IP Library › Granted Patent US 12,359,198
Granted Patent B2
US 12,359,198 · App. 17/501,421 · Granted Jul 15, 2025

High-efficacy crispri system and strong synthetic promoters for alphaproteobacteria and gammaproteobacteria

Inventors: Jason Peters (Madison, WI); Amy Banta (Madison, WI)
Assignee: Wisconsin Alumni Research Foundation
C12N15/111C12N9/22C12N15/63C12N2310/20C12N2800/10
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Quick Facts
Patent No.
US 12,359,198
App. No.
17/501,421
Granted
Jul 15, 2025
Kind
B2
Abstract

Described herein are synthetic inducible promoters including 5′-(UP element)-(−35 element)-(spacer element)-(−10 element)-(discriminator element)-3′. Also included are vectors, α-Proteobacteria strains and γ-Proteobacteria strains including the synthetic inducible promoters. A Mobile-CRISPRi plasmid and methods of partially or fully knocking-down expression of a gene in α-Proteobacteria or γ-Proteobacteria are also described. Further included are methods of making an α-Proteobacteria or γ-Proteobacteria strain.

Claims (29)

1. A synthetic inducible proteobacterial promoter comprising

5′-(UP element)-(−35 element)-(spacer element)-(−10 element)-(discriminator element)-3′; wherein

the (UP element) is SEQ ID NO: 14 or SEQ ID NO: 15;

the (−35 element) is TTTACA, TTGAAA, TTGAAT, or TTTAAA;

the (spacer element) is ATTGTGAGCGCTCACAAT (SEQ ID NO: 11), TTGTGAGCGCTCACAAT (SEQ ID NO: 12), or TGTGAGCGGATAACAAT (SEQ ID NO: 13);

the (−10 element) is TATAAN 26 wherein N 26 is A, C, G or T; and

the (discriminator element) is GTCTAGT or TCTAGT,

wherein the synthetic inducible promoter is inducible with isopropyl β-d-1-thiogalactopyranoside (IPTG), or lactose in proteobacteria strain,

wherein the proteobacteria strain is Zymomonas mobilis or Acinetobacter baumanii.

2. The synthetic inducible promoter of claim 1 , that is Promoter C (SEQ ID NO: 3), Promoter D (SEQ ID NO: 4), Promoter E (SEQ ID NO: 5) or Promoter F (SEQ ID NO: 6).

3. A vector comprising the synthetic inducible promoter of claim 1 .

4. A proteobacteria strain comprising the inducible promoter of claim 1 , wherein the proteobacteria strain is Zymomonas mobilis or Acinetobacter baumanii.

5. The proteobacteria strain of claim 4 , wherein the proteobacteria strain is Zymomonas mobilis.

6. A Mobile-CRISPRi plasmid, comprising, in operable communication,

an expression cassette for a dCas9 protein, the dCas9 protein expressed with an α-Proteobacteria or γ-Proteobacteria ribosome binding site; and

a gRNA expression cassette under control of the synthetic inducible promoter of claim 1 ,

wherein the spacer of the gRNA is fully matched or has a mismatch with a proteobacteria gene and wherein the gRNA-dCas9 complex produced from the Mobile-CRISPRi partially or fully blocks expression of the proteobacteria gene,

wherein the proteobacteria strain is Zymomonas mobilis or Acinetobacter baumanii.

7. The Mobile-CRISPRi plasmid of claim 6 , wherein the synthetic inducible promoter is Promoter C (SEQ ID NO: 3), Promoter D (SEQ ID NO: 4), Promoter E (SEQ ID NO: 5), or Promoter F (SEQ ID NO: 6).

8. The Mobile-CRISPRi plasmid of claim 6 , wherein the dCas9 is Streptococcus pyogenes dCas9, or a Homo sapiens optimized dCas9.

9. The Mobile-CRISPRi plasmid of claim 6 , wherein the proteobacteria is Zymomonas mobilis.

10. The Mobile-CRISPRi plasmid of claim 6 , wherein the proteobacteria gene is an essential metabolic gene, a stress response gene, or a stress resistance gene.

11. The Mobile-CRISPRi plasmid of claim 6 , wherein the gRNA perfectly matches the proteobacteria gene.

12. A population of Mobile-CRISPRi plasmids of claim 6 , wherein each individual species of the population comprises a gRNA library member comprising a unique gRNA spacer sequence.

13. A method of partially or fully knocking-down expression of a gene in proteobacteria, comprising transferring the Mobile-CRISPRi plasmid of claim 6 into the proteobacterium, and expressing the dCas9 protein and the gRNA, wherein the proteobacterium is Zymomonas mobilis or Acinetobacter baumanii.

14. The method of claim 13 , wherein the proteobacterium is Zymomonas mobilis.

15. A method of making a proteobacteria strain comprising a chromosomally inserted Mobile-CRISPRi expression cassette, comprising triparental mating a first E. coli donor strain and a second E. coli donor strain and a proteobacteria recipient, wherein the first E. coli donor strain comprises a plasmid encoding a Tn7 transposase, wherein the second E. coli donor strain comprises a Tn7 transposon encoding the Mobile-CRISPRi expression cassette, and wherein the Mobile-CRISPRi expression cassette comprises an expression cassette for a dCas9 protein, the dCas9 protein expressed with a α-Proteobacteria or γ-Proteobacteria ribosome binding site; and a gRNA expression cassette under control of the synthetic inducible promoter of claim 1 , wherein a gRNA-dCas9 complex produced from the Mobile-CRISPRi plasmid partially or fully blocks expression of a proteobacteria gene; and producing the proteobacteria strain comprising the chromosomally inserted Mobile-CRISPRi expression cassette, wherein the proteobacteria is Zymomonas mobilis or Acinetobacter baumannii.

16. The method of claim 15 , wherein the proteobacteria strain is Zymomonas mobilis.

17. The method of claim 6 , wherein the mismatched spacer of the gRNA has a one or two nucleotide mismatches with the proteobacteria gene.

Assignments (2)
CONFIRMATORY LICENSE Recorded Mar 18, 2022
From: UNIVERSITY OF WISCONSIN-MADISON
To: UNITED STATES DEPARTMENT OF ENERGY
Reel/Frame 060202/0314 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 19, 2021
From: BANTA, AMY; PETERS, JASON
To: WISCONSIN ALUMNI RESEARCH FOUNDATION
Reel/Frame 057833/0447 →
Continuity (2)
Provisional Application 63092182 · Oct 15, 2020
Related Publication 20220119810A1 · Apr 21, 2022
References Cited (67)
US 20210071179A1 · Peters et al. · 2021 [cited by applicant]
WO WO2004003139A2 · 2004 [cited by examiner]
WO WO2009073551A2 · 2009 [cited by examiner]
Yang Y et al. Biotechnol Biofuels. Mar. 14, 2019;12:52 (Year: 2019). [cited by examiner]
Vigouroux A et al. Microbiol Mol Biol Rev. Apr. 1, 2020;84(2):e00077-19 (Year: 2020). [cited by examiner]
Vera JM et al. Jul. 21, 2020;5(4):e00250-20 (Year: 2020). [cited by examiner]
Estrem ST et al. Proc Natl Acad Sci U S A. Aug. 18, 1998;95(17):9761-6 (Year: 1998). [cited by examiner]
NCBI MK775710.1 (NCBI Database, Jun. 26, 2019) (Year: 2019). [cited by examiner]
Peters JM et al. Nat Microbiol. Feb. 2019;4(2):244-250 (Year: 2019). [cited by examiner]
Seleem (Seleem M et al. Appl Microbiol Biotechnol. Jan. 2007;73(5):1123-7 (Year: 2007). [cited by examiner]
Chain (Chain PS et al. J Bacteriol. Aug. 2011;193(16):4274-5) (Year: 2011). [cited by examiner]
Munoz-Gomez (Munoz-Gomez SA et al. Elife. Feb. 25, 2019;8:e42535 (Year: 2019). [cited by examiner]
Zheng, Y. et al.; “Characterization and repurposing of the endogenous Type I-F CRISPR-Cas system of Zymomonas mobilis for genome engineering”; Nucleic Acids Research, vol. 47; 2019; pp. 11461-11475. [cited by applicant]
Baba, T. et al.; “Construction of [cited by applicant]
Banta, A. et al.; “A High-Efficacy CRISPR Interference System for Gene Function Discovery in Zymomonas mobilis”; Applied and Environmental Microbiology, vol. 86, Issue No. 23; 2020; 16 pages; doi: 10.1128/AEM.01621-20. [cited by applicant]
Belaïch, J-P. et al.; “Influence of Aeration and of Pantothenate on Growth Yields of Zymomonas mobilis”; Journal of Bacteriology. vol. 89, Issue No. 5; 1965; pp. 1195-1200. [cited by applicant]
Bikard, D. et al.; “Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system”; Nucleic Acids Research, vol. 41, Issue No. 15; 2013; pp. 7429-7437. [cited by applicant]
Blodgett, J. et al.; “Unusual transformations in the biosynthesis of the antibiotic phosphinothricin tripeptide”; Nature Chemical Biology, vol. 3, Issue No. 8; 2007; pp. 480-485. [cited by applicant]
Brenac, L. et al.; “Distinct functional roles for hopanoid composition in the chemical tolerance of Zymomonas mobilis”; Molecular Microbiology, vol. 112, Issue No. 5; 2019; pp. 1564-1575. [cited by applicant]
Choi, Y. et al.; “Metabolic engineering of microorganisms for the production of higher alcohols”; mBio, vol. 5, Issue No. 5; 2014; 10 pages; doi: 10.1128/mBio.01524-01514. [cited by applicant]
Conway, T.; “The Entner-Doudoroff pathway: history, physiology and 432 molecular biology”; FEMS Microbiology Reviews, vol. 9, Issue No. 1; 1992; pp. 1-27; doi: 10.1111/j.1574-6968.1992.tb05822.x. [cited by applicant]
Dawes, E. et al.; “The route of ethanol formation in Zymomonas mobilis”; The Biochemical Journal, vol. 98, Issue No. 3; 1966; pp. 795-803. [cited by applicant]
Estrem S. et al.; “Identification of an UP element consensus sequence for bacterial promoters”; PNAS USA, vol. 95, Issue No. 17; 1998; pp. 9761-9766. [cited by applicant]
Felczak, M. et al.; “Expression of Phosphofructokinase Is Not Sufficient to Enable Embden-Meyerhof-Parnas Glycolysis in Zymomonas mobilis ZM4”; Frontiers in Microbiology, vol. 10, Article 2270; 2019; 11 pages; doi: 10.3… [cited by applicant]
Ghosh, I. et al.; “OptSSeq explores enzyme expression and function landscapes to maximize isobutanol production rate”; Metabolic Engineering, vol. 52; 2019; pp. 324-340. [cited by applicant]
Hawkins, J. et al.; “Modulated efficacy CRISPRi reveals evolutionary conservation of essential gene expression-fitness relationships in bacteria”; Cell Systems; 2019; 15 pages; DOI: 10.1016/j.cels.2020.09.009. [cited by applicant]
Hawley, D. et al.; “Compilation and analysis of [cited by applicant]
He, M. et al.; “Zymomonas mobilis: a novel platform for future biorefineries”; Biotechnology for Biofuels, vol. 7, Article No. 101; 2014; 15 pages; DOI: https://doi.org/10.1186/1754-6834-7-101. [cited by applicant]
Hermans, M. et al.; “Content and composition of hopanoids in Zymomonas mobilis under various growth conditions”; Journal of Bacteriology, vol. 173, Issue No. 17; 1991; pp. 5592-5595. [cited by applicant]
Horbach, S. et al.; “Effect of azasqualene on hopanoid biosynthesis and ethanol tolerance of Zymomonas mobilis”; FEMS Microbiology Letters, vol. 79, Issue No. 2-3; 1991; pp. 347-350. [cited by applicant]
Jacobson, T. et al.; “2H and 13C metabolic flux analysis elucidates in vivo thermodynamics of the ED pathway in Zymomonas mobilis”; Metabolic Engineering, vol. 54; 2019; pp. 301-316; doi: 10.1016/j.ymben.2019.05.006. [cited by applicant]
Jost, M. et al.; “Titrating gene expression using libraries of systematically attenuated CRISPR guide RNAs”; Nature Biotechnology, vol. 38, Issue No. 3; 2020; pp. 355-364. [cited by applicant]
Kalnenieks, U. et al.; “Modeling of Zymomonas mobilis central metabolism for novel metabolic engineering strategies”; Frontiers in Microbiology, vol. 42; 2014; 7 pages; doi: 10.3389/fmicb.2014.00042. [cited by applicant]
Khan, S. et al.; “Broad-Host-Range Expression Vectors with Tightly Regulated Promoters and Their Use to Examine the Influence of TraR and TraM Expression on Ti Plasmid Quorum Sensing”; Applied and Environmental Microbio… [cited by applicant]
Kulkarni, G. et al.; “Specific hopanoid classes differentially affect free-living and symbiotic states of Bradyrhizobium diazoefficiens”; mBio, vol. 6, Issue No. 5; 2015; 9 pages; doi: 10.1128/mBio.01251-01215. [cited by applicant]
Lal, P. et al.; “Improving Mobilization of Foreign DNA into Zymomonas mobilis Strain ZM4 by Removal of Multiple Restriction Systems”; Applied and Environmental Microbiology, vol. 87, Issue No. 19; 2021; 16 pages; doi: 1… [cited by applicant]
Lee, K. et al.; “The genome-scale metabolic network analysis of Zymomonas mobilis ZM4 explains physiological features and suggests ethanol and succinic acid production strategies”; Microbial Cell Factories, vol. 9, Arti… [cited by applicant]
Liu, X. et al.; “High-throughput CRISPRi phenotyping identifies new essential genes in [cited by applicant]
Müh, U. et al.; “A Xylose-Inducible Expression System and a CRISPR Interference Plasmid for Targeted Knockdown of Gene Expression in Clostridioides difficile”; Journal of Bacteriology, vol. 201, Issue No. 14; 2019; 12 p… [cited by applicant]
Nouri, H. et al.; “A reconciliation of genome-scale metabolic network model of Zymomonas mobilis ZM4”; Scientific Reports, vol. 10, Article No. 7782; 2020; 11 pages. [cited by applicant]
Pédelacq, J-D. et al.; “Engineering and characterization of a superfolder green fluorescent protein”; Nature Biotechnology, vol. 24, Issue No. 1; 2006; pp. 79-88. [cited by applicant]
Peters, J. et al.; “A Comprehensive, CRISPR-based Functional Analysis of Essential Genes in Bacteria”; Cell, vol. 165; 2016; pp. 1493-1506. [cited by applicant]
Peters, J. et al.; “Enabling genetic analysis of diverse bacteria with Mobile-CRISPRi”; Nature Microbioogy, vol. 4; 2019; pp. 244-250. [cited by applicant]
Qi, L. et al.; “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression”; Cell, vol. 152; 2013; pp. 1173-1183. [cited by applicant]
Qiu, M. et al.; “Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production”; Biotechnology for Biofuels, vol. 13, Article No. 15; 2020; 14 pages; DOI: https://doi.org/10.1186/s13068-020-1654-x. [cited by applicant]
Qu, J. et al.; “Modulating pathogenesis with Mobile-CRISPRi”; Journal of Bacteriology, vol. 201, Issue No. 22; 2019; 9 pages; DOI: 10.1128/JB.00304-19. [cited by applicant]
Rath, D. et al.; “Efficient programmable gene silencing by Cascade”; Nucleic Acids Research, vol. 43; 2015; pp. 237-246. [cited by applicant]
Roberts, R. et al.; “REBASE—a database for DNA restriction and modification: enzymes, genes and genomes”; Nucleic Acids Research, vol. 43; 2015; pp. D298-D299. [cited by applicant]
Rousset, F. et al.; “Genome-wide CRISPR-dCas9 screens in [cited by applicant]
Sadler, J. et al.; “A perfectly symmetric lac operator binds the lac repressor very tightly”; PNAS USA, vol. 80, Issue No. 22; 1983; pp. 6785-6789. [cited by applicant]
Sáenz, J. et al.; “Hopanoids as functional analogues of cholesterol in bacterial membranes”; PNAS USA, vol. 112, Issue No. 38; 2015; pp. 11971-11976. [cited by applicant]
Schmerk, C. et al.; “Hopanoid production is required for low-pH tolerance, antimicrobial resistance, and motility in Burkholderia cenocepacia”; Journal of Bacteriology, vol. 193, Issue No. 23; 2011; pp. 6712-6723. [cited by applicant]
Silipo, A. et al.; “Covalently linked hopanoid-lipid A improves outer-membrane resistance of a Bradyrhizobium symbiont of legumes”; Nature Communications, vol. 5; 2014; 11 pages; doi: 10.1038/ncomms6106. [cited by applicant]
Skerker, J. et al.; “Dissecting a complex chemical stress: chemogenomic profiling of plant hydrolysates”; Molecular Systems Biology, vol. 9, Issue No. 674; 2013; 21 pages; doi: 10.1038/msb.2013.30. [cited by applicant]
Vigoroux, A. et al.; “CRISPR Tools to Control Gene Expression in Bacteria”; Microbiology and Molecular Biology Reviews, vol. 84, Issue No. 2; 2020; 18 pages; doi: 10.1128/MMBR.00077-19. [cited by applicant]
Wang, T. et al.; “Pooled CRISPR interference screening enables genome-scale functional genomics study in bacteria with superior performance”; Nature Communications, vol. 9, Article No. 2475; 2018; 15 pages; doi: https:/… [cited by applicant]
Wecker, M. et al.; “Production of Acetaldehyde by Zymomonas mobilis”; Applied Environmental Microbiology, vol. 53, Issue No. 12; 1987; pp. 2815-2820. [cited by applicant]
Welander, P. et al.; “Hopanoids play a role in membrane integrity and pH homeostasis in Rhodopseudomonas palustris TIE-1”; Journal of Bacteriology, vol. 191, Issue No. 19; 2009; pp. 6145-6156. [cited by applicant]
Wu, B. et al.; “Engineered Zymomonas mobilis tolerant to acetic acid and low pH via multiplex atmospheric and room temperature plasma mutagenesis”; Biotechnology for Biofuels, vol. 12, Article No. 10; 2019; 13 pages; DO… [cited by applicant]
Wu, C-H. et al.; “Methylation 453 at the C-2 position of hopanoids increases rigidity in native bacterial membranes”; eLife, vol. 4, e05663; 2015; doi: 10.7554/eLife.05663. [cited by applicant]
Yang, S. et al.; “Complete genome sequence and the expression pattern of plasmids of the model ethanologen Zymomonas mobilis ZM4 and its xylose-utilizing derivatives 8b and 2032”; Biotechnol biofuels, vol. 11; Article 1… [cited by applicant]
Yang, S. et al.; “Zymomonas mobilis as a model system for production of biofuels and biochemicals”; Microbial Biotechnology, vol. 9, Issue No. 6; 2016; pp. 699-717. [cited by applicant]
Yang, Y. et al.; “Prediction and characterization of promoters and ribosomal binding sites of Zymomonas mobilis in system biology era”; Biotechnology for Biofuels, vol. 12, Article No. 52; 2019; DOI: https://doi.org/10.… [cited by applicant]
Hawkins et al., “Mismatch-CRISPRi reveals the co-varying expression-fitness relationships of essential genes in [cited by applicant]
Jost, Marco et al. Titrating gene expression using libraries of systematically attenuated CRISPR guide RNAs, Nat Biotechnol. Mar. 2020 ; 38(3): 355-364. [cited by applicant]
Vigouroux, Antoine, et al. Tuning dCas9's ability to block transcription enables robust, noiseless knockdown of bacterial genes, Mol Syst Biol. (2018) 14: e7899, 1-14. [cited by applicant]
Hall et al., “Tools for Genetic Engineering and Gene Expression Control in [cited by applicant]