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 .
Priority
Acknowledgment is made of applicant’s claim for priority based on a provisional application filed as 63/110,683 on 11/06/2020.
All claims are given the priority date of 11/06/2020.
Application Status
Receipt is acknowledged of amendment, filed 05/01/2023. Claims 1-17, 20-22 and 24-31 are currently pending.
Information Disclosure Statement
Receipt of acknowledgment of the information disclosure statement filed on 04/27/2024 have been received and all references have been considered.
Claim Objections
Claim 15 is objected to because of the following informalities:
Claim 15 recites “the recipient cell.”. It would be remedial to amend the claims to recite “the microbial cell”.
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.
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 16, 30 and 31 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 16 is vague and indefinite in that the metes and bounds of the claim are unclear. The claim is unclear in that the claim recites “A microbial cell comprising the recombinant nucleic acid type I-F CRISPR-Cas system of claim 1, the system comprising” without any additional limitations of the claim rending the claim incomplete. It would be remedial to amend the claim to either remove the recitation of “the system comprising” or complete the limitations of the claim after the recitation of “the system comprising”.
The term “CRISPRi” in claim 30 renders the claim indefinite. The term “CRISPRi” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. The instant specification defines the “CRISPRi”, as presented in claims 30 and 31, as transferable CRISPR-based transcriptional interference (transferable CRISPRi) system wherein the cas2-3 gene is not present (Page 4, Lines 20-28). However, the specification does not teach whether the CRISPRi system includes all of the elements of the claim 1 CRISPR system and/or if the CRISPRi system is still a Type I CRISPR system.
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.
Claim 10 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 9 recites “wherein the type I-F cas operon lacks a functional copy of cas2-3 gene” whereas claim 10 recites “wherein the functional copy of cas2-3 gene is absent from the type I-F cas operon”. 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.
Claims 30 and 31 are 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 30 depends from claim 1 but only requires the use of part (a) without part (b), so it does not include all of the limitations system of claim 1 in the active steps of the method. 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
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.
Claims 1, 3 5, 6, 11-17, 20-22, 24 and 26-28 are rejected under 35 U.S.C. 103 as being unpatentable by Becher et al (BioTechniques, 29(5), 948–952; 2000) in view of Li et al (Cell Research, 26:1273-1287; 2016) and Xu et al (Cell Reports, Volume 29, Issue 6, 1707 - 1717.e3; November 5th, 2019) as evidenced by Zheng et al (Nucleic Acids Research, 2019, Vol. 47, No. 21 11461–11475).
Regarding claims 1 and 16, Becher teaches the integration vector, mini-CTX-LacZ, comprising an integrase enzyme (int), a nucleic acid sequence recognized as an integration site configured to recognize and attach the vector to a target attachment site within the microbial genome (attP) and two nucleic acid sequences configured to be Flp recombinase target sites on either side of a LacZ reporter gene (Page 948, Figure 1). Becher teaches that the FRT sites are included to improve the system to allow for subsequent in vivo removal of unwanted plasmid sequences from the genome (page 984, right column). Becher teaches the integration of the mini-CTX-LacZ vector into the PAO1 chromosome yielding strain PAO1 (lacZ) containing a chromosomally integrated promoterless lacZ cassette (Page 950, Column 2 bridging column 3).
Becher does not teach the plasmid comprising a Type I-F CRISPR cascade and a second plasmid comprising a crRNA and donor sequence.
Li teaches the CRISPR Type I-F cascade (Cas1, Cas3 (comprises Cas2-3), Cas8 (referred to as Csy1), Cas5 (referred to as Csy2), Cas7 (referred to as Csy3) and Cas6 (referred to as Csy 4)) within a plasmid (pAK1900 P. aeruginosa expression vector) for enabling an evasion from the host defense and leading to a reduced cytokine production and impaired immune response (Page 1275, Figure 1, Page 1284, Column 1 bridging Column 2, Page 1282, Column 1 briding column 2 and Supplemental Information Page 2). Zheng is only cited to show that the cas proteins of the Type I-F system are cas1–cas2/3–cas8(csy1)–cas5(csy2)–cas7(csy3)–cas6(csy4) in the P. aeruginosa UCBPP-PA14 strain (11465, Column 1).
Li does not teach a second plasmid comprising a crRNA and donor DNA sequence.
Xu teaches to achieve specific targeting to this genomic region by the native CRISPR-Cas machinery in PA154197 cells, an artificial mini-CRISPR encompassing the selected 32-bp internal sequence flanked by two 28-bp repeats was assembled (Page 1708, Column 2). Xu teaches a plasmid-based tool enabling expression and delivery of the mini-CRISPR into PA154197 cells comprising: the type I-F Cas operon (wherein the operon comprises cas1, cas2-3, cas8f, cas5, cas7 and cas6), Ptat for expression of the mini-CRISPR wherein the expression cassette was cloned into the pMS402 vector upstream of the lux genes along with the kanamycin resistance marker (Page 1708, Column 2 and Page 1709, Figure 1B). Xu teaches the lux genes along with the kanamycin resistance marker on the vector allows an antibiotic-luminescence dual selection of the transformants (1708, Column 2). Xu teaches the editing plasmid pAY5235 containing both a crRNA and a repair donor for mexB deletion (Page 1708, Column 2 and Page 1709, Figure 1). Xu teaches first administering the pAY5233 plasmid which contains the mini-CRISPR type I expression cassette along with the lux genes (Figure 1B) resulting in 100% reduction in expression of the mexB gene and subsequently delivering the editing plasmid (pAY5235) comprising the crRNA and a repair donor for mexB deletion yielding increased recovery rate of the PA154197 cells (Page 1708 Column 2 bridging Page 1709, columns 1 and 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Becher to include the CRISPR Type I-F cas proteins within a plasmid as taught by Li and the crRNA and donor DNA sequence within a second plasmid as taught by Xu because Becher teaches it is within the ordinary skill in the art to use integration of the mini-CTX-LacZ vector into the PAO1 chromosome yielding strain PAO1 (lacZ) containing a chromosomally integrated promoterless lacZ cassette, Li teaches the CRISPR Type I-Fcascade (Cas1, Cas3 (comprises Cas2-3), Cas8 (referred to as Csy1), Cas5 (referred to as Csy2), Cas7 (referred to as Csy3) and Cas6 (referred to as Csy 4)) within a plasmid (pAK1900 P. aeruginosa expression vector) for enabling an evasion from the host defense and leading to a reduced cytokine production and impaired immune response and Xu teaches first administering the pAY5233 plasmid which contains the mini-CRISPR type I expression cassette along with the lux genes (Figure 1B) resulting in 100% reduction in expression of the mexB gene and subsequently delivering the editing plasmid (pAY5235) comprising the crRNA and a repair donor for mexB deletion yielding increased recovery rate of the PA154197 cells.
One would have been motivated to make such a modification in order to receive the expected benefit of the Type I-F CRISPR cas system for enabling an evasion from the host defense and the recovery of PA154197 cells by using a plasmid comprising a crRNA and donor DNA sequence as taught by Li and Xu.
Regarding claims 3, 11 and 28, Becher teaches the integration vector, mini-CTX-LacZ, comprising an integrase enzyme (int), a nucleic acid sequence recognized as an integration site configured to recognize and attach the vector to a target attachment site within the microbial genome (attP) and two nucleic acid sequences configured to be Flp recombinase target sites on either side of a LacZ reporter gene (Page 948, Figure 1).
Regarding claims 5, 12, 17 and 22, Becher and Li do not teach the type I-F cas operon is the type I-F cas operon from P. aeruginosa strain PA154197.
Xu teaches in situ genome editing technique applicable in clinical and environmental isolates of the prototypic MDR pathogen P. aeruginosa by harnessing the endogenous type I-F CRISPR-Cas systems (Page 1707, Abstract). Xu teaches the use of PA154197 as a model to explore the native type I-F CRISPR-Cas-based genome editing in a clinical MDR P. aeruginosa genotype and its exploitation in the functional genomics of MDR (Page 1708, Column 1). Xu teaches the PA154197 strain Type I-F CRISPR cas system conferred resistance to antibiotics making the bacteria drug-resistant and thus removal of the Type I-F CRISPR cas system would allow for susceptibility to antibiotics (Page 1710, Figure 2 and Page 1711, Column 1 bridging Column 2 and Page 1712, Column 1).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Becher and Li to include the crRNA and donor DNA sequence within a second plasmid from the PA154197 strain as taught by Xu because Becher teaches it is within the ordinary skill in the art to use integration of the mini-CTX-LacZ vector into the PAO1 chromosome yielding strain PAO1 (lacZ) containing a chromosomally integrated promoterless lacZ cassette, Li teaches the CRISPR Type I-Fcascade (Cas1, Cas3 (comprises Cas2-3), Cas8 (referred to as Csy1), Cas5 (referred to as Csy2), Cas7 (referred to as Csy3) and Cas6 (referred to as Csy 4)) within a plasmid (pAK1900 P. aeruginosa expression vector) for enabling an evasion from the host defense and leading to a reduced cytokine production and impaired immune response and Xu teaches first administering the pAY5233 plasmid which contains the mini-CRISPR type I expression cassette along with the lux genes (Figure 1B) resulting in 100% reduction in expression of the mexB gene and subsequently delivering the editing plasmid (pAY5235) comprising the crRNA and a repair donor for mexB deletion yielding increased recovery rate of the PA154197 cells.
One would have been motivated to make such a modification in order to receive the expected benefit of the Type I-F CRISPR cas system for enabling an evasion from the host defense and the recovery of PA154197 cells by using a plasmid comprising a crRNA and donor DNA sequence as taught by Li and Xu.
Regarding claims 6 and 13, Becher teaches development of an integration proficient fCTX-based system for Pseudomonas aeruginosa, modeled after an approach that was previously described for mycobacterial integration vectors which included (i) inclusion of the fCTX int gene on the same vector containing attP and (ii) inclusion of a multiple cloning site (MCS) for facilitated cloning of DNA fragments (Page 948, Column 2 bridging Column 3). Becher teaches the integration vector, mini-CTX-LacZ, comprising an integrase enzyme (int), a nucleic acid sequence recognized as an integration site configured to recognize and attach the vector to a target attachment site within the microbial genome (attP) and two nucleic acid sequences configured to be Flp recombinase target sites on either side of a LacZ reporter gene (Page 948, Figure 1). Becher teaches that the FRT sites are included to improve the system to allow for subsequent in vivo removal of unwanted plasmid sequences from the genome (page 984, right column). Becher teaches the attB attachment site within the PAO chromosome for the recognition and integration of the plasmid at the attachment site (Page 948, Figure 1B).
Regarding claims 14 and 15, Becher and Li do not teach one or more CRISPR RNA nucleic acids are configured to target one or more transcriptional sites of the gene of interest and wherein the one or more transcriptional sites are selected from the group consisting of the RNA polymerase binding region, the transcription initiation region, the 5'-end of the coding region, the middle region of the gene, the 3'-end of the coding region, or a combination thereof, of the gene of interest in the recipient cell.
Xu teaches the crRNA targets a transcriptional site within the middle region (the C74-C105 region in mexB) of the mexB gene which resulted in deactivation of the gene and thus cell death (Page 1708, Column 2 and Page 1709, Figure 1B and Figure 1B).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Becher and Li to include the crRNA targets a transcriptional site within the middle region of the mexB gene as taught by Xu because Becher teaches it is within the ordinary skill in the art to use integration of the mini-CTX-LacZ vector into the PAO1 chromosome yielding strain PAO1 (lacZ) containing a chromosomally integrated promoterless lacZ cassette, Li teaches the CRISPR Type I-Fcascade (Cas1, Cas3 (comprises Cas2-3), Cas8 (referred to as Csy1), Cas5 (referred to as Csy2), Cas7 (referred to as Csy3) and Cas6 (referred to as Csy 4)) within a plasmid (pAK1900 P. aeruginosa expression vector) for enabling an evasion from the host defense and leading to a reduced cytokine production and impaired immune response and Xu teaches first administering the pAY5233 plasmid which contains the mini-CRISPR type I expression cassette along with the lux genes (Figure 1B) resulting in 100% reduction in expression of the mexB gene and subsequently delivering the editing plasmid (pAY5235) comprising the crRNA and a repair donor for mexB deletion yielding increased recovery rate of the PA154197 cells.
One would have been motivated to make such a modification in order to receive the expected benefit of the Type I-F CRISPR cas system for enabling an evasion from the host defense and the recovery of PA154197 cells by using a plasmid comprising a crRNA and donor DNA sequence as taught by Li and Xu.
Regarding claims 24, 26 and 27, Becher teaches the integration vector, mini-CTX-LacZ, comprising an integrase enzyme (int), a nucleic acid sequence recognized as an integration site configured to recognize and attach the vector to a target attachment site within the microbial genome (attP) and two nucleic acid sequences configured to be Flp recombinase target sites on either side of a LacZ reporter gene (Page 948, Figure 1). Becher teaches that the FRT sites are included to improve the system to allow for subsequent in vivo removal of unwanted plasmid sequences from the genome (page 984, right column). Becher teaches the integration of the mini-CTX-LacZ vector into the PAO1 chromosome yielding strain PAO1 (lacZ) containing a chromosomally integrated promoterless lacZ cassette (Page 950, Column 2 bridging column 3).
Becher does not teach the plasmid comprising a Type I-F CRISPR cascade and a second plasmid comprising a crRNA and donor sequence.
Li teaches the CRISPR Type I-Fcascade (Cas1, Cas3 (comprises Cas2-3), Cas8 (referred to as Csy1), Cas5 (referred to as Csy2), Cas7 (referred to as Csy3) and Cas6 (referred to as Csy 4)) within a plasmid (pAK1900 P. aeruginosa expression vector) for enabling an evasion from the host defense and leading to a reduced cytokine production and impaired immune response (Page 1275, Figure 1, Page 1284, Column 1 bridging Column 2, Page 1282, Column 1 briding column 2 and Supplemental Information Page 2). Zheng is only cited to show that the cas proteins of the Type I-F system are cas1–cas2/3–cas8(csy1)–cas5(csy2)–cas7(csy3)–cas6(csy4) in the P. aeruginosa UCBPP-PA14 strain (11465, Column 1).
Li does not teach a second plasmid comprising a crRNA and donor DNA sequence.
Xu teaches to achieve specific targeting to this genomic region by the native CRISPR-Cas machinery in PA154197 cells, an artificial mini-CRISPR encompassing the selected 32-bp internal sequence flanked by two 28-bp repeats was assembled (Page 1708, Column 2). Xu teaches a plasmid-based tool enabling expression and delivery of the mini-CRISPR into PA154197 cells comprising: the type I-F Cas operon (wherein the operon comprises cas1, cas2-3, cas8f, cas5, cas7 and cas6), Ptat for expression of the mini-CRISPR wherein the expression cassette was cloned into the pMS402 vector upstream of the lux genes along with the kanamycin resistance marker (Page 1708, Column 2 and Page 1709, Figure 1B). Xu teaches the lux genes along with the kanamycin resistance marker on the vector allows an antibiotic-luminescence dual selection of the transformants (1708, Column 2). Xu teaches the editing plasmid pAY5235 containing both a crRNA and a repair donor for mexB deletion (Page 1708, Column 2 and Page 1709, Figure 1). Xu teaches first administering the pAY5233 plasmid which contains the mini-CRISPR type I expression cassette along with the lux genes (Figure 1B) resulting in 100% reduction in expression of the mexB gene and subsequently delivering the editing plasmid (pAY5235) comprising the crRNA and a repair donor for mexB deletion yielding increased recovery rate of the PA154197 cells (Page 1708 Column 2 bridging Page 1709, columns 1 and 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Becher to include the CRISPR Type I-F cas proteins within a plasmid as taught by Li and the crRNA and donor DNA sequence within a second plasmid as taught by Xu because Becher teaches it is within the ordinary skill in the art to use integration of the mini-CTX-LacZ vector into the PAO1 chromosome yielding strain PAO1 (lacZ) containing a chromosomally integrated promoterless lacZ cassette, Li teaches the CRISPR Type I-Fcascade (Cas1, Cas3 (comprises Cas2-3), Cas8 (referred to as Csy1), Cas5 (referred to as Csy2), Cas7 (referred to as Csy3) and Cas6 (referred to as Csy 4)) within a plasmid (pAK1900 P. aeruginosa expression vector) for enabling an evasion from the host defense and leading to a reduced cytokine production and impaired immune response and Xu teaches first administering the pAY5233 plasmid which contains the mini-CRISPR type I expression cassette along with the lux genes (Figure 1B) resulting in 100% reduction in expression of the mexB gene and subsequently delivering the editing plasmid (pAY5235) comprising the crRNA and a repair donor for mexB deletion yielding increased recovery rate of the PA154197 cells.
One would have been motivated to make such a modification in order to receive the expected benefit of the Type I-F CRISPR cas system for enabling an evasion from the host defense and the recovery of PA154197 cells by using a plasmid comprising a crRNA and donor DNA sequence as taught by Li and Xu.
Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over Becher et al (BioTechniques, 29(5), 948–952; 2000) in view of Li et al (Cell Research, 26:1273-1287; 2016) and Xu et al (Cell Reports, Volume 29, Issue 6, 1707 - 1717.e3; November 5th, 2019) as evidenced by Zheng et al (Nucleic Acids Research, 2019, Vol. 47, No. 21 11461–11475), as applied to claims 1, 3 5, 6, 11-17, 20-22, 24 and 26-28, and further in view of Chugani et al (Proc. Natl. Acad. Sci. U.S.A. 107 (23) 10673-10678).
The teachings of Becher, Li and Xu are described above and applied as before.
Regarding claim 4, Becher, Li and Xu do not specifically teach the targeting vector comprises crRNA nucleic acids configured to disrupt one or more genes associated with expression of the Acyl-homoserine-lactone synthase enzyme.
Chugani teaches the transfection of the mini-CTX-lacZ and mini-CTX-lacZ-EB vectors into P. aeruginosa to allow chromosomal integration at attachment site attB (Page 10676, Column 2). Chugani teaches acyl-HSL regulation of gene expression in P. aeruginosa that does not follow the classical quorum-sensing tenet, in that it is not mediated by a LuxR-type transcription factor (Page 10673, Column 2). Chugani teaches that the disruption of the ant operon effects the production of acyl-HSL (Page 10676, Column 1).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Becher, Li and Xu to include the targeting vector configured to disrupt one or more genes associated with expression of the Acyl-homoserine-lactone synthase enzyme as taught by Chugani because Becher teaches it is within the ordinary skill in the art to use integration of the mini-CTX-LacZ vector into the PAO1 chromosome yielding strain PAO1 (lacZ) containing a chromosomally integrated promoterless lacZ cassette, Li teaches the CRISPR Type I-Fcascade (Cas1, Cas3 (comprises Cas2-3), Cas8 (referred to as Csy1), Cas5 (referred to as Csy2), Cas7 (referred to as Csy3) and Cas6 (referred to as Csy 4)) within a plasmid (pAK1900 P. aeruginosa expression vector) for enabling an evasion from the host defense and leading to a reduced cytokine production and impaired immune response, Xu teaches first administering the pAY5233 plasmid which contains the mini-CRISPR type I expression cassette along with the lux genes (Figure 1B) resulting in 100% reduction in expression of the mexB gene and subsequently delivering the editing plasmid (pAY5235) comprising the crRNA and a repair donor for mexB deletion yielding increased recovery rate of the PA154197 cells and Chugani teaches the disruption of the ant operon effects the production of acyl-HSL.
One would have been motivated to make such a modification in order to receive the expected benefit of reduced production of acyl-HSL by disruption of the ant operon as taught by Chugani.
Claims 7 and 25 are rejected under 35 U.S.C. 103 as being unpatentable over Becher et al (BioTechniques, 29(5), 948–952; 2000) in view of Li et al (Cell Research, 26:1273-1287; 2016) and Xu et al (Cell Reports, Volume 29, Issue 6, 1707 - 1717.e3; November 5th, 2019) as evidenced by Zheng et al (Nucleic Acids Research, 2019, Vol. 47, No. 21 11461–11475), as applied to claims 1, 3 5, 6, 11-17, 20-22, 24 and 26-28, and further in view of Reisch et al (Sci Rep 5, 15096 (2015)) and Hatoum-Aslan (Viruses. 2018 Jun 19; 10(6):335, Pages 1-11).
The teachings of Becker, Li and Xu are described above and applied as before.
Regarding claims 7 and 25, Becher, Li and Xu do not specifically teach a functional phage λ-red recombination system, comprising genes encoding λ-Red proteins Exo, Gam, and Beta, and an arabinose-inducible promoter.
Reisch teaches λ-Red prophage assisted recombineering has facilitated new and easy methods for defined insertions, deletions, and point-mutations (Page 1, Paragraph 1). Reisch teaches linear dsDNA is then introduced into cells that have the λ-Red genes bet, exo, and gam using an arabinose-inducible promoter for expression to facilitate genome integration (Page 1, Paragraph 1).
Reisch does not specifically teach the phage lambda red recombination system in a Type I-F cascade system, however, Hatoum-Aslan teaches phage lambda recombination systems can be used to help improve recombination and editing efficiency of the Type I CRISPR cas systems specifically in bacteria (Page 4, Paragraphs 1 and 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Becher, Li and Xu to include the a functional phage λ-red recombination system, comprising genes encoding λ-Red proteins Exo, Gam, and Beta, and an arabinose-inducible promoter as taught by Reisch because Becher teaches it is within the ordinary skill in the art to use integration of the mini-CTX-LacZ vector into the PAO1 chromosome yielding strain PAO1 (lacZ) containing a chromosomally integrated promoterless lacZ cassette, Li teaches the CRISPR Type I-Fcascade (Cas1, Cas3 (comprises Cas2-3), Cas8 (referred to as Csy1), Cas5 (referred to as Csy2), Cas7 (referred to as Csy3) and Cas6 (referred to as Csy 4)) within a plasmid (pAK1900 P. aeruginosa expression vector) for enabling an evasion from the host defense and leading to a reduced cytokine production and impaired immune response, Xu teaches first administering the pAY5233 plasmid which contains the mini-CRISPR type I expression cassette along with the lux genes (Figure 1B) resulting in 100% reduction in expression of the mexB gene and subsequently delivering the editing plasmid (pAY5235) comprising the crRNA and a repair donor for mexB deletion yielding increased recovery rate of the PA154197 cells, Reisch teaches linear dsDNA is then introduced into cells that have the λ-Red genes bet, exo, and gam using an arabinose-inducible promoter for expression to facilitate genome integration and Hatoum-Aslan teaches phage lambda recombination systems can be used to help improve recombination and editing efficiency of the Type I CRISPR cas systems specifically in bacteria.
One would have been motivated to make such a modification in order to receive the expected benefit of improved genome integration of the CRISPR Type I system by phage lambda recombination as taught by Reisch and Hatoum-Aslan.
Claims 2, 8-10 and 29-31 and rejected under 35 U.S.C. 103 as being unpatentable over Becher et al (BioTechniques, 29(5), 948–952; 2000) in view of Li et al (Cell Research, 26:1273-1287; 2016) and Xu et al (Cell Reports, Volume 29, Issue 6, 1707 - 1717.e3; November 5th, 2019) as evidenced by Zheng et al (Nucleic Acids Research, 2019, Vol. 47, No. 21 11461–11475), as applied to claims 1, 3 5, 6, 11-17, 20-22, 24 and 26-28, and further in view of Zheng et al (Nucleic Acids Research, 2019, Vol. 47, No. 21 11461–11475).
The teachings of Becher, Li and Xu are described above and applied as before.
Regarding claims 2 and 8, Becher, Li and Xu do not specifically teach a nucleic acid CRISPR-Cas removal vector, comprising one or more CRISPR RNA (crRNA) nucleic acids configured to delete an integrated type I-F cas system from the microbial cell.
Zheng teaches that endogenous systems of the microbial cell exhibited strong interference activity against the protospacer-bearing plasmids therefore DNA cleavage activity can be redirected to a PAM-flanking sequence on the chromosome for self-targeting and subsequent genome editing (Page 11466, Column 2). Zheng teaches the use of a self-targeting plasmid carrying an artificial CRISPR expression cassette of leader-repeat-spacer-repeat for the deletion of the non-essential cas2/3 gene from the type I-F cascade (Page 11467, Column 1). Zheng teaches the deletion of the CRISPR Type I-F cascade from the microbial cell to remove the genome editing array (Page 11470, Column 1 bridging Column 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Becher, Li and Xu to include the removal of the Type I-F CRISPR array after effective genome editing as taught by Zheng because Becher teaches it is within the ordinary skill in the art to use integration of the mini-CTX-LacZ vector into the PAO1 chromosome yielding strain PAO1 (lacZ) containing a chromosomally integrated promoterless lacZ cassette, Li teaches the CRISPR Type I-Fcascade (Cas1, Cas3 (comprises Cas2-3), Cas8 (referred to as Csy1), Cas5 (referred to as Csy2), Cas7 (referred to as Csy3) and Cas6 (referred to as Csy 4)) within a plasmid (pAK1900 P. aeruginosa expression vector) for enabling an evasion from the host defense and leading to a reduced cytokine production and impaired immune response, Xu teaches first administering the pAY5233 plasmid which contains the mini-CRISPR type I expression cassette along with the lux genes (Figure 1B) resulting in 100% reduction in expression of the mexB gene and subsequently delivering the editing plasmid (pAY5235) comprising the crRNA and a repair donor for mexB deletion yielding increased recovery rate of the PA154197 cells and Zheng teaches that endogenous systems of the microbial cell exhibited strong interference activity against the protospacer-bearing plasmids therefore DNA cleavage activity can be redirected to a PAM-flanking sequence on the chromosome for self-targeting and subsequent genome editing, therefore the removal of the non-essential cas2/3 genes reduced interference.
One would have been motivated to make such a modification in order to receive the expected benefit of reduced interreference from the removal of the cas2/3 gene as taught by Zheng.
Regarding claims 9 and 10, Becher and Li do not teach the type I-F cas operon lacks a functional copy of cas2-3 gene.
Zheng teaches that endogenous systems of the microbial cell exhibited strong interference activity against the protospacer-bearing plasmids therefore DNA cleavage activity can be redirected to a PAM-flanking sequence on the chromosome for self-targeting and subsequent genome editing (Page 11466, Column 2). Zheng teaches the use of a self-targeting plasmid carrying an artificial CRISPR expression cassette of leader-repeat-spacer-repeat for the deletion of the cas2/3 gene from the type I-F cascade (Page 11467, Column 1). Zheng teaches that the Type I cas2/3 gene can be removed and editing will still occur because it is non-essential (Page 11468, Column 1). Zheng also teaches the deletion of the CRISPR Type I-F cascade from the microbial cell to remove the genome editing array (Page 11470, Column 1 bridging Column 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Xu and Becher to include the removal of the Type I-F CRISPR array after effective genome editing as taught by Zheng because Becher teaches it is within the ordinary skill in the art to use integration of the mini-CTX-LacZ vector into the PAO1 chromosome yielding strain PAO1 (lacZ) containing a chromosomally integrated promoterless lacZ cassette, Li teaches the CRISPR Type I-Fcascade (Cas1, Cas3 (comprises Cas2-3), Cas8 (referred to as Csy1), Cas5 (referred to as Csy2), Cas7 (referred to as Csy3) and Cas6 (referred to as Csy 4)) within a plasmid (pAK1900 P. aeruginosa expression vector) for enabling an evasion from the host defense and leading to a reduced cytokine production and impaired immune response, Xu teaches first administering the pAY5233 plasmid which contains the mini-CRISPR type I expression cassette along with the lux genes (Figure 1B) resulting in 100% reduction in expression of the mexB gene and subsequently delivering the editing plasmid (pAY5235) comprising the crRNA and a repair donor for mexB deletion yielding increased recovery rate of the PA154197 cells and Zheng teaches that endogenous systems of the microbial cell exhibited strong interference activity against the protospacer-bearing plasmids therefore DNA cleavage activity can be redirected to a PAM-flanking sequence on the chromosome for self-targeting and subsequent genome editing, therefore the removal of the non-essential cas2/3 genes reduced interference.
One would have been motivated to make such a modification in order to receive the expected benefit of reduced interreference from the removal of the cas2/3 gene as taught by Zheng.
Regarding claim 29, Becher, Li and Xu do not teach removing the CRISPR-Cas system from the recipient cell by contacting the recipient cell with a nucleic acid CRISPR-Cas removal vector.
Zheng teaches that endogenous systems of the microbial cell exhibited strong interference activity against the protospacer-bearing plasmids therefore DNA cleavage activity can be redirected to a PAM-flanking sequence on the chromosome for self-targeting and subsequent genome editing (Page 11466, Column 2). Zheng teaches the use of a self-targeting plasmid carrying an artificial CRISPR expression cassette of leader-repeat-spacer-repeat for the deletion of the cas2/3 gene from the type I-F cascade (Page 11467, Column 1). Zheng teaches the deletion of the CRISPR Type I-F cascade from the microbial cell to remove the genome editing array (Page 11470, Column 1 bridging Column 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Becher, Li and Xu to include the removal of the Type I-F CRISPR array after effective genome editing as taught by Zheng because Becher teaches it is within the ordinary skill in the art to use integration of the mini-CTX-LacZ vector into the PAO1 chromosome yielding strain PAO1 (lacZ) containing a chromosomally integrated promoterless lacZ cassette, Li teaches the CRISPR Type I-Fcascade (Cas1, Cas3 (comprises Cas2-3), Cas8 (referred to as Csy1), Cas5 (referred to as Csy2), Cas7 (referred to as Csy3) and Cas6 (referred to as Csy 4)) within a plasmid (pAK1900 P. aeruginosa expression vector) for enabling an evasion from the host defense and leading to a reduced cytokine production and impaired immune response, Xu teaches first administering the pAY5233 plasmid which contains the mini-CRISPR type I expression cassette along with the lux genes (Figure 1B) resulting in 100% reduction in expression of the mexB gene and subsequently delivering the editing plasmid (pAY5235) comprising the crRNA and a repair donor for mexB deletion yielding increased recovery rate of the PA154197 cells and Zheng teaches that endogenous systems of the microbial cell exhibited strong interference activity against the protospacer-bearing plasmids therefore DNA cleavage activity can be redirected to a PAM-flanking sequence on the chromosome for self-targeting and subsequent genome editing, therefore the removal of the non-essential cas2/3 genes reduced interference.
One would have been motivated to make such a modification in order to receive the expected benefit of removal of the Type I-F cascade reduces interference and future genome editing as taught by Zheng.
Regarding claims 30 and 31, The instant specification defines the “CRISPRi”, as presented in claims 30 and 31, as transferable CRISPR-based transcriptional interference (transferable CRISPRi) system wherein the cas2-3 gene is not present (Page 4, Lines 20-28). Therefore, the claim is being interpreted as the same CRISPR system with all the same components of instant claim 1, however, without the cas2-3 gene.
Becher, Li and Xu do not teach the type I-F cas operon lacks a functional copy of cas2-3 gene.
Zheng teaches that endogenous systems of the microbial cell exhibited strong interference activity against the protospacer-bearing plasmids therefore DNA cleavage activity can be redirected to a PAM-flanking sequence on the chromosome for self-targeting and subsequent genome editing (Page 11466, Column 2). Zheng teaches the use of a self-targeting plasmid carrying an artificial CRISPR expression cassette of leader-repeat-spacer-repeat for the deletion of the cas2/3 gene from the type I-F cascade (Page 11467, Column 1). Zheng teaches that the Type I cas2/3 gene can be removed and editing will still occur because it is non-essential (Page 11468, Column 1). Zheng also teaches the deletion of the CRISPR Type I-F cascade from the microbial cell to remove the genome editing array (Page 11470, Column 1 bridging Column 2). Zheng teaches the Type I-F system was further applied for CRISPRi upon Cas2/3 depletion, which has been demonstrated to successfully silence the chromosomally integrated mCherry gene with its fluorescence intensity reduced by up to 88% (Page 11461, Abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Becher, Li and Xu to include the removal of the Type I-F CRISPR array after effective genome editing as taught by Zheng because Becher teaches it is within the ordinary skill in the art to use integration of the mini-CTX-LacZ vector into the PAO1 chromosome yielding strain PAO1 (lacZ) containing a chromosomally integrated promoterless lacZ cassette, Li teaches the CRISPR Type I-Fcascade (Cas1, Cas3 (comprises Cas2-3), Cas8 (referred to as Csy1), Cas5 (referred to as Csy2), Cas7 (referred to as Csy3) and Cas6 (referred to as Csy 4)) within a plasmid (pAK1900 P. aeruginosa expression vector) for enabling an evasion from the host defense and leading to a reduced cytokine production and impaired immune response, Xu teaches first administering the pAY5233 plasmid which contains the mini-CRISPR type I expression cassette along with the lux genes (Figure 1B) resulting in 100% reduction in expression of the mexB gene and subsequently delivering the editing plasmid (pAY5235) comprising the crRNA and a repair donor for mexB deletion yielding increased recovery rate of the PA154197 cells and Zheng teaches that endogenous systems of the microbial cell exhibited strong interference activity against the protospacer-bearing plasmids therefore DNA cleavage activity can be redirected to a PAM-flanking sequence on the chromosome for self-targeting and subsequent genome editing, therefore the removal of the non-essential cas2/3 genes reduced interference.
One would have been motivated to make such a modification in order to receive the expected benefit of reduced interreference from the removal of the cas2/3 gene as taught by Zheng.
Conclusion
No claims are allowed.
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/ALEXANDRA ROSE LIPPOLIS/ Examiner, Art Unit 1637
/CELINE X QIAN/ Primary Examiner, Art Unit 1637