WO2018067846A1 - Methods of crispr mediated genome modulation in v. natriegens - Google Patents

Methods of crispr mediated genome modulation in v. natriegens Download PDF

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WO2018067846A1
WO2018067846A1 PCT/US2017/055386 US2017055386W WO2018067846A1 WO 2018067846 A1 WO2018067846 A1 WO 2018067846A1 US 2017055386 W US2017055386 W US 2017055386W WO 2018067846 A1 WO2018067846 A1 WO 2018067846A1
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nucleic acid
cell
method
acid sequence
natriegens
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George M. Church
Henry Hung-yi LEE
Nili OSTROV
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President And Fellows Of Harvard College
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Publication of WO2018067846A1 publication Critical patent/WO2018067846A1/en

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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
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    • C12N15/90Stable introduction of foreign DNA into chromosome
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    • C12N2795/00Bacteriophages
    • C12N2795/00011Bacteriophages
    • C12N2795/00041Use of virus, viral particle or viral elements as a vector
    • C12N2795/00043Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Abstract

Methods and compositions are provided for modulating expression of a target nucleic acid sequence within a non-E. coli cell. The method includes providing the cell with a guide RNA comprising a portion that is complementary to all or a portion of the target nucleic acid sequence, and providing the cell a Cas protein, wherein the guide RNA and the Cas protein co-localize at the target nucleic acid sequence and wherein the Cas protein modulate the expression of the target nucleic acid sequence.

Description

METHODS OF CRISPR MEDIATED GENOME MODULATION IN V.

NATRIEGENS RELATED APPLICATION DATA

This application claims priority to U.S. Provisional Application No. 62/404,518 filed on October 5, 2016 and U.S. Provisional Application No. 62/455,668 filed on February 7, 2017 which are hereby incorporated herein by reference in its entirety for all purposes. STATEMENT OF GOVERNMENT INTERESTS

This invention was made with government support under Grant No. DE-FG02- 02ER63445 from the United States Department of Energy. The government has certain rights in the invention. FIELD

The present invention relates in general to methods of genome modulation in the organism V. natrigens, such as by using CRISPR system. BACKGROUND

Methods of genome modulation are known and have been carried out in E. coli, S. enterica, Pseudomonas putida KT2440, Pseudomonas syringae, Pseudomonas aerginosa, Y. pseudotuberculosis, M. tuberculosis, S. cerevisiae and a growing number of organisms. SUMMARY

According to one aspect, the present disclosure provides a method of altering a target nucleic acid sequence within a non-E. coli cell including providing a cell with a functioning beta-like recombinase and a donor nucleic acid sequence, wherein the donor nucleic acid sequence is inserted into the target nucleic acid sequence as a result of the functioning beta- recombinase. In one embodiment, the present disclosure provides that the non-E. coli cell is Vibrio natriegens. In one embodiment, the present disclosure provides that the beta-like recombinase is identified in a horizontal gene transfer element such as a phage. In another embodiment, the present disclosure provides that the beta-like recombinase is identified in a horizontal gene transfer element such as an Integrative and Conjugative Element (ICE). In one embodiment, the present disclosure provides that the beta-like recombinase is identified in a horizontal gene transfer element such as a conjugative plasmid. In another embodiment, the present disclosure provides that the beta-like recombinase is identified in a horizontal gene transfer element such as a Vibrio spp. phage. In yet another embodiment, the present disclosure provides that the beta-like recombinase is identified in a horizontal gene transfer element such as a Vibrio spp. Integrative and Conjugative Element (ICE). In one embodiment, the present disclosure provides that the beta-like recombinase is s065. In one embodiment, the present disclosure provides that additional recombination assisting proteins are provided to the cell. In another embodiment, the present disclosure provides that additional recombination assisting proteins are provided to the cell including the exonuclease s066, and a host nuclease inhibitor such as gam, and a single-strand DNA binding (SSB) protein s064 (Uniprot: A0A0X1L3H7). In yet another embodiment, the present disclosure provides that additional recombination assisting proteins are provided to the cell including s066, and gam to create a single-stranded intermediate from a double stranded nucleic acid donor. In one embodiment, the present disclosure provides that the donor nucleic acid sequence is introduced into the cell as a single stranded nucleic acid. In one embodiment, the present disclosure provides that the donor nucleic acid sequence is introduced into the cell as a double stranded nucleic acid. In one embodiment, the present disclosure provides that the cell has been genetically modified to include a foreign nucleic acid sequence encoding the recombinase. In another embodiment, the present disclosure provides that the cell has been genetically modified to include a foreign nucleic acid sequence encoding the recombinase, exonuclease, and host nuclease inhibitor. In yet another embodiment, the present disclosure provides that the cell has been genetically modified to include a foreign nucleic acid sequence encoding the s065, exonuclease, and host nuclease inhibitor. In still another embodiment, the present disclosure provides that the cell has been genetically modified to include a foreign nucleic acid sequence encoding the s065, s066, and host nuclease inhibitor. In one embodiment, the present disclosure provides that the cell has been genetically modified to include a foreign nucleic acid sequence encoding the s065, s066, and gam. In one embodiment, the present disclosure provides that the donor nucleic acid sequence is provided to the cell by electroporation.

According to another aspect, the present disclosure provides a method of altering a target nucleic acid sequence within a Vibrio natriegens cell including providing the Vibrio natriegens cell with a functioning beta-like recombinase and a donor nucleic acid sequence, wherein the donor nucleic acid sequence is inserted into the target nucleic acid sequence as a result of the functioning beta-recombinase.

In one embodiment, the present disclosure provides that the beta-like recombinase is identified in a horizontal gene transfer element such as a phage. In another embodiment, the present disclosure provides that the beta-like recombinase is identified in a horizontal gene transfer element such as an Integrative and Conjugative Element (ICE). In one embodiment, the present disclosure provides that the beta-like recombinase is identified in a horizontal gene transfer element such as a conjugative plasmid. In another embodiment, the present disclosure provides that the beta-like recombinase is identified in a horizontal gene transfer element such as a Vibrio spp. phage. In yet another embodiment, the present disclosure provides that the beta-like recombinase is identified in a horizontal gene transfer element such as a Vibrio spp. Integrative and Conjugative Element (ICE). In one embodiment, the present disclosure provides that the beta-like recombinase is s065. In one embodiment, the present disclosure provides that additional recombination assisting proteins are provided to the cell. In another embodiment, the present disclosure provides that additional recombination assisting proteins are provided to the cell including the exonuclease s066, a host nuclease inhibitor such as gam, and SSB. In yet another embodiment, the present disclosure provides that additional recombination assisting proteins are provided to the cell including s066, and gam to create a single-stranded intermediate from a double stranded nucleic acid donor. In one embodiment, the present disclosure provides that the donor nucleic acid sequence is introduced into the cell as a single stranded nucleic acid. In one embodiment, the present disclosure provides that the donor nucleic acid sequence is introduced into the cell as a double stranded nucleic acid. In one embodiment, the present disclosure provides that the cell has been genetically modified to include a foreign nucleic acid sequence encoding the recombinase. In another embodiment, the present disclosure provides that the cell has been genetically modified to include a foreign nucleic acid sequence encoding the recombinase, exonuclease, host nuclease inhibitor, and SSB. In yet another embodiment, the present disclosure provides that the cell has been genetically modified to include a foreign nucleic acid sequence encoding the s065, exonuclease, host nuclease inhibitor, and SSB. In still another embodiment, the present disclosure provides that the cell has been genetically modified to include a foreign nucleic acid sequence encoding the s065, s066, s064, and host nuclease inhibitor. In one embodiment, the present disclosure provides that the cell has been genetically modified to include a foreign nucleic acid sequence encoding the s065, s066, s064, and gam. In one embodiment, the present disclosure provides that the donor nucleic acid sequence is provided to the cell by electroporation.

According to another aspect, the present disclosure provides a method of altering a target nucleic acid sequence within a Vibrio natriegens cell including providing the Vibrio natriegens cell with a functioning s065 recombinase and a donor nucleic acid sequence, wherein the donor nucleic acid sequence is inserted into the target nucleic acid sequence as a result of the functioning s065.

In one embodiment, the present disclosure provides that additional recombination assisting proteins are provided to the cell. In another embodiment, the present disclosure provides that additional recombination assisting proteins are provided to the cell including the exonuclease s066, and a host nuclease inhibitor such as gam. In yet another embodiment, the present disclosure provides that additional recombination assisting proteins are provided to the cell including s066, and gam to create a single-stranded intermediate from a double stranded nucleic acid donor. In one embodiment, the present disclosure provides that the donor nucleic acid sequence is introduced into the cell as a single stranded nucleic acid. In one embodiment, the present disclosure provides that the donor nucleic acid sequence is introduced into the cell as a double stranded nucleic acid. In one embodiment, the present disclosure provides that the cell has been genetically modified to include a foreign nucleic acid sequence encoding the recombinase. In another embodiment, the present disclosure provides that the cell has been genetically modified to include a foreign nucleic acid sequence encoding the recombinase, exonuclease, and host nuclease inhibitor. In yet another embodiment, the present disclosure provides that the cell has been genetically modified to include a foreign nucleic acid sequence encoding the s065, exonuclease, host nuclease inhibitor, and SSB. In still another embodiment, the present disclosure provides that the cell has been genetically modified to include a foreign nucleic acid sequence encoding the s065, s066, s064, and host nuclease inhibitor. In one embodiment, the present disclosure provides that the cell has been genetically modified to include a foreign nucleic acid sequence encoding the s065, s066, s064, and gam. In one embodiment, the present disclosure provides that the donor nucleic acid sequence is provided to the cell by electroporation.

According to another aspect, the present disclosure provides a genetically modified Vibrio natriegens cell comprising a foreign nucleic acid sequence encoding a beta-like recombinase.

In one embodiment, the present disclosure provides that the beta-like recombinase is s065. In another embodiment, the present disclosure provides that the genetically modified Vibrio natriegens cell further includes a foreign donor nucleic acid sequence. In yet another embodiment, the present disclosure provides that the genetically modified Vibrio natriegens cell further includes a foreign donor nucleic acid sequence inserted into plasmid or genomic DNA within the Vibrio natriegens cell.

According to one aspect, the present disclosure provides a method of modulating expression of a target nucleic acid sequence within a non-E. coli cell. The method includes providing the cell with a guide RNA comprising a portion that is complementary to all or a portion of the target nucleic acid sequence, providing the cell a Cas protein, wherein the guide RNA and the Cas protein co-localize at the target nucleic acid sequence and wherein the Cas protein modulate the expression of the target nucleic acid sequence.

According to another aspect, the present disclosure provides a method of altering a target nucleic acid sequence within a non-E. coli cell. The method include providing the cell with a guide RNA comprising a portion that is complementary to all or a portion of the target nucleic acid sequence, providing the cell a Cas protein, and providing the cell a donor nucleic acid sequence, wherein the guide RNA and the Cas protein co-localize at the target nucleic acid sequence, wherein the Cas protein cleaves the target nucleic acid sequence and the donor nucleic acid sequence is inserted into the target nucleic acid sequence in a site specific manner.

In some embodiments, the non-E. coli cell is Vibrio natriegens. In some embodiments, the Cas protein is a Cas9 protein. In other embodiments, the Cas9 is a Cas9 nickase or a nuclease null Cas9 (dCas9). In some embodiments, the Cas9 is further fused with a transcription repressor or activator. In other embodiments, the guide RNA and/or Cas protein are provided on a vector. In one embodiment, the vector is a plasmid. In some embodiments, a plurality of guide RNAs that are complementary to different target nucleic acid sequences are provided to the cell and wherein expressions of different target nucleic acid sequences are modulated. In certain embodiments, expression of Cas protein is inducible. In some embodiments, the cell has been genetically modified to include a foreign nucleic acid sequence. In some embodiments, the foreign nucleic acid sequence encodes a reporter protein. In one embodiment, the reporter protein is GFP. In some embodiments, the providing step comprising providing nucleic acid sequences encoding the guide RNA and/or the Cas protein to the cell by transfection or electroporation. In other embodiments, the guide RNA, Cas protein and donor nucleic acid sequence are provided on a vector. In one embodiment, the vector is a plasmid. In some embodiments, the guide RNA, Cas protein and donor nucleic acid sequence are provided on plasmids and provided to the cell by electroporation. In some embodiments, the donor nucleic acid sequence is introduced into the cell as a single stranded nucleic acid. In other embodiments, the donor nucleic acid sequence is introduced into the cell as a double stranded nucleic acid. According to another aspect, the present disclosure provides a nucleic acid construct. In one embodiment, the nucleic acid construct encodes a guide RNA comprising a portion that is complementary to a target nucleic acid sequence in Vibrio natriegens. In another embodiment, the nucleic acid construct encodes a Cas protein. In yet another embodiment, the nucleic acid construct encodes a donor nucleic acid sequence for insertion into a target nucleic acid sequence in Vibrio natriegens.

According to another aspect, the present disclosure provides a non-E. coli cell. In one embodiment, the cell comprises a guide RNA comprising a portion that is complementary to all or a portion of the target nucleic acid sequence, and a Cas protein, wherein the guide RNA and the Cas protein co-localize at the target nucleic acid sequence and modulates the expression of the target nucleic acid sequence in the cell. In another embodiment, the cell comprises a guide RNA comprising a portion that is complementary to all or a portion of the target nucleic acid sequence, a Cas protein, and a donor nucleic acid sequence, wherein the guide RNA and the Cas protein co-localize at the target nucleic acid sequence, wherein the Cas protein cleaves the target nucleic acid sequence and the donor nucleic acid sequence is inserted into the target nucleic acid sequence in a site specific manner. In one embodiment, the non-E. coli cell is Vibrio natriegens.

According to one aspect, the present disclosure provides a method of improving the growth rate of a non-E. coli cell by suppressing the expression of a target gene of the non-E. coli cell. In certain embodiments, a plurality of target gene expression is suppressed. In one embodiment, the expression of the target gene is suppressed by transcriptional repression. In another embodiment, the expression of the target gene is suppressed by mutagenization of the target gene. In yet another embodiment, the expression of the target gene is suppressed by providing the cell with a guide RNA comprising a portion that is complementary to all or a portion of a gene sequence, and providing the cell a Cas protein, wherein the guide RNA and the Cas protein co-localize at the gene sequence and suppress the target gene expression. In one embodiment, the non-E. coli cell is Vibrio natriegens. In one embodiment, the Cas protein is a Cas9 protein. In other embodiments, the Cas9 is a Cas9 nickase or a nuclease null Cas9 (dCas9). In certain embodiment, the Cas9 is further fused with a transcription repressor. In one embodimnet, the guide RNA and Cas protein are each provided to the cell via a vector comprising nucleic acid encoding the guide RNA and the Cas protein. In one embodiment, the vector is a plasmid. In some embodiments, a plurality of guide RNAs that are complementary to different gene sequences are provided to the cell and wherein expressions of different target genes are suppressed. In certain embodiments expression of Cas protein is inducible. In one embodiment, the providing step comprising providing nucleic acid sequences encoding the guide RNA and the Cas protein to the cell by transfection or electroporation. In some embodiments, the target gene comprises genes in Table 3. In other embodiments, the target gene comprises ATP-dependent DNA helicase RecQ, N-acyl-L- amino acid amidohydrolase, a hypothetical protein fused to ribosomal protein S6 glutaminyl transferase, ABC transporter2C periplasmic spermidine putrescine-binding protein PotD, a putative protease, Na+/H+ antiporter NhaP, methyl-accepting chemotaxis protein, transporter2C putative, biotin synthesis protein BioC, alkaline serine protease, glutamate aspartate transport system permease protein GltJ, thiamin ABC transporter2C transmembrane component, or putrescine utilization regulator. In some embodiments, the guide RNA includes complementary sequences in Table 4 for use in target gene suppression.

Further features and advantages of certain embodiments of the present disclosure will become more fully apparent in the following description of the embodiments and drawings thereof, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The foregoing and other features and advantages of the present embodiments will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which:

Fig. 1 is a graph depicting data regarding resistant colonies as a result of recombineering of single-stranded oligonucleotides in V. natriegens using λ-Beta and SXT s065. The single-stranded oligonucleotide reverts a spectinomycin with a premature stop codon into a functional spectinomycin gene on plasmid.

Fig. 2 is a graph depicting data regarding resistant colonies as a result of recombineering with s065 and oligonucleotides targeting the forward (leading strand) or reverse (lagging strand) of DNA replication. A single-stranded oligonucleotide recombines with a spectinomycin gene, on a plasmid, with a premature stop codon to convert it into a functional spectinomycin gene.

Fig. 3 is a graph depicting data regarding resistant colonies as a result of recombineering based on the amount of oligonucleotide where an increased oligo amount used for s065-mediated recombination in V. natriegens. A single-stranded oligonucleotide recombines with a spectinomycin gene, on a plasmid, with a premature stop codon to convert it into a functional spectinomycin gene.

Fig. 4 is a graph depicting data regarding resistant colonies as a result of recombineering based on the number of phosphorothioates on the oligonucleotide added to enhance stability of the oligonucleotides in vivo. A single-stranded oligonucleotide recombines with a spectinomycin gene, on a plasmid, with a premature stop codon to convert it into a functional spectinomycin gene.

Fig. 5 depicts results of recombination on a chromosome and information as a result of Sanger sequencing of V. natriegens pyrF mutant colonies isolated by 5-FOA selection following ssDNA oligonucleotide recombination. The single-stranded oligonucleotide introduces a premature stop codon into the chromosomally encoded pyrF gene.

Fig. 6 depicts results of gene deletion by insertion of a double-stranded DNA cassette carrying an antibiotic marker with flanking homology arms into the V. natriegens genome using proteins s065 and s066 from SXT.

Figs. 7A-7B depict results of titration of Vibrio natriegens induction systems. Fig. 7A depicts the result of induction of the lactose promoter by IPTG. Fig. 7B depicts the result of induction of the arabinose promoter by Larabinose. Data are shown as mean±SD (N^3).

Fig. 8 depicts the result of targeted gene inhibition of chromosomally integrated GFP in Vibrio natriegens using dCas9 according to an embodiment of the present disclosure. Guide RNA (gRNA) were designed to target the template or nontemplate strand of GFP. Data are shown as mean±SD (N^3).

Fig. 9 is a graph depicting the temperature at which electroporation of plasmids in V. natriegens is performed.“Cold” temperature is 4°C for electroporation.“Room temperature” is 25°C for electroporation.

Figs. 10A-C depict quantifying V. natriegens generation time in rich and glucose- supplemented minimal media across a broad range of temperatures. Fig. 10A depicts bulk growth measurements of V. natriegens and E. coli across various temperatures (in LB3 and LB, respectively). M9 for V. natriegens was supplemented with 2% (w/v) NaCl. Glucose (0.4% w/v final) was used as a carbon source. Data shown are mean±SD (N=24). Fig. 10B depicts single-cell growth rate measurement based on conditions Fig. 10C. Data shown are mean±SD (N^12). Fig. 10C depicts representative time course images of V. natriegens (top, LB3 media) and E. coli (bottom, LB media) growing at 37°C for 93 minutes. Images were taken at 100x magnification. Figs. 11A-B depict V. natriegens genome and replication dynamics. Fig. 11A depicts two circular chromosomes are depicted. From outside inward: two outer circles represent protein-coding genes on the plus and minus strand, respectively, color coded by RAST annotation. The third circle represents G+C content relative to mean G+C content of the respective chromosome, using a sliding window of 3,000 bp. tRNA and rRNA genes are shown in the fourth and fifth circles, respectively. Below, the percentage of each RAST category relative to all annotated genes. Fig. 11B depicts filtered sequence coverage (black) and GC-skew (green) for each chromosome, as measured for exponentially growing V. natriegens in LB3 at 37°C. Origin (red) and terminus (blue) are denoted.

Figs. 12A-G depict fitness profiling of all protein-coding genes in V. natriegens by CRISPRi. Fig. 12A depicts schematics of pooled CRISPRi screen. Distribution of relative fitness (RF) is shown for passage one and passage three of competitively grown cultures (gray, dCas9 with guides; white, guides only). Fig. 12B depicts relative fitness of V. natriegens genes after passage three. Genes that are essential for fast growth (1070 genes total) are highlighted: essentials (purple, 604 genes, RF≤ 0.529, p≤ 0.001, non-parametric) are determined after passage one. Genes specifically required for fast growth (gold, 466 genes, RF≤ 0.781, p≤ 0.05, non-parametric) are determined after passage three. Fig. 12C depicts relative fitness of V. natriegens genes after passage one. Ribosomal genes (black). Essential genes denoted by dotted boxed region. Fig. 12D depicts overlap of putative essential V. natriegens genes with essentials found in E. coli and V. cholerae. Fig. 12E depicts relative fitness of ribosomal proteins, in the absence (open circles) or presence of V. natriegens expressing dCas9 (closed circles). Filled grey square indicates essentiality in V. natriegens (Vn, current study), V. cholerae (Vc) or E. coli (Ec). Fig. 12F depicts RAST categories for essential and fast growth gene sets. Number of essential (purple) and fast growing (gold) genes are shown out of all annotated V. natriegens genes (white). Asterisks indicates statistical enrichment (p < 0.05, BH-adjusted). Fold increase in each RAST category between fast growth subset and essentials (black circles). Fig. 12G depicts spatial distribution of essential genes (outer circle, purple) and genes required for fast growth (inner circle, gold) on V. natriegens chromosomes.

Fig. 13 depicts plasmid transformation in V. natriegens. Bright field (left) and fluorescence images (right) of V. natriegens colonies transformed with plasmids carrying the following replicons (a) colE1 (b) SC101 (c) RSF1010. All plasmid carry constitutive GFP expression cassette pLtetO-GFP.

Figs. 14A-G depict optimization of DNA transformation. Fig. 14A depicts cell viability in sorbitol, used as an osmoprotectant (representative data). Transformation efficiencies were optimized for the following criteria: (Fig. 14B) Voltage. (Fig. 14C) Recovery media. (Fig. 14D) Amount of input plasmid DNA. (Fig. 14E) Recovery time. (Fig. 14F) competent cell storage: transformation efficiencies of electrocompetent cells stored at - 80°C over time. (day 0: freshly prepared electrocompetent cells). Unless otherwise indicated, transformations were performed using 50ng plasmid DNA with recovery time of 45min at 37°C in SOC3 media. Data are shown as mean±SD (N≥2). Fig. 14G depicts rapid DNA amplification in V. natriegens. Single colonies of V. natriegens or E. coli were used to inoculate 3mL liquid LB3 or LB, respectively. Cultures were grown for 5 hours at 37°C and plasmid DNA was extracted and quantified. Data are shown as mean±SD (N^3).

Figs. 15A-C depict CTX bacteriophage replication and infectivity. Fig. 15A depicts V. natriegens transformants of CTX-Km RF (left) and recombinant vector, pRST, carrying the replicative CTX origin (right). Fig. 15B depicts transduction of V. natriegens (left) and V. cholerae O395 (right) by CTX-KmVcΦ bacteriophage produced by V. cholerae O395. Fig. 15C depicts transduction of V. natriegens (left) and V. cholerae O395 (right) by CTX-KmVnΦ bacteriophage produced by V. natriegens. Figs. 16A-C depict establishing CRISPR/Cas9 functionality in V. natriegens. Fig. 16A depicts nuclease activity of Cas9. Guide-dependent lethality was observed upon cutting of chromosomal targets. Data are shown as mean±SD (N^3). Colonies of V. natriegens with chromosomal integration of GFP were not detected (N.D.) when Cas9 and a GFP-targeting guide was coexpressed. Fig. 16B depicts dCas9 inhibition of chromosomally-integrated GFP. Guide RNAs (gRNAs) were designed to target the template (T) or non-template (NT) strand of GFP proximal to the transcriptional start site. Data are shown as mean±SD (N^3). Greater inhibition observed when targeting the non-template (NT, >13-fold) over template (T, 3.7- fold) strand, in line with previous reports (Qi, L. S. et al. Repurposing CRISPR as an RNA- guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013)). Significant GFP repression was observed without induction, indicating basal expression of dCas9. To maximize consistency in subsequent experiments, further experiments thus used the presence and absence of dCas9 in lieu of induction. Fig. 16C depicts a small scale pooled CRISPRi screen. CRISPRi assay in wild-type V. natriegens expressing dCas9 was performed by co-targeting five genes: growth-neutral genes (flgCVn flagellar subunit and two for GFP), putative essential genes (lptFVn, an essential gene in E. coli critical for the lipopolysaccharide transport system), and a negative control (the E.coli sequence for gene lptFEc). The pooled cell library was grown as a single batch culture under competitive growth conditions at 37°C, and gRNA abundance was quantified by sequencing at several time points. Fold change for each target is computed as the normalized gRNA abundance to reads per million and expressed as a ratio relative to initial conditions.

Depletion was only observed for the putative essential V. natriegens gene (lptFVn), demonstrating specificity and sensitivity of this pooled screen. These data establishes CRISPR in V. natriegens and illustrates the utility of a pooled CRISPRi screen. Fig. 17 depicts distribution of relative fitness scores for all V. natriegens protein- coding genes, as generated by pooled CRISPRi screen. Control (-dCas9) shown in green, inhibition assay (+dCas9) shown in blue. Data shown for three serial passages.

Fig. 18 depicts growth rates of various V. natriegens. The figure shows the time in minutes it takes for various strains to grow to exponential phase (optical density measured at 600nm of ~0.2).

DETAILED DESCRIPTION

Aspects of the present disclosure are directed to recombineering methods in non-E. coli microbes, such as Vibrio natriegens. Aspects of the present disclosure are directed to the use of one or more recombinases for recombineering methods in non-E. coli microbes, such as Vibrio natriegens. Aspects of the present disclosure utilize recombineering materials and methods known to those of skill in the art. Recombineering or recombination-mediated genetic engineering is a genetic and molecular biology technique that utilizes the recombination system of a cell, such as homologous recombination. Materials and methods useful for recombineering are described in Ellis, H. M., D. Yu, T. DiTizio & D. L. Court, (2001) High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc. Natl. Acad. Sci. USA 98: 6742-6746; Lajoie, M.J. et al., 2013. Genomically recoded organisms expand biological functions. Science, 342(6156), pp.357–360; Wang, H.H. et al. Programming cells by multiplex genome engineering and accelerated evolution. Nature 460, 894-898 (2009); Thomason, L.C. et al., 2014. Recombineering: genetic engineering in bacteria using homologous recombination. Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.], 106, pp.1.16.1– 39; Hmelo, L.R. et al., 2015. Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange. Nature protocols, 10(11), pp.1820–1841; Luo, X. et al., 2016. Pseudomonas putida KT2440 markerless gene deletion using a combination of λ Red recombineering and Cre/loxP site-specific recombination. FEMS microbiology letters, 363(4). Available at: http://dx.doi.org/10.1093/femsle/fnw014; and Swingle, B. et al., 2010. Recombineering Using RecTE from Pseudomonas syringae. Applied and environmental microbiology, 76(15), pp.4960–4968 each of which are hereby incorporated by reference in its entirety.

In E. coli, expression of λ Red Beta (also referred to as β or bet), a recombinase protein found on the λ-phage genome, potentiates recombineering by ~10,000-fold as described in Yu, D. et al., 2000. An efficient recombination system for chromosome engineering in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America, 97(11), pp. 5978–5983 hereby incorporated by reference in its entirety. Aspects of the present disclosure are directed to the identification and use of recombinases that can be used in non-E. coli organisms, such V. natriegens.

According to one aspect, recombinases may be identified for their ability to function in a recombineering method. Exemplary recombinases include those known as s065. See Chen et al., BMC Molecular Biology (2011) 12:16 hereby incorporated by reference in its entirety. The SXT mobile genetic element was originally isolated from an emerging epidemic strain of Vibrio cholerae (serogroup O139), which causes the severe diarrheal disease cholera. Formerly referred to as a conjugative transposon, SXT is now classified as being a type of integrating conjugative element (ICE). The SXT genome contains three consecutive coding DNA sequences (CDSs; s064, s065 and s066) arranged in an operon-like structure, which encode homologues of 'phage-like' proteins involved in DNA recombination. The encoded S064 protein (SXT-Ssb) is highly homologous to bacterial single strand DNA (ssDNA) binding proteins (Ssb); S065 (SXT-Bet) is homologous to the Bet single stranded annealing protein (SSAP) from bacteriophage lambda (lambda-Bet, which is also referred to as a DNA synaptase or recombinase); and S066 (SXT-Exo) shares homology with the lambda Exo/YqaJ family of alkaline exonucleases.

Aspects of the present disclosure are directed to the use of one or more recombinases to promote DNA recombination within V. natriegens. According to one aspect, exemplary recombinases include s065, beta (lambda) which is the alkaline exonuclease from bacteriophage lambda, which themselves are capable of promoting single-stranded DNA recombination with oligonucleotides. According to one aspect, exemplary helper proteins include s066, exo (lambda), an exonuclease from bacteriophage lambda, and gam (lambda), a host-nuclease inhibitor protein from bacteriophage lambda, as well as single-strand DNA binding protein such as s064 which are required for stabilization and recombination of single and double-stranded DNA. Aspects of the present disclosure are directed to methods of using s065, beta (lambda) or lambda recombinases, s066, s064, and gam to promote genetic recombination of the V. natriegens genomic DNA, i.e. between single stranded oligonucleotides and the V. natriegens genomic DNA, i.e. chromosomal DNA.

Vibrio natriegens (previously Pseudomonas natriegens and Beneckea natriegens) is a Gram negative, nonpathogenic marine bacterium isolated from salt marshes. It is purported to be one of the fastest growing organisms known with a generation time between 7 to 10 minutes. According to one aspect, Vibrio natriegens is characterized, cultured and utilized for genetic engineering methods as described in bioRxiv (June 12, 2016) doi: http://dx.doi.org/10.1101/058487 hereby incorporated by reference in its entirety. Vibrio natriegens includes two chromosomes of 3,248,023 bp and 1,927,310 bp that together encode 4,578 open reading frames. Vibrio natriegens may be genetically modified using tranformation protocols and compatible plasmids, such as a plasmid based on the RSF1010 operon, or a phage such as vibriophage CTX. Transformation of Vibrio natriegens with the CTX-Km RF yielded transformants which suggests that the CTX replicon is compatible in this host. A new plasmid, pRST, was constructed by fusing the specific replication genes from CTX-Km RF to a Escherichia coli plasmid based on the conditionally replicating R6k origin, thus adding a lowcopy shuttle vector to the list of available genetic tools for Vibrio natriegens. This plasmid may be used in combination with the pRSF plasmid as a dual plasmid system in Vibrio natriegens for complex regulation of proteins and high-throughput manipulation of diverse DNA libraries.

Aspects of the present disclosure are directed to methods of recombineering in non-E. coli organisms, such as V. natriegens using beta-like recombinases. An exemplary beta-like recombinases is s065 from the SXT mobile element found in Vibrio cholerae. See Beaber, J.W., Hochhut, B. & Waldor, M.K., 2002. Genomic and functional analyses of SXT, an integrating antibiotic resistance gene transfer element derived from Vibrio cholerae. Journal of bacteriology, 184(15), pp.4259–4269 hereby incorporated by reference in its entirety.

Aspects of the present disclosure are directed to recombineering methods using linear DNA substrates that are either double-stranded (dsDNA) or single-stranded (ssDNA). Aspects of the present disclosure are directed to recombineering methods using a double- stranded DNA (dsDNA) cassette. Aspects of the present disclosure are directed to methods as described herein of recombineering of plasmid borne DNA with single stranded oligonucleotides. Aspects of the present disclosure are directed to methods as described herein of recombineering of chromosomal DNA with single stranded oligonucleotides. Aspects of the present disclosure are directed to methods as described herein of recombineering of chromosomal DNA with a double-stranded DNA cassette. According to certain aspects, s065 is used as a recombinase in the recombineering methods. According to certain aspects, Vibrio natriegens is used as the organism or cell. According to certain aspects, the methods may include the use of other components, proteins or enzymes in a recombineering system, expressed from their respective genes or otherwise provided such as s066 from SXT with or without the protein gam expressed from λ-phage.

Aspects of the present disclosure are directed to recombineering methods used to create gene replacements, deletions, insertions, and inversions, as well as, gene cloning and gene/protein tagging (His-tags etc.) For gene replacements or deletions, aspects may utilize a cassette encoding a drug-resistance gene, such as one that is made by PCR using bi-partite primers. These primers consist of (from 5’^3’) 50 bases of homology to the target region, where the cassette is to be inserted, followed by 20 bases to prime the drug resistant cassette. The exact junction sequence of the final construct is determined by primer design. Methods to provide a cell with a nucleic acid, whether single stranded or double stranded or other genetic element are known to those of skill in the art and include electroporation. Selection and counterselection techniques are known to those of skill in the art.

The present disclosure provides methods of recombineering to perform knock-out and knock-in of genes in V. natriegens to create mutants with desired characteristics. For example, deletion of genes that catabolize DNA result in V. natriegens mutants that have improved plasmid yield and stability as described in Weinstock, M.T. et al., 2016. Vibrio natriegens as a fast-growing host for molecular biology. Nature methods, 13(10), pp.849–851 hereby incorporated by refernece in its entirety.

The present disclosure provides methods of performing multiplex oligo recombination (MAGE or multiplex automated genome engineering as is known in the art) using recombineering for accelerated evolution in V. natriegens as described in Wang, H.H. et al., 2009. Programming cells by multiplex genome engineering and accelerated evolution. Nature, 460(7257), pp.894–898 hereby incorporated by reference in its entirety.

The present disclosure provides methods for using recombineering to optimize metabolic pathways as described in Wang, H.H. et al., 2009. Programming cells by multiplex genome engineering and accelerated evolution. Nature, 460(7257), pp.894–898 hereby incorporated by reference in its entirety.

The present disclosure provides methods for using recombineering to recode V. natriegens genome for virus resistance, incorporation of nonstandard amino acids, and genetic isolation as described in Ma, N.J. & Isaacs, F.J., 2016. Genomic Recoding Broadly Obstructs the Propagation of Horizontally Transferred Genetic Elements. Cell systems, 3(2), pp.199–207; Ostrov, N. et al., 2016. Design, synthesis, and testing toward a 57-codon genome. Science, 353(6301), pp.819–822; and Lajoie, M.J. et al., 2013. Genomically recoded organisms expand biological functions. Science, 342(6156), pp.357–360 each of which are hereby incorporated by reference in its entirety.

According to certain aspects, recombineering components or proteins for carrying out recombineering methods in V. natriegens as described herein may be provided on a plasmid (trans) or integrated into the chromosome (cis) to create a variety of recombineering V. natriegens strains, such as those found for recombineering E. coli strains as described in world wide website redrecombineering.ncifcrf.gov/strains--plasmids.html.

Recombineering methods as described herein may be carried out using a basic protocol of growing cultures or cells such as by overnight culturing; subculturing cells in desired growth media; inducing production of recombinase within the cell or cells or providing the cell or cells with a recombinase; and introducing the single strand DNA or double strand DNA into the cell or cells, whereby the recombinase promotes recombination of the single-stranded DNA or double-stranded DNA into target DNA within the cell or cells.

According to current understanding of the recombinase mediated recombination as herein described, Beta binds single-stranded DNA (ssDNA) donor and single-stranded binding proteins in the host to facilitate homing of the single-stranded DNA donor to its homologous region in the target DNA. This single-stranded DNA donor anneals as an Okazaki fragment of DNA replication, and is incorporated into the genome during cell replication. According to the present disclosure, Beta is a phage protein and its natural function is to operate during phage (vs. bacterial) replication. When lambda phages infect a cell, they insert linear DNA, and in lytic replication this DNA is then circularized and replicated as a circular genome (first as theta-replication, then through rolling-circle). In order to make the circular form, the linear DNA from the initial insertion (and from cut concatemers from rolling-circle) have a repeated“cos” sequence at each end, and these sequences are rendered single stranded so that the two ends can hybridize and form a circle (“cos” ends =“cohesive” ends). Without intending to be bound by scientific theory, Beta may operate to help anneal these single-stranded cos ends. In recombineering, this capacity of Beta is used in a non-natural context– to help anneal oligos to the lagging strand during bacterial replication. (See, Thomason, L.C. et al., 2014, Recombineering: genetic engineering in bacteria using homologous recombination, Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.], 106, pp.1.16.1–39;

Sharan, S.K. et al., 2009, Recombineering: a homologous recombination-based method of genetic engineering, Nature protocols, 4(2), pp.206–223; Hirano, N. et al., 2011, Site-specific recombinases as tools for heterologous gene integration, Applied microbiology and biotechnology, 92(2), pp.227–239; Mosberg, J.A., Lajoie, M.J. & Church, G.M., 2010, Lambda red recombineering in Escherichia coli occurs through a fully single-stranded intermediate, Genetics, 186(3), pp.791–799, hereby incorporated by reference in their entireties).

According to the present disclosure, it is shown that s065 performs better than Beta for recombination in Vibrio natriegens. Recombination can also be performed with a double- stranded DNA donor, as detailed herein. This requires at least one additional protein, Exo, which is thought to digest the double-stranded DNA into a single-strand which recombines as detailed herein. Expression of the protein, Gam, inhibits endogenous digestion of this donor DNA. According to the present disclosure, it is shown that s066 and gam, in addition to s065, mediate the double-stranded recombination. Without intending to be bound by scientific theory, the improved performance of s065 is likely due to its molecular interactions with the single-stranded binding proteins in Vibrio natriegens. The fast growth rate is an attractive feature of working with Vibrio natriegens, but likely not directly responsible for s065 recombination.

According to the present disclosure, s065 is for single-stranded DNA recombination in V. natriegens for both DNA on plasmids and DNA on the chromosome. According the present disclosure, optimizing the single-stranded DNA oligos in the following way improves recombination with s065: a. the oligos are 90 base pairs long, b. the oligos target the lagging strand of DNA replication, c. the oligos are added at >100uM for electroporation, and d. the oligos are protected by multiple phosphorothioate bonds.

According to the present disclosure, s066 + gam (in addition to s065) is for double- stranded DNA recombination. The double-stranded DNA is protected by phosphorothioates at one or both 5' ends. ( See, J. A. Mosberg, M. J. Lajoie, G. M. Church, Lambda Red Recombineering in Escherichia coli Occurs Through a Fully Single-Stranded Intermediate, Genetics, November 1, 2010 vol.186 no.3, 791-799, hereby incorporated by reference in its entirety).

According to one exemplary aspect, electrocuvettes are provided with up to 5uL of DNA (>= 50uM of single-stranded DNA oligo and about 1ug of double-stranded DNA oligo with 500bp homology arms) and are placed on ice. Cells are washed in 1M cold sucrose or sorbitol, and cells are concentrated 200x by volume. Electroporation is carried out with the following settings: 0.4kV, 1kΩ, 25uF; time constants may be >12ms, The cells are recovered from the electrocuvette in rich media. The cells are plated and incubated for colony formation.

CAS9 DESCRIPTION

RNA guided DNA binding proteins are readily known to those of skill in the art to bind to DNA for various purposes. Such DNA binding proteins may be naturally occurring. DNA binding proteins having nuclease activity are known to those of skill in the art, and include naturally occurring DNA binding proteins having nuclease activity, such as Cas9 proteins present, for example, in Type II CRISPR systems. Such Cas9 proteins and Type II CRISPR systems are well documented in the art. See Makarova et al., Nature Reviews, Microbiology, Vol.9, June 2011, pp.467-477 including all supplementary information hereby incorporated by reference in its entirety.

In general, bacterial and archaeal CRISPR-Cas systems rely on short guide RNAs in complex with Cas proteins to direct degradation of complementary sequences present within invading foreign nucleic acid. See Deltcheva, E. et al. CRISPR RNA maturation by trans- encoded small RNA and host factor RNase III. Nature 471, 602-607 (2011); Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proceedings of the National Academy of Sciences of the United States of America 109, E2579-2586 (2012); Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821 (2012); Sapranauskas, R. et al. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic acids research 39, 9275- 9282 (2011); and Bhaya, D., Davison, M. & Barrangou, R. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annual review of genetics 45, 273-297 (2011). A recent in vitro reconstitution of the S. pyogenes type II CRISPR system demonstrated that crRNA (“CRISPR RNA”) fused to a normally trans- encoded tracrRNA (“trans-activating CRISPR RNA”) is sufficient to direct Cas9 protein to sequence-specifically cleave target DNA sequences matching the crRNA. Expressing a gRNA homologous to a target site results in Cas9 recruitment and degradation of the target DNA. See H. Deveau et al., Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. Journal of Bacteriology 190, 1390 (Feb, 2008).

Three classes of CRISPR systems are generally known and are referred to as Type I, Type II or Type III). According to one aspect, a particular useful enzyme according to the present disclosure to cleave dsDNA is the single effector enzyme, Cas9, common to Type II. See K. S. Makarova et al., Evolution and classification of the CRISPR-Cas systems. Nature reviews. Microbiology 9, 467 (Jun, 2011) hereby incorporated by reference in its entirety. Within bacteria, the Type II effector system consists of a long pre-crRNA transcribed from the spacer-containing CRISPR locus, the multifunctional Cas9 protein, and a tracrRNA important for gRNA processing. The tracrRNAs hybridize to the repeat regions separating the spacers of the pre-crRNA, initiating dsRNA cleavage by endogenous RNase III, which is followed by a second cleavage event within each spacer by Cas9, producing mature crRNAs that remain associated with the tracrRNA and Cas9. TracrRNA-crRNA fusions are contemplated for use in the present methods.

According to one aspect, the enzyme of the present disclosure, such as Cas9 unwinds the DNA duplex and searches for sequences matching the crRNA to cleave. Target recognition occurs upon detection of complementarity between a“protospacer” sequence in the target DNA and the remaining spacer sequence in the crRNA. Importantly, Cas9 cuts the DNA only if a correct protospacer-adjacent motif (PAM) is also present at the 3’ end.

According to certain aspects, different protospacer-adjacent motif can be utilized. For example, the S. pyogenes system requires an NGG sequence, where N can be any nucleotide. S. thermophilus Type II systems require NGGNG (see P. Horvath, R. Barrangou, CRISPR/Cas, the immune system of bacteria and archaea. Science 327, 167 (Jan 8, 2010) hereby incorporated by reference in its entirety and NNAGAAW (see H. Deveau et al., Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. Journal of bacteriology 190, 1390 (Feb, 2008) hereby incorporated by reference in its entirety), respectively, while different S. mutans systems tolerate NGG or NAAR (see J. R. van der Ploeg, Analysis of CRISPR in Streptococcus mutans suggests frequent occurrence of acquired immunity against infection by M102-like bacteriophages. Microbiology 155, 1966 (Jun, 2009) hereby incorporated by refernece in its entirety. Bioinformatic analyses have generated extensive databases of CRISPR loci in a variety of bacteria that may serve to identify additional useful PAMs and expand the set of CRISPR-targetable sequences (see M. Rho, Y. W. Wu, H. Tang, T. G. Doak, Y. Ye, Diverse CRISPRs evolving in human microbiomes. PLoS genetics 8, e1002441 (2012) and D. T. Pride et al., Analysis of streptococcal CRISPRs from human saliva reveals substantial sequence diversity within and between subjects over time. Genome research 21, 126 (Jan, 2011) each of which are hereby incorporated by reference in their entireties.

In S. pyogenes, Cas9 generates a blunt-ended double-stranded break 3bp upstream of the protospacer-adjacent motif (PAM) via a process mediated by two catalytic domains in the protein: an HNH domain that cleaves the complementary strand of the DNA and a RuvC-like domain that cleaves the non-complementary strand. See Jinek et al., Science 337, 816-821 (2012) hereby incorporated by reference in its entirety. Cas9 proteins are known to exist in many Type II CRISPR systems including the following as identified in the supplementary information to Makarova et al., Nature Reviews, Microbiology, Vol.9, June 2011, pp.467- 477: Methanococcus maripaludis C7; Corynebacterium diphtheriae; Corynebacterium efficiens YS-314; Corynebacterium glutamicum ATCC 13032 Kitasato; Corynebacterium glutamicum ATCC 13032 Bielefeld; Corynebacterium glutamicum R; Corynebacterium kroppenstedtii DSM 44385; Mycobacterium abscessus ATCC 19977; Nocardia farcinica IFM10152; Rhodococcus erythropolis PR4; Rhodococcus jostii RHA1; Rhodococcus opacus B4 uid36573; Acidothermus cellulolyticus 11B; Arthrobacter chlorophenolicus A6; Kribbella flavida DSM 17836 uid43465; Thermomonospora curvata DSM 43183; Bifidobacterium dentium Bd1; Bifidobacterium longum DJO10A; Slackia heliotrinireducens DSM 20476; Persephonella marina EX H1; Bacteroides fragilis NCTC 9434; Capnocytophaga ochracea DSM 7271; Flavobacterium psychrophilum JIP0286; Akkermansia muciniphila ATCC BAA 835; Roseiflexus castenholzii DSM 13941; Roseiflexus RS1; Synechocystis PCC6803; Elusimicrobium minutum Pei191; uncultured Termite group 1 bacterium phylotype Rs D17; Fibrobacter succinogenes S85; Bacillus cereus ATCC 10987; Listeria innocua;Lactobacillus casei; Lactobacillus rhamnosus GG; Lactobacillus salivarius UCC118; Streptococcus agalactiae A909; Streptococcus agalactiae NEM316; Streptococcus agalactiae 2603;

Streptococcus dysgalactiae equisimilis GGS 124; Streptococcus equi zooepidemicus MGCS10565; Streptococcus gallolyticus UCN34 uid46061; Streptococcus gordonii Challis subst CH1; Streptococcus mutans NN2025 uid46353; Streptococcus mutans; Streptococcus pyogenes M1 GAS; Streptococcus pyogenes MGAS5005; Streptococcus pyogenes

MGAS2096; Streptococcus pyogenes MGAS9429; Streptococcus pyogenes MGAS10270; Streptococcus pyogenes MGAS6180; Streptococcus pyogenes MGAS315; Streptococcus pyogenes SSI-1; Streptococcus pyogenes MGAS10750; Streptococcus pyogenes NZ131; Streptococcus thermophiles CNRZ1066; Streptococcus thermophiles LMD-9; Streptococcus thermophiles LMG 18311; Clostridium botulinum A3 Loch Maree; Clostridium botulinum B Eklund 17B; Clostridium botulinum Ba4657; Clostridium botulinum F Langeland;

Clostridium cellulolyticum H10; Finegoldia magna ATCC 29328; Eubacterium rectale ATCC 33656; Mycoplasma gallisepticum; Mycoplasma mobile 163K; Mycoplasma penetrans; Mycoplasma synoviae 53; Streptobacillus moniliformis DSM 12112; Bradyrhizobium BTAi1; Nitrobacter hamburgensis X14; Rhodopseudomonas palustris BisB18; Rhodopseudomonas palustris BisB5; Parvibaculum lavamentivorans DS-1;

Dinoroseobacter shibae DFL 12; Gluconacetobacter diazotrophicus Pal 5 FAPERJ;

Gluconacetobacter diazotrophicus Pal 5 JGI; Azospirillum B510 uid46085; Rhodospirillum rubrum ATCC 11170; Diaphorobacter TPSY uid29975; Verminephrobacter eiseniae EF01-2; Neisseria meningitides 053442; Neisseria meningitides alpha14; Neisseria meningitides Z2491; Desulfovibrio salexigens DSM 2638; Campylobacter jejuni doylei 26997;

Campylobacter jejuni 81116; Campylobacter jejuni; Campylobacter lari RM2100;

Helicobacter hepaticus; Wolinella succinogenes; Tolumonas auensis DSM 9187;

Pseudoalteromonas atlantica T6c; Shewanella pealeana ATCC 700345; Legionella pneumophila Paris; Actinobacillus succinogenes 130Z; Pasteurella multocida; Francisella tularensis novicida U112; Francisella tularensis holarctica; Francisella tularensis FSC 198; Francisella tularensis tularensis; Francisella tularensis WY96-3418; and Treponema denticola ATCC 35405. The Cas9 protein may be referred by one of skill in the art in the literature as Csn1. An exemplary S. pyogenes Cas9 protein sequence is provided in Deltcheva et al., Nature 471, 602-607 (2011) hereby incorporated by reference in its entirety.

Modification to the Cas9 protein is contemplated by the present disclosure. CRISPR systems useful in the present disclosure are described in R. Barrangou, P. Horvath, CRISPR: new horizons in phage resistance and strain identification. Annual review of food science and technology 3, 143 (2012) and B. Wiedenheft, S. H. Sternberg, J. A. Doudna, RNA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331 (Feb 16, 2012) each of which are hereby incorporated by reference in their entireties.

According to certain aspects, the DNA binding protein is altered or otherwise modified to inactivate the nuclease activity. Such alteration or modification includes altering one or more amino acids to inactivate the nuclease activity or the nuclease domain. Such modification includes removing the polypeptide sequence or polypeptide sequences exhibiting nuclease activity, i.e. the nuclease domain, such that the polypeptide sequence or polypeptide sequences exhibiting nuclease activity, i.e. nuclease domain, are absent from the DNA binding protein. Other modifications to inactivate nuclease activity will be readily apparent to one of skill in the art based on the present disclosure. Accordingly, a nuclease- null DNA binding protein includes polypeptide sequences modified to inactivate nuclease activity or removal of a polypeptide sequence or sequences to inactivate nuclease activity. The nuclease-null DNA binding protein retains the ability to bind to DNA even though the nuclease activity has been inactivated. Accordingly, the DNA binding protein includes the polypeptide sequence or sequences required for DNA binding but may lack the one or more or all of the nuclease sequences exhibiting nuclease activity. Accordingly, the DNA binding protein includes the polypeptide sequence or sequences required for DNA binding but may have one or more or all of the nuclease sequences exhibiting nuclease activity inactivated.

According to one aspect, a DNA binding protein having two or more nuclease domains may be modified or altered to inactivate all but one of the nuclease domains. Such a modified or altered DNA binding protein is referred to as a DNA binding protein nickase, to the extent that the DNA binding protein cuts or nicks only one strand of double stranded DNA. When guided by RNA to DNA, the DNA binding protein nickase is referred to as an RNA guided DNA binding protein nickase. An exemplary DNA binding protein is an RNA guided DNA binding protein nuclease of a Type II CRISPR System, such as a Cas9 protein or modified Cas9 or homolog of Cas9. An exemplary DNA binding protein is a Cas9 protein nickase. An exemplary DNA binding protein is an RNA guided DNA binding protein of a Type II CRISPR System which lacks nuclease activity. An exemplary DNA binding protein is a nuclease-null or nuclease deficient Cas9 protein. According to an additional aspect, nuclease-null Cas9 proteins are provided where one or more amino acids in Cas9 are altered or otherwise removed to provide nuclease-null Cas9 proteins. According to one aspect, the amino acids include D10 and H840. See Jinek et al., Science 337, 816-821 (2012). According to an additional aspect, the amino acids include D839 and N863. According to one aspect, one or more or all of D10, H840, D839 and H863 are substituted with an amino acid which reduces, substantially eliminates or eliminates nuclease activity. According to one aspect, one or more or all of D10, H840, D839 and H863 are substituted with alanine. According to one aspect, a Cas9 protein having one or more or all of D10, H840, D839 and H863 substituted with an amino acid which reduces, substantially eliminates or eliminates nuclease activity, such as alanine, is referred to as a nuclease-null Cas9 (“Cas9Nuc”) and exhibits reduced or eliminated nuclease activity, or nuclease activity is absent or substantially absent within levels of detection. According to this aspect, nuclease activity for a Cas9Nuc may be undetectable using known assays, i.e. below the level of detection of known assays.

According to one aspect, the Cas9 protein, Cas9 protein nickase or nuclease null Cas9 includes homologs and orthologs thereof which retain the ability of the protein to bind to the DNA and be guided by the RNA. According to one aspect, the Cas9 protein includes the sequence as set forth for naturally occurring Cas9 from S. thermophiles or S. pyogenes and protein sequences having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% homology thereto and being a DNA binding protein, such as an RNA guided DNA binding protein.

An exemplary CRISPR system includes the S. thermophiles Cas9 nuclease (ST1 Cas9) (see Esvelt KM, et al., Orthogonal Cas9 proteins for RNA-guided gene regulation and editing, Nature Methods., (2013) hereby incorporated by reference in its entirety).An exemplary CRISPR system includes the S. pyogenes Cas9 nuclease (Sp. Cas9), an extremely high-affinity (see Sternberg, S.H., Redding, S., Jinek, M., Greene, E.C. & Doudna, J.A. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62-67 (2014) hereby incorporated by reference in its entirety), programmable DNA-binding protein isolated from a type II CRISPR-associated system (see Garneau, J.E. et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468, 67-71 (2010) and Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821 (2012) each of which are hereby incorporated by reference in its entirety). According to certain aspects, a nuclease null or nuclease deficient Cas 9 can be used in the methods described herein. Such nuclease null or nuclease deficient Cas9 proteins are described in Gilbert, L.A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442-451 (2013); Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature biotechnology 31, 833-838 (2013); Maeder, M.L. et al. CRISPR RNA-guided activation of endogenous human genes. Nature methods 10, 977-979 (2013); and Perez-Pinera, P. et al. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nature methods 10, 973-976 (2013) each of which are hereby incorporated by reference in its entirety. The DNA locus targeted by Cas9 (and by its nuclease-deficient mutant,“dCas9” precedes a three nucleotide (nt) 5´-NGG-3´“PAM” sequence, and matches a 15–22-nt guide or spacer sequence within a Cas9-bound RNA cofactor, referred to herein and in the art as a guide RNA. Altering this guide RNA is sufficient to target Cas9 or a nuclease deficient Cas9 to a target nucleic acid. In a multitude of CRISPR-based biotechnology applications (see Mali, P., Esvelt, K.M. & Church, G.M. Cas9 as a versatile tool for engineering biology. Nature methods 10, 957-963 (2013); Hsu, P.D., Lander, E.S. & Zhang, F. Development and Applications of CRISPR-Cas9 for Genome Engineering. Cell 157, 1262- 1278 (2014); Chen, B. et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479-1491 (2013); Shalem, O. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84-87 (2014); Wang, T., Wei, J.J., Sabatini, D.M. & Lander, E.S. Genetic screens in human cells using the CRISPR- Cas9 system. Science 343, 80-84 (2014); Nissim, L., Perli, S.D., Fridkin, A., Perez-Pinera, P. & Lu, T.K. Multiplexed and Programmable Regulation of Gene Networks with an Integrated RNA and CRISPR/Cas Toolkit in Human Cells. Molecular cell 54, 698-710 (2014); Ryan, O.W. et al. Selection of chromosomal DNA libraries using a multiplex CRISPR system. eLife 3 (2014); Gilbert, L.A. et al. Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Cell (2014); and Citorik, R.J., Mimee, M. & Lu, T.K. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. Nature biotechnology (2014) each of which are hereby incorporated by reference in its entirety), the guide is often presented in a so-called sgRNA (single guide RNA), wherein the two natural Cas9 RNA cofactors (gRNA and tracrRNA) are fused via an engineered loop or linker.

According to one aspect, the Cas9 protein is an enzymatically active Cas9 protein, a Cas9 protein wild-type protein, a Cas9 protein nickase or a nuclease null or nuclease deficient Cas9 protein. Additional exemplary Cas9 proteins include Cas9 proteins attached to, bound to or fused with functional proteins such as transcriptional regulators, such as transcriptional activators or repressors, a Fok-domain, such as Fok 1, an aptamer, a binding protein, PP7, MS2 and the like.

According to certain aspects, the Cas9 protein may be delivered directly to a cell by methods known to those of skill in the art, including injection or lipofection, or as translated from its cognate mRNA, or transcribed from its cognate DNA into mRNA (and thereafter translated into protein). Cas9 DNA and mRNA may be themselves introduced into cells through electroporation, transient and stable transfection (including lipofection) and viral transduction or other methods known to those of skill in the art. The Cas9 protein complexed with the guide RNA, known as a ribonucleotide protein (RNP) complex, may also be introduced to the cells via electroporation, injection, or lipofection.

GUIDE RNA DESCRIPTION

Embodiments of the present disclosure are directed to the use of a CRISPR/Cas system and, in particular, a guide RNA which may include one or more of a spacer sequence, a tracr mate sequence and a tracr sequence. The term spacer sequence is understood by those of skill in the art and may include any polynucleotide having sufficient complementarity with a target nucleic acid sequence to hybridize with the target nucleic acid sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. The guide RNA may be formed from a spacer sequence covalently connected to a tracr mate sequence (which may be referred to as a crRNA) and a separate tracr sequence, wherein the tracr mate sequence is hybridized to a portion of the tracr sequence. According to certain aspects, the tracr mate sequence and the tracr sequence are connected or linked such as by covalent bonds by a linker sequence, which construct may be referred to as a fusion of the tracr mate sequence and the tracr sequence. The linker sequence referred to herein is a sequence of nucleotides, referred to herein as a nucleic acid sequence, which connect the tracr mate sequence and the tracr sequence. Accordingly, a guide RNA may be a two component species (i.e., separate crRNA and tracr RNA which hybridize together) or a unimolecular species (i.e., a crRNA-tracr RNA fusion, often termed an sgRNA).

According to certain aspects, the guide RNA is between about 10 to about 500 nucleotides. According to one aspect, the guide RNA is between about 20 to about 100 nucleotides. According to certain aspects, the spacer sequence is between about 10 and about 500 nucleotides in length. According to certain aspects, the tracr mate sequence is between about 10 and about 500 nucleotides in length. According to certain aspects, the tracr sequence is between about 10 and about 100 nucleotides in length. According to certain aspects, the linker nucleic acid sequence is between about 10 and about 100 nucleotides in length.

According to one aspect, embodiments described herein include guide RNA having a length including the sum of the lengths of a spacer sequence, tracr mate sequence, tracr sequence, and linker sequence (if present). Accordingly, such a guide RNA may be described by its total length which is a sum of its spacer sequence, tracr mate sequence, tracr sequence, and linker sequence (if present). According to this aspect, all of the ranges for the spacer sequence, tracr mate sequence, tracr sequence, and linker sequence (if present) are incorporated herein by reference and need not be repeated. A guide RNA as described herein may have a total length based on summing values provided by the ranges described herein. Aspects of the present disclosure are directed to methods of making such guide RNAs as described herein by expressing constructs encoding such guide RNA using promoters and terminators and optionally other genetic elements as described herein.

According to certain aspects, the guide RNA may be delivered directly to a cell as a native species by methods known to those of skill in the art, including injection or lipofection, or as transcribed from its cognate DNA, with the cognate DNA introduced into cells through electroporation, transient and stable transfection (including lipofection) and viral transduction.

DONOR DESCRIPTION

The term“donor nucleic acid” include a nucleic acid sequence which is to be inserted into genomic DNA according to methods described herein. The donor nucleic acid sequence may be expressed by the cell.

According to one aspect, the donor nucleic acid is exogenous to the cell. According to one aspect, the donor nucleic acid is foreign to the cell. According to one aspect, the donor nucleic acid is non-naturally occurring within the cell. FOREIGN NUCLEIC ACIDS DESCRIPTION

Foreign nucleic acids (i.e. those which are not part of a cell’s natural nucleic acid composition) may be introduced into a cell using any method known to those skilled in the art for such introduction. Such methods include transfection, transduction, viral transduction, microinjection, lipofection, nucleofection, nanoparticle bombardment, transformation, conjugation and the like. One of skill in the art will readily understand and adapt such methods using readily identifiable literature sources.

CELLS

Cells according to the present disclosure include any cell into which foreign nucleic acids can be introduced and expressed as described herein. It is to be understood that the basic concepts of the present disclosure described herein are not limited by cell type. In some embodiments, the cell is a eukaryotic cell or prokaryotic cell. In some embodiments, the prokaryotic cell is a non-E. coli cell. In an exemplary embodiment, the non-E. coli cell is Vibrio natriegens.

VECTORS

Vectors are contemplated for use with the methods and constructs described herein. The term“vector” includes a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors used to deliver the nucleic acids to cells as described herein include vectors known to those of skill in the art and used for such purposes. Certain exemplary vectors may be plasmids, lentiviruses or adeno-associated viruses known to those of skill in the art. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, doublestranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a“plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g. retroviruses, lentiviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with t