tagging as an alternative to physical shearing methods such as those taught by Kennedy and discussed above (Chen [00085]). In addition, one having ordinary skill in the art would have recognized that the known techniques in the cited references could have been combined with predictable results because the known techniques in the cited references predictably result in the addition of adapters to nucleic acids.
Conclusion
13. Claims 22, 27 and 28 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
14. No claims are allowed.
15. Any inquiry concerning this communication or earlier communications from the examiner should be directed to BRIAN ELLIS YOUNG whose telephone number is (703)756-5397. The examiner can normally be reached M-T 0800 - 1630.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Heather Calamita can be reached at (571) 272-2876. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/BRIAN ELLIS YOUNG/Examiner, Art Unit 1684
/HEATHER CALAMITA/Supervisory Patent Examiner, Art Unit 1684
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