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 .
This action is in response to the amendment filed 05/18/2026, in which claims 9, 15, 16 and 30 were amended and claims 1-8, 10-14, 17, 20-22, 24-29 and 31 are previously presented.
Claims 1-17, 20-22 and 24-31 are currently pending.
Applicant’s arguments have been thoroughly reviewed, but are not persuasive for the
reasons that follow. Any rejection and objections not reiterated in this action have been
withdrawn. This action is FINAL.
Claim Objections
The previous objection to claim 15 has been withdrawn in view of Applicant’s amendments to the claims filed on 05/18/2026.
Claim Rejections - 35 USC § 112
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.
Response to Arguments - Claim Rejections - 35 USC § 112
The previous rejection of claim 10 under 35 U.S.C. 112(d) has been maintained in view of no amendment or argument being placed on the record as of the latest filing on 05/18/2026.
The previous rejection of claims 16, 30 and 31 under 35 U.S.C. 112(b) has been withdrawn in view of Applicant’s amendments to the claims filed on 05/18/2026.
The previous rejection of claims 30 and 31 under 35 U.S.C. 112(d) has been withdrawn in view of Applicant’s amendments to the claims filed on 05/18/2026.
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.
Claim Rejections - 35 USC § 103
The previous rejection of claims 1, 3 5, 6, 11-17, 20-22, 24 and 26-28 under 35 U.S.C. 103 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) has been maintained in view of Applicant’s reply filed on 05/18/2026.
The previous rejection of claim 4 under 35 U.S.C. 103 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) and further in view of Chugani et al (Proc. Natl. Acad. Sci. U.S.A. 107 (23) 10673-10678) has been maintained in view of Applicant’s reply filed on 05/18/2026.
The previous rejection of claims 7 and 25 under 35 U.S.C. 103 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) 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) has been maintained in view of Applicant’s reply filed on 05/18/2026.
The previous rejection of claims 2, 8-10 and 29-31 under 35 U.S.C. 103 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) and further in view of Zheng et al (Nucleic Acids Research, 2019, Vol. 47, No. 21 11461–11475) has been maintained in view of Applicant’s reply filed on 05/18/2026.
Applicant’s arguments have been fully considered but have not been found to be persuasive.
Applicant argues the problem to be solved by the claimed CRISPR-Cas genome editing system, specifically, that the claimed genome editing system solves the problem of how to provide generic, transferable and integrative type I CRISPR-Cas based genome editing system that can be used in diverse microbial hosts, including hosts that do not naturally contain a functional endogenous type I-F CRISPR-Cas system. Applicant continues that Type I CRISPR-Cas systems, although the most abundant CRISPR-Ca systems in bacteria and archaea and highly promising due to their cascade-based DNA targeting activity, had generally been used only in specific microbial hosts that already contain an active and well characterized endogenous CRISPR-Cas system therefore, these approaches could not be reliably extended to other strains that lack CRISPR-Cas systems.
However, the current claim limitations of independent claim 1 do not recite that the original microbial cell lacks its own endogenous CRISPR-Cas system but rather broadly claims “a microbial cell, comprising (i) a type I-F cas operon, comprising the cas genes cas1, cas2-3, cas8f, cas5, cas7 and cas6, and a nucleic acid sequence configured to promote transcription of the cas genes in the microbial cell; (ii) one or more genes encoding an integrase enzyme; (iii) 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; (iv) two nucleic acid sequences configured to be Flp recombinase target sites; and (v) one or more reporter genes and a nucleic acid sequence configured to promote transcription of the reporter gene(s) upon integration into the microbial cell genome; wherein the vector is configured for integration into the genome of the microbial cell via attachment at the target attachment site; and (b) a nucleic acid editing vector comprising one or more CRISPR RNA (crRNA) nucleic acids and repair donors, configured to change, add, and/or delete one or more target genomic sites in the microbial cell through the type I-F CRISPR-Cas system”. Therefore, Applicant’s arguments are not found to be persuasive.
Applicant outlines that claim 1 defines a genome editing system for changing, adding, and/or deleting one or more genes in a microbial cell. The genome editing system defined by claim 1 requires a nucleic acid type I-F cas system vector and a nucleic acid editing vector, specifically, the nucleic acid type I-F cas system vector requires: (i) a type I-F cas operon, including the cas genes cas1, cas2-3, cas8f, cas5, cas7, and cas6, together with a nucleic acid sequence configured to promote transcription of the cas genes; (ii) one or more genes encoding an integrase enzyme; (iii) 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; (iv) two nucleic acid sequences configured to be Flp recombinase target sites; and (v) one or more reporter genes and a nucleic acid sequence configured to promote transcription of the reporter gene(s) after integration into the microbial genome; and the nucleic acid editing vector requires one or more CRISPR RNA (crRNA) nucleic acids and repair donors, configured to change, add, and/or delete one or more target genomic sites in the microbial cell through the type I-F CRISPR-Cas system; which was shown in the schematic illustrations of Figures 1A, 1B and 1D.
Applicant argues Becher is not concerned with solving the problem addressed by the claimed systems, i.e., providing a transferable and integrative type I-F CRISPR-Cas genome editing system that allows a microbial host to acquire and express functional type I-F CRISPR-Cas machinery and Becher does not teach or suggest inserting a complete type I-F cas operon into the mini-CTX vector, does not teach cas1, cas2-3, cas8f, cas5, cas7, or cas6, does not teach a nucleic acid sequence configured to promote transcription of those cas genes, does not teach conferring a functional type I-F CRISPR-Cas system on a recipient microbial host, and does not teach use of a separate programmed crRNA and repair donor vector to change, add, or delete a genomic sequence through an installed type I-F system. Applicant continues to argue Li is not concerned with solving the problem addressed by the claimed systems, i.e., providing a transferable and integrative type I-F CRISPR-Cas genome-editing system that allows a microbial host to acquire and express functional type I-F CRISPR-Cas machinery and Li does not teach a mini-CTX-based vector, does not teach inserting a complete type I-F cas operon into an integrative vector, does not teach using Int, attP, and FRT sites to install a functional type I-F Cascade into a recipient microbial genome, and does not teach a separate editing vector carrying programmed crRNA and repair donor sequences for changing, adding, or deleting genomic sequences. Li also does not teach using a transferable cas system to enable CRISPR-Cas editing in CRISPR-free or CRISPR-deficient strains. Applicant argues Xu is not concerned with solving the problem addressed by the claimed systems, i.e., providing a transferable and integrative type I-F CRISPR-Cas genome-editing system that allows a microbial host lacking a functional endogenous CRISPR-Cas system to acquire and express functional type I-F CRISPR-Cas machinery and Xu does not direct one of skill in the art to the specific selections required by the claims, because Xu’s system is for a different purpose: editing the CRISPR-Cas machinery that is already naturally present and functional in the host cell. Applicant continues to argue that Xu does not teach a mini-CTX-IF vector, does not teach inserting a complete type I-F cas operon into an integrative vector, does not teach a type I-F cas system vector including the full cas operon together with an integrase, attP integration site, FRT sites, and a Ptat-lacZ reporter arranged for chromosomal integration, and does not teach installing a functional type I-F Cascade into a CRISPR-free or CRISPR-deficient microbial host.
In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case, Applicant addresses each prior art individually instead of in combination as it is written.
Applicant further argues that the claimed genome editing systems are not obvious over Becher, Li and Xu because the Examiner relies on impermissible hindsight reconstruction of Applicant’s findings. Applicant argues that, as discussed above, none of the cited references provide any teachings or guidance for constructing a transferable and integrative type I-F CRISPR Cas genome editing system that allows a microbial host, including a CRISPR-free or CRISPR-deficient host, to acquire and express a functional type I-F CRISPR-Cas system.
In response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971). Further, with regards to Applicant’s argument that the cited references do not teach the limitations that the microbial host must be CRISPR-free or a CRISPR-deficient host and as discussed above, the current claim limitations of independent claim 1 do not recite that the original microbial cell lacks its own endogenous CRISPR-Cas system.
Applicant argues that Becher does not evaluate or suggest that use of the backbone to transfer a complete functional CRISPR-Cas system into a microbial host and also does not teach or suggest that the mini-CTX vector could be modified to carry, integrate, and functionally express a complete multi-gene type I-F cas operon. Applicant continues to argue that Li and Xu do not make up for the deficiencies of Becher, specifically that one of skill in the art would not extrapolate from a combination of Becher and Li’s studies of the natural biological role of an endogenous type I-F CRISPR-Cas system in P. aeruginosa PA14 to arrive at the claimed transferable genome editing system for use in diverse microbial strains that do not posses them endogenously and Li also does not teach a separate editing vector carrying programmed crRNA and repair donor sequences.
However, in response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case, Becher is relied on for its teachings that 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. Therefore, 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
Applicant argues the same argument as previously discussed that Xu’s system depends on the host already having a functional type I-F CRISPR-Cas locus. However, as discussed above, the current claim limitations of independent claim 1 do not recite that the original microbial cell lacks its own endogenous CRISPR-Cas system.
Applicant argues further impermissible hindsight reasoning as that the Examiner’s relies on Becher, Li and Xu that one of ordinary skill in the art presented with a reporter-fusion integration vector, an endogenous virulence-regulation study, and a native CRISPR-Cas editing method, would have selected and combined only those elements needed to reconstruct Applicant’s claimed system. Applicant continues to argue that the prior art did not provide a path to the claimed system and cited references do not address these obstacles or provide a roadmap for converting Becher’s reporter-fusion vector into a transferable type I-F cas system vector. Applicant argues that there was no teaching or guidance from Becher, Li and/or Xu, that a complete, multi-component type I-F CRISPR-Cas system could be transferred into a heterologous microbial host, stably integrated, expressed and used for genome editing.
As discussed above, in response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971). In addition, in response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case, Becher is relied on for its teachings that 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. Therefore, 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.
Applicant argues the primer and probe sequences required by the claims as amended have superior and unexpected properties that could not have been predicted from the cited art. Applicant continues to argue that due to having unexpected results, and in view of case law (Plantronic, Inc. v. Aliph, Inc., 24 F.3d 1343, 1355, (Fed.Cir., 2013), that objective indicia of non-obviousness must be considered before reaching an obviousness conclusion in order to guard against hindsight bias. Applicant argues that experimental data demonstrate that the claimed system achieved results not suggested or predictable from Becher, Li or Xu such as when the transferable I-F and λ-I-f cas systems were introduced into the CRISPR-free model strain PAO1, all recovered clones appeared blue, indicating successful chromosomal integration and lacZ expression; whole genome sequencing or a representative clone showed site-specific integration at the attB site and only a 4-bp synonymous substitution, demonstrating highly efficient and precises integration; expression of all cas genes were detected in the PAO1 strain containing the transferable I-F system, but not in a control strain lacking the cas operon; the examples further demonstrates that integration did not significantly affect bacterial physiology, as there was no difference in cell growth, proteolytic activity, biofilm formation, C. elegans filling, antibiotic susceptibility, colony morphology, or motility and transcriptome analysis identified only one gene with 2-fold upregulation. Applicant further argues that the data demonstrates functional DNA interference such as the introduction of a self-targeting plasmid targeting rhII caused a greater than 10-5 reduction in conjugation efficiency in the PAO1 strain containing the transferable I-F system, but not in the control strain lacking the cas operon; thus, the results showed that the transferred I-F cas system was not merely integrated, but was highly active in executing genome cleavage in a CRISPR-free host. Applicant argues that the data also demonstrates that the transferable system allowed editing across different genetic backgrounds, specifically, in PA14 which had poor intrinsic homologous recombination capacity, addition of the λ-Red system substantially increased rhII deletion efficiency from about 15.6% to about 60.4% and this system also allowed editing in unsequenced clinical isolates PA151671 and PA132533, and in another Pseudomonas species, P. putida KT2440, where deletion of algR was achieved with 10/10, i.e., 100% editing efficiency. Applicant thus argues that these results could not have been predicted from the combination of Becher, Li and Xu and none of these references provides guidance that a complete PA154197 type I-F cas operon could be transferred into a CRISPR-free or CRISPR-deficient host, stably integrated, expressed, assembled into a functional cascade, used for high-efficiency DNA interference, and then combined with a separate editing vector to achieve precise genome editing.
In response to applicant's argument that the invention showed unpredictable results when the invention was used in a microbial cell that was CRISPR-free or CRISPR-deficient, the fact that the inventor has recognized another advantage which would flow naturally from following the suggestion of the prior art cannot be the basis for patentability when the differences would otherwise be obvious. See Ex parte Obiaya, 227 USPQ 58, 60 (Bd. Pat. App. & Inter. 1985). As previously discussed, the current claim limitations of independent claim 1 do not recite that the original microbial cell lacks its own endogenous CRISPR-Cas system. Also discussed above, in response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971). Applicant argues that secondary consideration must be used in order to protect from hindsight reasoning bias, however, there can not be secondary consideration for unpredictable results when the “unpredictable results” are predictable. Furthermore, arguments of unexpected results are unpersuasive when they are not commensurate in scope with the claims.
Applicant argues that claim 4 is not obvious over the combination of references, specifically Becher, Li, Xu and Chugani, because Chugani does not cure the deficiencies of Becher, Li and Xu as described above. Applicant further argues that for at least the reasons described above in connection with Becher, Li and Xu, claims 1 and 4 are non-obvious over the combination.
Applicant’s arguments as they pertained to claim 1 were not found persuasive and therefore, are not persuasive to withdraw the rejection of claim 4 and the rejection is maintained.
Applicant argues that claims 7 and 25 are not obvious over the combination of references, specifically Becher, Li, Xu and Reisch, because Reisch does not cure the deficiencies of Becher, Li and Xu as described above. Applicant further argues that for at least the reasons described above in connection with Becher, Li and Xu, claims 1, 7 and 25 are non-obvious over the combination.
Applicant’s arguments as they pertained to claim 1 were not found persuasive and therefore, are not persuasive to withdraw the rejection of claims 7 and 25 and the rejection is maintained.
Applicant argues that claims 2, 8-10 and 29-31 are not obvious over the combination of references, specifically Becher, Li, Xu and Zheng, because Zheng does not cure the deficiencies of Becher, Li and Xu as described above. Applicant further argues that for at least the reasons described above in connection with Becher, Li and Xu, claims 1, 2, 8-10 and 29-31 are non-obvious over the combination.
Applicant’s arguments as they pertained to claim 1 were not found persuasive and therefore, are not persuasive to withdraw the rejection of claims 2, 8-10 and 29-31 and the rejection is maintained.
Conclusion
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/ALEXANDRA ROSE LIPPOLIS/Examiner, Art Unit 1637
/CELINE X QIAN/Primary Examiner, Art Unit 1637
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