Methods and compositions for improved IE-type CRISPR-based gene silencing

Abstract

CRISPR-based interference has become common in a variety of applications, from genetic circuits to dynamic metabolic control. In Escherichia coli (E. coli), the natural CRISPR Cascade system can be harnessed for silencing by deletion of the cas3 nuclease along with the expression of guide RNA arrays, allowing for the silencing of multiple genes from a single transcript.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 990,172, filed March 16, 2020, which is incorporated by reference in its entirety.

[0002] Statement on Federally Sponsored Research This invention was made with Government support under 12043956 awarded by the Office of Naval Research and EE0007563 awarded by DOE EERE. The Government has certain rights in this invention.

[0003] Sequence Listing Reference This application contains a Sequence Listing, filed electronically in ASCII format as 49186-45_ST25.txt, created on March 10, 2020, which is 28,943 bytes in size and is incorporated by reference in its entirety. [Background technology]

[0004] Gene silencing is a powerful tool and CRISPR-based methods have increased the simplicity of this approach (Adli, M. The CRISPR tool kit for genome editing and beyond. Nat. Commun. 9, 1911 (2018)). In Escherichia coli (E. coli), the native multiprotein Cascade (type I CRISPR) system could be engineered for use in gene silencing, which required deletion of the nuclease components and overexpression of genes responsible for processing the CRISPR array and target DNA binding. One advantage of using the modified Cascade system is the targeting of multiple genes by expression of a single transcript containing multiple protospacers, which are then processed into individual guide RNAs (Luo, ML, Mullis, AS, Leenay, RT & Beisel, CL Repurposing endogenous type I CRISPR-Cas systems for programmable gene repression. Nucleic Acids Research vol. 43 674-681 (2015)). Summary of the Invention [Means for solving the problem]

[0005] CRISPR-based interference has become common in a variety of applications, from genetic circuits to dynamic metabolic control. Instability of the guide array mediated by Cas1 / 2 endonuclease has been identified as a challenge in some cases. In E. coli, the native CRISPR Cascade system can be utilized for silencing by deletion of the cas3 nuclease along with expression of guide RNA arrays, allowing silencing of multiple genes from a single transcript. [Brief description of the drawings]

[0006] [Figure 1A] FIG. 2 is a schematic diagram of a guide array. [Figure 1B]FIG. 13 shows an example of a protospacer defect in a guide array. [Figure 1C] FIG. 13. Protospacer modifications quantified by PCR. [Figure 1D] FIG. 1 shows stability of guide arrays as a function of guide array and host strain. [Figure 1E] FIG. 1 is a schematic diagram for complementing fabI silencing with pFABI. [Figure 1F] 1 is a graph showing the number of colonies. [Figure 1G] 1 is a graph showing guide arrays and stability of guide arrays by strains transformed with pFABI.

[0007] [Diagram 2] 1 shows an exemplary collection of plasmids of the present invention.

[0008] [Diagram 3] 1 shows exemplary strains of the present invention.

[0009] [Figure 4] An exemplary sgRNA guide sequence and primers are outlined for construction, with spacers in italics.

[0010] [Diagram 5] 1 shows an outline of an exemplary synthetic DNA of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] general definition

[0012] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an "expression vector" includes a single expression vector as well as multiple expression vectors, whether the same (e.g., the same operon), reference to a "microorganism" includes a single microorganism as well as multiple microorganisms, etc.

[0013] As used herein, the terms "heterologous DNA", "heterologous nucleic acid sequence", and the like refer to a nucleic acid sequence in which at least one of the following is true: (a) the nucleic acid sequence is foreign to a given host microorganism (i.e., not found in nature); (b) the sequence may be found in nature in a given host microorganism, but in a non-natural (e.g., greater than expected) amount; or (c) the nucleic acid sequence contains two or more subsequences that are not found in the same relationship to each other in nature. For example, with respect to example (c), a recombinantly produced heterologous nucleic acid sequence has two or more sequences from unrelated genes arranged to create a new functional nucleic acid, such as a non-natural promoter driving gene expression.

[0014] Species and other phylogenetic identifications are in accordance with classifications known to those skilled in the art of microbiology.

[0015] Enzymes are listed herein with reference to their UniProt identification numbers, which are well known to those skilled in the art. The UniProt database can be accessed at http: / / www.UniProt.org / . When a genetic modification of a gene product, i.e., an enzyme, is referred to herein, including in the claims, it is understood that the genetic modification is of the nucleic acid sequence, such as or including a gene, that normally codes for the given gene product, i.e., the enzyme.

[0016] Where methods and steps described herein indicate particular events occurring in a particular order, one of ordinary skill in the art will recognize that the particular order of steps may be varied and that such variations are in keeping with variations of the invention. Additionally, certain steps may be performed simultaneously in a parallel process where possible, or may be performed sequentially.

[0017] The meaning of the abbreviations is as follows: As is clear from the usage, "C" means Celsius or degrees, DCW means dry cell weight, "s" means second(s), "min" means minute(s), "h", "hr" or "hrs" means hour(s), "psi" means pounds per square inch, "nm" means nanometers, "d" means day(s), "μL" or "uL" or "ul" means psi, "nm" means nanometers, "d" means day(s), "μL" or "uL" or "ul" means psi, "nm" means nanometers, "d" means day(s), "μL" or "uL" or "ul" means psi, "nm" means nanometers, "d" means day(s), "nm" means psi, "nm" means nanometers ... "µM" means microliter(s), "mL" means milliliter(s), "L" means liter(s), "mm" means millimeter(s), "nm" means nanometer(s), "mM" means millimole, "µM" or "uM" means micromole, "M" means mole, "mmol" means millimole(s), "µmol" or "µMol" means micromole(s), "g" means gram(s), "μg" or "ug" means microgram(s), "ng" means nanogram(s), "PCR" means polymerase chain reaction, "OD" means optical density, "OD600" means optical density measured at a photon wavelength of 600 nm, "kDa" means kilodalton, "g" means the gravitational constant, "bp" means base pair(s), "kbp" means kilobase pair(s), "%w / v" means weight / volume percent, "%v / v" means volume / volume percent, "IPTG" means isopropyl-μ-D-thiogalactopyranoside, "aTc" means anhydrotetracycline, "RBS" means ribosome binding site, "rpm" means revolutions per minute, "HPLC" means high performance liquid chromatography, and "GC" means gas chromatography.

[0018] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0019] microorganisms The features described and claimed herein may be provided in a microorganism selected from the lists herein, or another suitable microorganism, which also includes one or more natural, introduced, or enhanced product bioproduction pathways. Thus, in some embodiments, the microorganism includes an endogenous product production pathway (which may, in some such embodiments, be enhanced), while in other embodiments, the microorganism does not include an endogenous product production pathway.

[0020] More specifically, based on the various criteria described herein, suitable microbial hosts for the biological production of chemical products can generally include, but are not limited to, the organisms described in the Methods section.

[0021] The host or source microorganism for any of the genes or proteins described herein may be selected from the following list of microorganisms: Citrobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus, Aspergillus, Saccharomyces, Schizosaccharomyces, Zygosaccharomyces, Lactobacillus subtilis ... Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia, Salmonella, Bacillus, Streptomyces, and Pseudomonas. In some embodiments, the host microorganism is an E. coli microorganism.

[0022] Summary of Invention Aspects Generally, unstable guide arrays are eliminated through the use of genetically modified microorganisms featuring a deleted or mutated endogenous cas3 nuclease, the Cascade operon is overexpressed, and at least one CRISPR / Cascade gRNA can be expressed to result in reduced expression of at least one gene.

[0023] In some embodiments, the microorganisms and any methods using the microorganisms may include the use of genetically modified microorganisms having a deletion or mutation of the endogenous cas1 gene.

[0024] In some embodiments, the microorganisms and any methods using the microorganisms may include the use of a genetically modified microorganism further characterized by a Cascade operon under the control of a PhoB-activated inducible promoter.

[0025] In some embodiments, the microorganisms and any methods using the microorganisms may include the use of genetically modified microorganisms that are Escherichia coli (E. coli) microorganisms.

[0026] In some embodiments, the microorganisms and any methods using the microorganisms may function to reduce expression of the genes fabI, gltA1, gltA2, udhA, zwf, or combinations thereof.

[0027] Detailed Description Unstable guide arrays can result from expression of the Cas1 / 2 endonuclease complex. Deletion of cas1 reduces the instability of the guide array. Constant Cas1 / 2 endonuclease activity leads to loss of protospacers from the guide array. The guide array can then become ineffective at silencing, which can be amplified by selection. Replacing the constitutive promoter driving Cascade complex expression with a tightly regulated inducible promoter improves the stability of the guide array.

[0028] A method of conditionally silencing genes in a genetically modified microorganism, comprising providing a genetically modified microorganism characterized by deletion or mutation of endogenous cas3 nuclease, Cascade operon, and at least one CRISPR / Cascade gRNA, also eliminates unstable guide arrays. The method comprises growing a genetically modified microorganism under conditions in which expression of CRISPR / Cascade gRNA reduces expression of at least one gene of the genetically modified microorganism. The microorganisms of the present invention and methods of using these microorganisms may include any combination of deletion or selective mutation of endogenous cas3 nuclease gene or conditional expression of Cascade operon. Either or both of these conditions results in increased stability of the guide array.

[0029] A guide array may contain a single gRNA that results in increased transcriptional silencing of a single gene upon conditional expression of the array. Alternatively, a guide array may contain two or more gRNAs that result in transcriptional silencing of two or more genes. A single guide array may contain means for simultaneously regulating one, two, three, four, five or more genes. Alternatively, a genetically modified microorganism may contain two or more guide arrays simultaneously, each of which may be conditionally expressed, resulting in transcriptional silencing of one or more genes.

[0030] In one embodiment, the method may include the use of genetically modified microorganisms with endogenous cas1 gene deletion or mutation. Cas1 gene deletion or mutation can be combined with both conditions to provide optimal guide array stability, which is combined with both endogenous cas3 nuclease gene deletion or selective mutation, or Cascade operon conditional expression. However, it is understood that any combination of these three factors (cas3 deletion / mutation, cas1 deletion / mutation, or conditional Cascade operon expression) will actually increase guide array stability.

[0031] Deletion or mutation of the cas3 and / or cas1 endogenous genes refers simply to any modification of the endogenous gene that makes it impossible to express this endogenous gene. The deletion or mutation may occur through modification of the gene regulatory sequence, or the coding sequence of the gene itself, or through other means that prevent the expression of a particular endogenous gene in a genetically modified microorganism.

[0032] The phrases conditionally express, conditionally overexpress, inducible promoter, or tightly repressed inducible promoter refer to the means of regulating gene expression.Gene expression can be conditionally regulated by introducing stimuli or removing necessary nutrients or other substances.Tightly repressed promoter sequence refers to the fact that while the promoter is under the repression conditions described, the regulation of gene expression does not occur strictly, and inducibility refers to the fact that the promoter can respond to externally applied signals.

[0033] A guide array refers to any configuration that allows for the expression of gRNAs specific to a target, in this case a gene that is transcriptionally silenced under specific conditions.

[0034] Another aspect of the present invention is described by comparing guide array expression in genetically modified microorganisms with any combination of deletion or selective mutation of the endogenous cas3 nuclease gene, or conditional expression of the Cascade operon, in contrast to genetically modified microorganisms lacking these features. These features serve to enhance the stability of the guide array and therefore the transcriptional gene silencing of the target gene.

[0035] In one embodiment, the method may include the use of a genetically modified microorganism further characterized by a Cascade operon under the control of a PhoB-activated inducible promoter. It is understood that any inducible promoter other than PhoB is encompassed by the present invention.

[0036] In one embodiment, the method may include the use of a genetically modified microorganism that is an E. coli microorganism. However, it is understood that the genes to be regulated, deleted or mutated, and the operons and guide arrays to be expressed are applicable to any known microorganism.

[0037] In one aspect, the method can function to reduce expression of genes that are fabI, gltA1, gltA2, udhA, zwf, or combinations thereof. These genes have been identified as candidates for gene regulation in the genetically modified microorganisms described herein, but it is understood that the method and microorganism is broadly applicable to any gene identified as desirable to be selectively regulated. For example,

[0038] example For the purposes of promoting an understanding of the principles of the present disclosure, reference will be made to preferred embodiments and specific language will be used to describe the same. Nonetheless, no limitation of the scope of the disclosure is intended thereby, and it will be understood that such modifications and further alterations of the disclosure as set forth herein are contemplated as would normally occur to one of ordinary skill in the art to which the disclosure pertains.

[0039] material and method

[0040] In Figure 2, exemplary plasmids of the invention are summarized. In Figure 3, exemplary microbial strains of the invention are summarized. In Figure 4, a list of exemplary sgRNA guide sequences and the primers used to construct them are summarized. In Figure 5, exemplary synthetic DNAs of the invention are summarized. Spacers are in italics.

[0041] Reagents and media: Unless otherwise stated, all materials and reagents were of the highest grade possible and were purchased from Sigma (St. Louis, MO). Low-salt Luria medium, Lennox formulation, was used for growth, construction and colony isolation of routine strains and plasmids. Chloramphenicol, ampicillin, and tetracycline were used at final working concentrations of 20 μg / mL, 100 μg / mL, and 5 μg / mL, respectively. Puromycin selection was performed using a final working concentration of 200 μg / mL with LB supplemented with 50 mM potassium phosphate buffer (pH = 8.0) to maintain the pH for proper selection.

[0042] Strains and Plasmids: The pCASCADE array plasmids were constructed using PCR assembly of smaller arrays as previously reported. For the pCASCADE plasmids constructed in this study, see Figures 2-5 for sequence and primer details. A plasmid, pFABI, was constructed to allow constitutive expression from the codon-optimized fabI gene using the strong synthetic EM7 promoter. Plasmid DNA containing the promoter and gene was obtained from Twist Biosciences (San Francisco, CA). E. cloni 10G strain was obtained from Lucigen. Strains DLF_Z0025, DLF_Z0045 and DLF_Z0047 were generated as previously reported. All strains generated in this study were constructed using standard recombination. The recombination plasmid pSIM5 and the tet-sacB selection / counterselection marker cassette were a kind gift from Donald Court (NCI, https: / / redrecombineering.ncifcrf.gov / court-lab.html). DLF_Z0047ΔsbcD::ampR was constructed by direct integration and gene replacement with linear donor DNA containing the appropriate antibiotic marker. The donor was prepared by PCR of a synthetic ampicillin resistance cassette (ampR2) with primers del_sbcD_p1 and del_sbcD_p2.DLF_Z0047 and recA1::ampR was constructed similarly, but the integration incorporated a G160D mutation into the recA gene rather than a deletion. Strains DLF_Z0047Δcas1::purR and DLF_Z0047Δcas2::purR were constructed by direct integration and gene replacement with linear donor DNA. Strains DLF_S0047 and DLF_S0025 were constructed from DLF_Z0047 and DLF_Z0025, respectively, using reassortment and tet-sacB-based selection-counterselection to replace the sspB gene and promoter in front of the Cascade operon. All genetic modifications were confirmed by PCR and sequencing. Sequencing was performed by either Genewiz (Morrisville, NC) or Eurofins (Louisville, KY).Plasmid transformation was accomplished using standard methods.

[0043] Guide stability testing: Plasmid DNA minipreps and sequencing were performed using standard methods. The following two primers were used to amplify the guide array from the pCASCADE plasmid gRNA-forward: 5'-GGGAGACCACAACGG-3' (SEQ ID NO: 60), gRNA-reverse: 5'-CGCAGTCGAACGACCG-3' (SEQ ID NO: 61). Colony PCR was performed as follows: 2X EconoTaq Master mix (Lucigen) was used in a 10 μL PCR reaction consisting of 5 μL of 2X EconoTaq Master mix (Lucigen), 1 μL of each primer (10 uM concentration), 3 μL of dH2O and a small portion of a colony. PCR parameters were an initial denaturation at 98°C for 2 minutes, followed by 35 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, with a final extension at 72°C for 5 minutes. PCR products were then analyzed by agarose gel electrophoresis.

[0044] Referring now to Figure 1A-1B, we sequenced the first few guide array plasmids suspected to be guide deficient. As an example (Figure 1A), we transformed the guide array plasmid containing the protospacer into a host strain (DLF_Z0047) engineered with degron tags capable of proteolysis of FabI (enoyl-ACP reductase), GltA (citrate synthase) and UdhA (soluble transhydrogenase) to silence the gltAp1 (G1), gltAp2 (G2) and udhA (U) promoters. A single colony was selected and used to inoculate a 5 mL culture (Luria medium), and after overnight growth, the culture was plated to isolate single colonies. 24 clones were isolated and the guide array plasmid was mini-prepared and sequenced. Seventeen plasmids had the expected sequence and retained all three protospacers (Fig. 1B, top sequences), whereas the other seven had mutations that deleted the two protospacers (G1 and U) adjacent to the central protospacer G2. Four of these modified clones retained both the 5' and 3' flanking repeat sequences, whereas the other three also deleted either the 5' or 3' repeat sequences adjacent to the G2 protospacer.

[0045] As shown in Figure 1C, the next step was to evaluate the stability of a single guide (G2) and three additional guide arrays: FG2, FG1G2 and FG1G2U, where "F" is a protospacer targeting the fabI promoter. Again, strain DLF_Z0047 was used, again starting with a single colony used to inoculate a 5 mL culture (Luria medium). After overnight growth, the cultures were plated to isolate single colonies. In this case, four clones were isolated from each of the four cultures and colony PCR rather than sequencing was utilized to assess the stability of the guide arrays. The results are shown in Figure 1C. In this case, the single G2 guide proved stable, but larger arrays of two to four protospacers produced amplicons with differing degrees of instability, consistent with the loss of one to three protospacers.

[0046] Referring now to FIG. 1D, due to the success of PCR as a tool to assess stability, the next step in the assessment of guide array stability for a larger assortment of guide arrays in several different host strains was investigated. These included F, G1, G2 and U protospacers, as well as protospacer Z targeting the zwf promoter Z. Strains evaluated included E. cloni 10G, a commercially available recA1 cloning strain (Lucigen), as well as DLF_Z0025, a control host utilized for two-stage dynamic metabolic control lacking proteolytic degron tags on any metabolic enzymes, DLF_Z0045, DLF_Z0047 (FGU as described above), which has degron tags for GltA and UDhA, as well as a derivative of DLF_Z0047 containing a recA1 mutant (recAG160D), an sbCD gene deletion (a component of the SbcCD endonuclease that recognizes hairpin and palindromic sequences present in the guide array) and deletions of cas1 and cas2. The results are shown in FIG. 1D.

[0047] The guide arrays were stable in the cloning strains. This result was not unexpected as these constructs were originally constructed using E. cloni 10G and the original plasmids without protospacer deletions were confirmed by sequencing. Protospacer deletions were first noted in DLF_Z0025 for a small group of arrays. DLF_Z0025 has been modified for constitutive expression of the Cascade operon (Figure 1A). Increased instability was detected in host strains DLF_Z0045 and DLF_Z0047. Neither integration of the recA1 mutation nor deletion of sbcD reduced the protospacer deletion in the DLF_Z0047 background. In the case of recA, this is consistent with previous studies showing that only 20 bp of homology allows recombination in E. coli, but more than 50 bp of homologous sequence is required for significant recombination, with the protospacer being 30 bp long. In contrast, deletion of cas1 or cas2 improves array stability, with deletion of cas1 having minimal protospacer deletion.

[0048] The results of the cas1 deletion are consistent with Cas1 / 2 endonuclease being responsible for protospacer loss, with Cas1 being the nuclease component. This activity is consistent with previously reported activity in protospacer acquisition. The fact that very low protospacer loss was still observed with the Cas1 mutation indicates the possibility of a second alternative mechanism of protospacer loss, or an imprecision in our PCR assay. However, as seen in Figure 1D, guide arrays containing F protospacers were significantly less stable than those that did not. This protospacer specificity is not consistent with a generalized endonuclease activity, and F-containing arrays have an increased propensity for protospacer loss, but it prompts further investigation.

[0049] FabI may be a strictly essential enzyme, and despite the fact that the guide array is under inducible expression, leaky expression would result in growth inhibition, and the guide array lacking the F protospacer would have a selective advantage in strains where the Cascade operon (including cas1 and cas2) is overexpressed. This is also consistent with the general observation that transformation of guide array plasmids with F protospacers results in lower colony numbers than other arrays. We constructed a plasmid (pFABI, Figure 1E) that allows expression of FabI from an alternative constitutive promoter that is not silenced by the F protospacer. We then evaluated the impact of co-transformation of pFABI with guide array plasmids containing the F protospacer on colony numbers as well as array stability. As seen in Figures 1F-1G, co-transformation of pFABI increased colony numbers as well as array stability. These data are consistent with growth inhibition due to leaky silencing of fabI, and therefore with a selective advantage for arrays lacking the F protospacer.

[0050] Taken together, the results discussed above support a model in which steady-state Cas1 / 2 endonuclease activity leads to the deletion of protospacers from guide arrays. Silencing arrays with protospacers targeting essential genes may result in growth inhibition due to leaky expression of guides when the Cascade operon is overexpressed, even slightly. Arrays lacking toxic protospacers can be amplified by selection in routine culture. There are several options to improve the stability of the arrays. First, simply deleting cas1 should improve stability, and gene silencing should be unaffected since Cas1 is not required for the silencing function of the Cascade operon. This approach would require two modifications to future silencing strains, deletion of cas3 and cas1 (Figure 1A). However, in light of the toxicity observed in the case of constant fabI silencing, we also evaluated a second option, in which we deleted cas3 and expressed the Cascade operon using a tightly regulated hypophosphate-inducible promoter rather than the originally reported constitutive promoter (Biobrick J23100). To implement and test this approach, we constructed DLF_S0047, identical to DLF_Z0047, containing degron tags on FabI, GltA and UdhA, but the constitutive J23100 promoter (Figure 1A) was replaced by a tightly regulated hypophosphate-inducible modified yibD gene promoter preceded by a strong synthetic transcriptional tZ terminator. As seen in Figure 1D, the instability of the array was eliminated using DLF_S0047. We also constructed DLF_S0025 as a new stable strain for future engineering for dynamic metabolic control.

[0051] The use of Cascade for CRISPR interference would benefit from tighter control of expression of the Cascade operon (cas1 / 2), if not deletion of cas1 / 2, or at least assessment of guide stability.

[0052] The disclosed embodiments are non-limiting Although various embodiments of the present invention have been shown and described herein, it is emphasized that such embodiments are provided by way of example only. Numerous variations, modifications, and substitutions can be made in the various embodiments thereof without departing from the invention herein. In particular, for any classification or other classification of compounds, nucleic acid sequences, functional enzymes, polypeptides, elements, or other compositions, including specific proteins, including metabolic pathway enzymes or intermediates, described or otherwise presented herein in a list, table, or other classification, unless otherwise specified for any reason, each such classification is intended to provide a basis for and serve to identify various subset embodiments, the broadest scope of which includes all subsets of such classifications by excluding one or more members (or subsets) of each described classification. Furthermore, when any range is described herein, unless otherwise specified, the range includes all values ​​therein and all subranges therein.

[0053] Also, more generally, in accordance with the disclosure, discussion, examples and embodiments herein, conventional molecular biology, cell biology, microbiology, and recombinant DNA techniques within the skill of the art may be employed. Such techniques are fully explained in the literature. See, for example, Sambrook and Russell, "Molecular Cloning: A Laboratory Manual," Third Edition 2001 (volumes 1-3), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Animal Cell Culture, RIFreshney, ed., 1986. These published sources are incorporated herein by reference.

[0054] The following published sources of information are incorporated herein by reference for explanatory purposes useful in conjunction with the invention described herein, e.g., methods for the industrial bioproduction of chemical products from sugar sources, and also industrial systems that can be used to achieve such conversions (for bioreactor design, see Biochemical Engineering Fundamentals, 2006). nd Ed. JE Bailey and DF Allis, McGraw Hill, New York, 1986, e.g., Chapter 9, pages 533-657; for analysis of process and separation techniques, see, e.g., Unit Operations of Chemical Engineering, 5 th Ed., WLMcCabe et al., McGraw Hill, New York 1993; for teachings of separation techniques, see, e.g., Equilibrium Staged Separations, PC Wankat, Prentice Hall, Englewood Cliffs, NJ USA, 1988).

[0055] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety. [Sequence List Free Text]

[0056] Sequence Listings 1-8 <223> Synthetic guide array constructs Sequence Listing 9 <223> Synthetic sgRNA guide sequences Sequence Tables 10-11 <223> Synthetic primer Sequence Listing 12 <223> Synthetic sgRNA guide sequences Sequence Tables 13-16 <223> Synthetic primer Sequence Listing 17 <223> Synthetic sgRNA guide sequences Sequence Table 18-21 <223> Synthetic primer Sequence Listing 22 <223> Synthetic sgRNA guide sequences Sequence List 23-26 <223> Synthetic primer Sequence Listing 27 <223> Synthetic sgRNA guide sequences Sequence List 28-31 <223> Synthetic primer Sequence Listing 32 <223> Synthetic sgRNA guide sequences Sequence List 33-36 <223> Synthetic primer Sequence Listing 37 <223> Synthetic sgRNA guide sequences Sequence List 38-41 <223> Synthetic primer Sequence Listing 42 <223> Synthetic sgRNA guide sequences Sequence list 43-46 <223> Synthetic primer Sequence Listing 47 <223> Synthetic sgRNA guide sequences Sequence list 48-51 <223> Synthetic primer Sequence Table 52-54 <223> Synthetic plasmids Sequence List 55-56 <223> Synthetic sequences Sequence Listing 57-59 <223> Synthetic plasmids Sequence table 60-61 <223> synthesis

Claims

1. A genetically modified microorganism, comprising: Deletion or mutation of the endogenous cas3 nuclease gene and the endogenous cas1 gene; A Cascade operon operably linked to a promoter that allows for conditional overexpression of the Cascade operon; and A guide array in which at least one CRISPR / Cascade gRNA can be conditionally expressed, wherein expression of the guide array results in reduced expression of at least one gene; A genetically modified microorganism characterized by improved stability of the guide array when compared to a genetically modified microorganism lacking a deletion or mutation of the endogenous cas3 nuclease or conditional expression of the Cascade operon.

2. 2. The genetically modified microorganism of claim 1, wherein the Cascade operon is overexpressed under the control of a tightly repressed inducible promoter.

3. 3. The genetically modified microorganism of claim 2, wherein the tightly repressed inducible promoter is PhoB activated.

4. The genetically modified microorganism of claim 1 , wherein the genetically modified microorganism is an E. coli microorganism.

5. 2. The genetically modified microorganism of claim 1, wherein the expression-reduced gene is selected from the group consisting of fabI, gltA1, gltA2, udhA, and zwf.

6. 1. A method for conditionally silencing a gene in a genetically modified microorganism, comprising: Deletion or mutation of the endogenous cas3 nuclease gene and the endogenous cas1 gene; Cascade operon; and At least one guide array comprising at least one CRISPR / Cascade gRNA To provide a genetically modified microorganism, Growing the genetically modified microorganism under conditions in which expression of the CRISPR / Cascade gRNA results in reduced expression of at least one gene of the genetically modified microorganism; A method comprising:

7. 7. The method of claim 6, wherein the genetically modified microorganism is further characterized by a Cascade operon under the control of a PhoB-activated inducible promoter.

8. 7. The method of claim 6, wherein the genetically modified microorganism is an E. coli microorganism or the under-expressed genes are selected from the group consisting of fabI, gltA1, gltA2, udhA, and zwf.

Images