The authors thank the following undergraduate and high school students for their work to generate OSCAR mutants in Fusarium verticillioiodes: Anjellica Miller, Athar Naseer, Xiu Lin, Katelyn Woodburry, Chelsea Patterson, Kathleen Robertson, Krystina Bradley, Ashton Rogers, Alexis McKensie, Manny Hernandez, Ashli Crepsac, Jeff Delong, Christian King, Gi Jeong, Maria Belding, Christy Burre, Daniel O&#39;Meara, Lauren (Victoria) Cook, Jake Goodman, Sampriti De, Oge Okoye, Alyssa Beckstead, Garrett Hibbs, Nick Goldstein, Caroline Twum, Chris Benson, Louis Stokes, Hannah Itell, Jane Hulse, Jasim Mohammed, James Loggins, Kelli Russell, Gre&#39;Nisha Jones, Kristin Sheaffer, Mariam Hammady, Ava Wilson, Katrina Bazemore, Toney Harper, Karlin McGhee, Mohmed Momin, Rima Momin, Thi Ngoc Le and Angel Pham.

1. Primer Design for PCR Amplification of Gene Flanks
<ol>
	<li>Download to a word processing file the genomic region of the gene of interest (GOI) including the open reading frame (ORF) and at least 2 kb flanking the gene on each side from FungiDB or other genomic data resource.</li>
	<li>Highlight the ORF intended for deletion and label start and stop codons.</li>
	<li>Identify and highlight adjacent ORFs within the downloaded sequence.</li>
	<li>Use the 2 kb 5&#39; end of the GOI ORF and the primer design tool (see Materials List) to design PCR primer pair O1 and O2 to generate a minimum size product of 1 kb. Take care to not impact adjacent ORFs.
	<ol>
		<li>Paste the 2 kb 5&#39; flank into the &#34;Sequence Entry&#34; window. Click on &#34;Show Custom Parameters&#34; box. Enter the following custom design parameters: primer TM 58 (min), 60 (opt) and 62 (max); Primer GC 40 (min), 50 (opt) and 60 (max); Primer Size 22 (min), 24 (opt) and 26 (max); Amplicon Size 1250 (min), 1500 (opt) and 2000 (max).</li>
		<li>Choose the result that places the reverse primer (O2) closest to the ORF. Capture a screen shot of the assays and copy and paste the primer sequences into the word processing document.</li>
		<li>Repeat the process for 3&#39; flank to generate primers O3 and O4, this time choose the result placing the forward primer (O3) closest to the target ORF.</li>
	</ol>
	</li>
	<li>Add appropriate attach sites to 5&#39; ends of primers 1 through 4 (see Table 1).</li>
	<li>Also design and order ORF specific primers to be used for deletion confirmation in section 4 below. Parameters should be the same as in step 1.4.1 except use Amplicon Size 500 (min), 750 (opt) and 1000 (max) adjusting for length of ORF if necessary. Finally, for confirmation of homologous recombinants, generate 5&#39; out and 3&#39; out primers found just outside the flanks within the chromosome. These &#34;out&#34; primers will be used with hygR (210) and hygF (850), respectively (Fig 2).</li>
	<li>Order primers.</li>
</ol>
2. Production of OSCAR Constructs
<ol>
	<li>Using a hi-fidelity Taq, carry out two reactions, one each for the 5&#39; and 3&#39; flanks, using primers (100 &#181;M)&#160;O1 and O2 in one reaction and primers O3 and O4 in the second. Reaction mixture contains the following: 29.5 &#181;L sterile distilled water (SDW), 5 &#181;L 10x LA PCR buffer, 8 &#181;L 2.5 mM dNTP mix, 5 &#181;L 25 mM MgCl2, 1 &#181;L template (50 ng/&#181;L), 0.5 &#181;L hi-fidelity Taq, 0.5 &#181;L Primer O1 and 0.5 &#181;L Primer O2 for 5&#39; flank (or 0.5 &#181;L Primer O3 and 0.5 &#181;L Primer O4 for 3&#39; flank) Use reaction conditions are as follows: 94 &#176;C for 1 min, then 30 cycles of 94 &#176;C for 30 s, 60 &#176;C for 30 s, 72 &#176;C for 2 min, then final steps of 72 &#176;C for 5 min and then hold indefinitely at 10 &#176;C.</li>
	<li>Run 0.8% agarose 1x TAE (40 mM Tris acetate pH 8.3, 1 mM EDTA) gel electrophoresis for approximately 125 Vh in a standard minigel apparatus to determine product size and relative concentration. Post stain the gel with non-ethidium stain according to the manufacturer instructions. Visualize on a UV illuminator.</li>
	<li>If 5&#39; and 3&#39; flank products are in roughly even concentration, combine them and use an affinity column kit (or PEG precipitation 90 &#181;L combined PCR products, 240 &#181;L TE buffer pH 7.5, 160 &#181;L of 30% polyethylene glycol PEG8000 30 mM MgCl2) to co-purify the PCR products according to the manufacturer&#39;s protocol. If they are not of equal concentration combine all of low concentration product with an estimated equal amount of product from the higher yield reaction.</li>
	<li>Use a spectrophotometer to measure purified DNA concentration.</li>
	<li>Carry out BP reaction by mixing the following: 1 &#181;L of 60 ng/&#181;L pOSCAR, 2 &#181;L of 60 ng/&#181;L pA-Hyg-OSCAR, 1 &#181;L of 60 ng/&#181;L combined flanks, 1 &#181;L of clonase. Incubate for 16 h at 25 &#176;C. Terminate the reaction by adding 0.5 &#181;L proteinase K and incubate for 10 min at 37 &#176;C.</li>
	<li>Transform high competence E. coli cells. Both commercially available competent cells and home-made DH5&#945; CaCl2 competent cells were used successfully as recipients as described by the manufacturer&#39;s instructions. Plate on low sodium (0.5 g/L NaCl) LB containing 100 &#181;g/mL spectinomycin (Spec).</li>
	<li>Identify the correct construct by performing DNA minipreps10 of colonies generated above. Perform double digestion with HindIII and KpnI as follows: pipette 5 &#181;L DNA, 2 U of each enzyme, 2 &#181;L appropriate 10x buffer with SDW to a 20 &#181;L total volume. This releases the insert (approximately 4.5 kb with 1.5 kb gene flanks) from the vector (ca. 7 kb) and cuts any sites in flanks which can give predictable band sizes.</li>
	<li>Sequence11 select clones showing an appropriate banding pattern.</li>
</ol>
3. Agrobacterium tumefaciens Mediated Transformation (ATMT) of Fungi
<ol>
	<li>Transform Agrobacterium tumefaciens strain AGL1 with OSCAR deletion construct12.</li>
	<li>Transform fungus with AGL1 containing the GOI OSCAR deletion plasmid. To do this carry out the following steps:
	<ol>
		<li>Prepare a fungal spore suspension with sterile water at 2 x 106 spores/mL. Quantify spores on a hemocytometer or automated cell counter. Place 500 &#181;L of this in a 1.5 mL microcentrifuge tube. This set up is for 4 transformation plates.</li>
		<li>Use a blue sterile disposable loop to add an easily visible gob (should cover about 20% of the loop diameter) of the deletion plasmid containing AGL1 from 2-3 day old LB-Spec 100 medium (see Materials List for composition) containing plates and mix into the spore suspension. Vortex till bacterial cells are well dispersed (ca. 5 min medium speed).</li>
		<li>Pipette 100 &#181;L of the conidia-Agro suspension onto the center of a cellulose membrane filter placed on each of four 6 cm Petri dishes containing co-cultivation medium12.</li>
		<li>Spread the suspension with sterile glass beads (about 4) to cover the whole surface of the membrane filter. Alternatively spread the suspension using a traditional spreader tool. Allow the membrane filter to dry in a hood for approximately 10 min, wrap with paraffin film. Repeat steps 3.2.2 and 3.2.3 to set up multiple transformations.</li>
		<li>Incubate the plate for 2 days at room temperature, inverted.</li>
		<li>Transfer the membrane filter onto 6 cm Aspergillus selection medium12 plates containing hygromycin B (Hyg) 150 &#181;g/mL, 200 mM cefotaxime and 100 &#181;g/mL moxalactam. Incubate at room temperature for 5-7 days before isolating Hyg resistant colonies.</li>
		<li>With sterile toothpicks, transfer putative transformants onto 6 cm Potato Dextrose Agar (PDA)&#160;plates containing 150 &#181;g/mL Hyg, 100 &#181;g/mL kanamycin.</li>
	</ol>
	</li>
</ol>
4. Deletion Mutant Identification by PCR
<ol>
	<li>Extract DNA for PCR from transformants by thermolysis13.
	<ol>
		<li>Once transformants form colonies on the PDA from step 3.2.7, use a sterile toothpick to transfer a small amount (2 mm x 2 mm) of mycelium or yeast cells from the colony into 100 &#181;L of lysis solution (50 mM sodium phosphate, pH 7.4, 1 mM EDTA, 5% glycerol) in a microcentrifuge tube.</li>
		<li>Incubate the mixture at 85-90 &#176;C for 20-30 min. Store the crude extract containing genomic DNA at -20 &#176;C until use in step 4.2.</li>
	</ol>
	</li>
	<li>Carry out 4 PCR reactions per transformant, one each to detect homologous recombination of the 5&#39; and 3&#39; flanks, respectively, one for the selectable marker (hygromycin phosphotransferase in this case) and one for the GOI ORF. 
	NOTE: We generally use inexpensive low-fidelity Taq for these reactions. Reaction conditions are as described&#160;in step 2.1 above and contain SDW 20 &#181;L, 10x Taq buffer 2.5 &#181;L, dNTPS (10 mM) 0.5 &#181;L, homemade Taq14, 0.5 &#181;L, Primer A (100 &#181;M) 0.5 &#181;L, Primer B (100 &#181;M) 0.5 &#181;L, Template 0.5 &#181;L. Lower concentration primer stocks (10 &#181;M) can be used equally well.</li>
	<li>Run an agarose gel (e.g. 0.8%) of the PCR products. Deletion mutants will produce the Hyg band but not an ORF product. Ectopic integrants will show both bands. Wild type will produce only the ORF band.</li>
	<li>Permanently store deletion mutants (and an ectopic transformant or two for controls) for future use. 
	NOTE: 15% glycerol is used to store our strains long-term at -80 &#176;C.</li>
</ol>

<ol><li>Paz, Z., García-Pedrajas, M. D., Andrews, D. L., Klosterman, S. J., Baeza-Montañez, L., Gold, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((One+step+construction+of+Agrobacterium-Recombination-ready-plasmids+%28OSCAR%29%2C+an+efficient+and+robust+tool+for+ATMT+based+gene+deletion+construction+in+fungi%5BTitle%5D)+AND+%22Fungal+Genet+Biol%22%5BJournal%5D)">One step construction of Agrobacterium-Recombination-ready-plasmids (OSCAR), an efficient and robust tool for ATMT based gene deletion construction in fungi.</a> Fungal Genet Biol. 48 (7), 677-684 (2011).</li>
<li>Gibson, D. G., Young, L., Chuang, R. Y., Venter, J. C., Hutchison, C. A., Smith, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nat Methods. 6 (5), 343-345 (2009).</li>
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<li>Xue, C., Wu, D., Condon, B. J., Bi, Q., Wang, W., Turgeon, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+gene+knockout+in+the+maize+pathogen+Setosphaeria+turcica+using+Agrobacterium+tumefaciens-mediated+transformation%5BTitle%5D)+AND+%22Phytopathology%22%5BJournal%5D)">Efficient gene knockout in the maize pathogen Setosphaeria turcica using Agrobacterium tumefaciens-mediated transformation.</a> Phytopathology. 103 (6), 641-647 (2013).</li>
<li>Xu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+high-throughput+gene+disruption+methodology+for+the+entomopathogenic+fungus+Metarhizium+robertsii%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">A high-throughput gene disruption methodology for the entomopathogenic fungus Metarhizium robertsii.</a> PloS One. 9 (9), (2014).</li>
<li>Crutcher, F. K., Liu, J., Puckhaber, L. S., Stipanovic, R. D., Bell, A. A., Nichols, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FUBT%2C+a+putative+MFS+transporter%2C+promotes+secretion+of+fusaric+acid+in+the+cotton+pathogen+Fusarium+oxysporum+f.+sp.+vasinfectum%5BTitle%5D)+AND+%22Microbiology%22%5BJournal%5D)">FUBT, a putative MFS transporter, promotes secretion of fusaric acid in the cotton pathogen Fusarium oxysporum f. sp. vasinfectum.</a> Microbiology. 161, 875-883 (2015).</li>
<li>Yu, X., Wang, Y., Pan, J., Wei, D., Zhu, X. <a target="_blank" href="http://link.springer.com/article/10.1007/s13213-015-1055-8">High frequency of homologous gene disruption by single-stranded DNA in the taxol-producing fungus Pestalotiopsis microspora.</a> Ann Microbiol. 65 (4), 2151-2160 (2015).</li>
<li>Korn, M., Schmidpeter, J., Dahl, M., Müller, S., Voll, L. M., Koch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Genetic+Screen+for+Pathogenicity+Genes+in+the+Hemibiotrophic+Fungus+Colletotrichum+higginsianum+Identifies+the+Plasma+Membrane+Proton+Pump+Pma2+Required+for+Host+Penetration%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">A Genetic Screen for Pathogenicity Genes in the Hemibiotrophic Fungus Colletotrichum higginsianum Identifies the Plasma Membrane Proton Pump Pma2 Required for Host Penetration.</a> PloS One. 10 (5), e0125960(2015).</li>
<li>Chettri, P. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Regulation+of+dothistromin+toxin+biosyn+by+the+pine+needle+pathogen+Dothistroma+septosporum%3A+a++presented+in+the+partial+fulfilment+of+the+requirements+for+the+degree+of+Doctor+of+Philosophy+%28%29+in+Genetics+at+Massey+University%2C+Manawatu%2C+New+Zealand%22">Regulation of dothistromin toxin biosynthesis by the pine needle pathogen Dothistroma septosporum: a thesis presented in the partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatu, New Zealand </a>. , Massey University. Manawatu, New Zealand. (2014).</li>
<li>Chen Zhou,, Yujun Yang,, Jong, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mini-prep+in+ten+minutes%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">Mini-prep in ten minutes.</a> Biotechniques. 8 (2), 172(1990).</li>
<li>Sanger, F., Nicklen, S., Coulson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+sequencing+with+chain-terminating+inhibitors%5BTitle%5D)+AND+%22P+Natl+Acad+SciUSA%22%5BJournal%5D)">DNA sequencing with chain-terminating inhibitors.</a> P Natl Acad SciUSA. 74 (12), 5463-5467 (1977).</li>
<li>Khang, C. H., Park, S. Y., Rho, H. S., Lee, Y. H., Kang, S. Filamentous fungi (Magnaporthe grisea and Fusarium oxysporum). <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKhang%2C+CH+%22Agrobacterium+Protocols%22">Agrobacterium Protocols</a>. Wang, K. an 2, Humana Press Inc. Totowa, NJ. 403-420 (2007).</li>
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The authors have nothing to disclose.

One Step Construction of Agrobacterium-Recombination-ready-plasmids (OSCAR) has been successfully employed with an ever-increasing number of Ascomycota fungi. The method should also easily be applicable to the Basidiomycota and species from other fungal phyla (with appropriate promoters driving selectable marker genes), assuming Agrobacterium mediated transformation and homologous recombination are possible. Additional marker vectors have been generated to diversify choices of anti-fungal compound as we...

Genetic dissection is a powerful methodology for determining the functional importance of individual or combinations of genes. A standard approach to understand the role of specific genes is production of single gene mutants unaltered in any other gene. The most powerful and least potentially confounding approach is complete and precise deletion of a gene of interest&#39;s open reading frame (GOI ORF) without damage to any other gene function.
Because standard ligation approaches for deletion plasmid generation require multiple steps, the rational for OSCAR1 was to produce a more rapid in vitro approach. Figure 1 depicts the assembly process in the OSCAR approach. The method described here has the advantage of combining rapid construction of individual gene deletion vectors in a single multipart reaction in combination with subsequent Agrobacterium tumefaciens mediated transformation (ATMT). OSCAR is very rapid and compares well with other strategies such as use of Gibson assembly in yeast2. The OSCAR method has been used successfully with several Ascomycota species of fungi. These species include: Fusarium verticillioides (unpublished), Verticillium dahliae3, Setosphaeria turcica4, Metarhizium robertsii5, Fusarium oxysporum f. sp. vasinfectum6, Pestalotiopsis microspora7, Colletotrichum higginsianum8, and Dothistroma septosporum9 and Sarocladium zeae (unpublished).
This protocol provides step-by-step instruction for the method including primer design, flank PCR amplification, the OSCAR BP reaction, deletion construct structure confirmation, transformation of Agrobacterium with the construct followed by ATMT based transfer of the deletion construct into the fungal cells, and finally differentiating fungal deletion mutants from those with ectopically integrated deletion constructs.

<table><tbody><tr><td>FungiDB</td><td></td><td></td><td>Database/ http://fungidb.org/fungidb/</td></tr><tr><td>IDT PrimerQuest</td><td>IDT</td><td></td><td>Primer design online software/ http://www.idtdna.com/Primerquest/Home/Index</td></tr><tr><td>Microsoft Word</td><td></td><td></td><td>Sequence file manipulation</td></tr><tr><td>Low Na LB Spec 100 medium</td><td></td><td></td><td>&lt;em&gt;E. coli&lt;/em&gt; transformant selection, composition: 1% tryptone, 0.05% NaCl, 0.5% yeast extract, 1.5% agar if for solid medium</td></tr><tr><td>Co-cultivation medium</td><td></td><td></td><td>ATMT transformation induction (Reference 12)</td></tr><tr><td>Aspergillus minimal medium with Hygromycin</td><td></td><td></td><td>Fungal transformant selection</td></tr><tr><td>PDA medium</td><td>Acumedia</td><td>7149A</td><td>Single spore slant tubes</td></tr><tr><td>PDA-Hyg-Kan medium</td><td></td><td></td><td>Fungal ransformant isolation, PDA containing 150 &amp;mu;g/mL hygromycin B and 100 &amp;mu;g/mL Kanamycin</td></tr><tr><td>Glass beads</td><td>Genlantis</td><td>C400100</td><td>Plate spreading</td></tr><tr><td>Nitrocellulose filters (47 mm)</td><td>Fisher</td><td>09-719-555</td><td>Co-culturing for ATMT</td></tr><tr><td>Various centifuge tubes</td><td></td><td></td><td>multiple preps</td></tr><tr><td>Petri plates (various)</td><td></td><td></td><td>Culturing of bacteria and Fungi</td></tr><tr><td>pA-Hyg OSCAR</td><td>Addgene</td><td>29640</td><td>Selectable marker vector</td></tr><tr><td>pOSCAR</td><td>Addgene</td><td>29639</td><td>Assembly vector</td></tr><tr><td>DH5&amp;#945; One Shot Competent &lt;em&gt;E. coli &lt;/em&gt;cells</td><td>Life Technologies&amp;nbsp;</td><td>12297-016</td><td>BP reaction transformation</td></tr><tr><td>ccdB survival E. coli cells</td><td>Life Technologies&amp;nbsp;</td><td>A10460</td><td>Maintenance of pOSCAR</td></tr><tr><td>Wooden transfer sticks</td><td></td><td></td><td>Colony streaking</td></tr><tr><td>Toothpicks</td><td></td><td></td><td>Colony picking</td></tr><tr><td>Microcentrifuge</td><td></td><td></td><td>Pelleting Bacteria, &lt;em&gt;etc.&lt;/em&gt;</td></tr><tr><td>Preparative centrifuge</td><td></td><td></td><td>Fungal spore collection</td></tr><tr><td>Dissecting microscope</td><td></td><td></td><td>Single spore isolation</td></tr><tr><td>Automated Cell Counter</td><td></td><td></td><td>Spore suspension calculation</td></tr><tr><td>Compound microscope</td><td></td><td></td><td>Hemocytometer cell counting</td></tr><tr><td>QIAquick PCR Purification Kit&amp;nbsp;</td><td>Qiagen</td><td>28104</td><td>PCR gene flank produict purification</td></tr><tr><td>TaKaRa LA &lt;em&gt;Taq&lt;/em&gt;&amp;nbsp;</td><td>Takara Bio USA</td><td>RR002A</td><td>Hi Fidelity &lt;em&gt;taq &lt;/em&gt;polymerase for OSCAR flank generation</td></tr><tr><td>Hygromycin B</td><td>InvivoGen</td><td>ant-hg-5</td><td></td></tr><tr><td>Spectinomycin</td><td>Sigma</td><td>22189-32-8</td><td></td></tr><tr><td>Cefotaxime</td><td>TCI America</td><td>C2224</td><td></td></tr><tr><td>Kanamycin</td><td></td><td>11815032</td><td></td></tr><tr><td>Moxalactam</td><td>Sigma-Aldrich</td><td>43963</td><td></td></tr><tr><td>GelRed</td><td>&amp;nbsp;Phenix Research Products</td><td>RGB-4103</td><td>Post staining agarose gels</td></tr><tr><td>Qiagen QIAquick PCR Purification Kit (Cat. No. 28104)&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>(OneShot_ Mach1TM T1R or One Shot_ OmniMAX&amp;trade; 2 T1R from Invitrogen)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>C862003</td><td></td></tr><tr><td>Gateway BP Clonase II Enzyme mix</td><td>Thermo Fisher Scientific</td><td>11789020</td><td>Used to assemble deletion construct in pOSAR</td></tr></tbody></table>

rapid deletion production in fungi via agrobacterium mediated transformation of oscar deletion constructs

Precise deletion of gene(s) of interest, while leaving the rest of the genome unchanged, provides the ideal product to determine that particular gene&#39;s function in the living organism. In this protocol the OSCAR method of precise and rapid deletion plasmid construction is described. OSCAR relies on the cloning system in which a single recombinase reaction is carried out containing the purified PCR-amplified 5&#39; and 3&#39; flanks of the gene of interest and two plasmids, pA-Hyg OSCAR (the marker vector) and pOSCAR (the assembly vector). Confirmation of the correctly assembled deletion vector is carried out by restriction digestion mapping followed by sequencing. Agrobacterium tumefaciens is then used to mediate introduction of the deletion construct into fungal spores (referred to as ATMT). Finally, a PCR assay is described to determine if the deletion construct integrated by homologous or non-homologous recombination, indicating gene deletion or ectopic integration, respectively. This approach has been successfully used for deletion of numerous genes in Verticillium dahliae and in Fusarium verticillioides among other species.

The OSCAR method, in a single reaction, generates a plasmid containing the flanks of the target gene to be deleted surrounding the selectable marker cassette. The production of deletion constructs using OSCAR is very efficient. The system can, however, produce partial constructs containing some but not all three fragments (the two gene flanks and the selectable marker). Generally, the majority of E. coli transformants contain the correct OSCAR construct. For example, Figu...

Watch this Scientific Journal Video about Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs at JoVE.com

Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs

Gene deletion mutants generated through homologous recombination are the gold standard for gene function studies. The OSCAR (One Step Construction of Agrobacterium-Recombination-ready-plasmids) method for rapid generation of deletion constructs is described. Agrobacterium mediated fungal transformation follows. Finally, a PCR based confirmation method of gene deletions in fungal transformants is presented.

Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs

Precise deletion of gene(s) of interest, while leaving the rest of the genome unchanged, provides the ideal product to determine that particular ...

rapid-deletion-production-fungi-via-agrobacterium-mediated

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9.9K Views.  NPRC, USDA-ARS.  Gen deletie mutanten gegenereerd door homologe recombinatie zijn de gouden standaard voor genfunctie studies. De OSCAR (One Step Construction of Agrobacterium-Recombination-ready-plasmids) methode voor snelle generatie van deletieconstructen wordt beschreven. Agrobacterium gemedieerde schimmel transformatie volgt. Tenslotte wordt een PCR-gebaseerde bevestigingsmethode van gendeleties in zwamtransformanten gepresenteerd. 

Video: Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic characteristics, this unconventional yeast is not only a good model for the study of the fundamental nature of fungal differentiation but is also a promising microbial platform for biochemical production and various biotechnological applications, which require extensive genetic manipulations. However, genetic manipulations of Y. lipolytica have been limited due to the lack of an efficient and stable genetic transformation system as well as very high rates of non-homologous recombination that can be mainly attributed to the KU70 gene. Here, we report an easy and rapid protocol for the efficient genetic transformation and for gene deletion in Y. lipolytica Po1g. First, a protocol for the efficient transformation of exogenous DNA into Y. lipolytica Po1g was established. Second, to achieve the enhanced double-crossover homologous recombination rate for further deletion of target genes, the KU70 gene was deleted by transforming a disruption cassette carrying 1 kb homology arms. Third, to demonstrate the enhanced gene deletion efficiency after deletion of the KU70 gene, we individually deleted 11 target genes encoding alcohol dehydrogenase and alcohol oxidase using the same procedures on the KU70 knockout platform strain. It was observed that the rate of precise homologous recombination increased substantially from less than 0.5% for deletion of the KU70 gene in Po1g to 33%-71% for the single gene deletion of the 11 target genes in Po1g KU70&#916;. A replicative plasmid carrying the hygromycin B resistance marker and the Cre/LoxP system was constructed, and the selection marker gene in the yeast knockout strains was eventually removed by expression of Cre recombinase to facilitate multiple rounds of targeted genetic manipulations. The resulting single-gene deletion mutants have potential applications in biofuel and biochemical production.

We herein report methods on the molecular genetic manipulation of the Yarrowia lipolytica Po1g strain for improved gene deletion efficiency. The resulting engineered Y. lipolytica strains have potential applications in biofuel and biochemical production.

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic ...

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell&#39;s DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag.

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol for transferring gene deletion mutations from one Escherichia coli strain to another by using generalized transduction with the bacteriophage P1. This method requires that the mutation be selectable (e.g., based on gene disruptions using antibiotic cassette insertions). Such antibiotic cassettes can be mobilized from a donor strain and introduced into a recipient strain of interest to quickly and easily generate a gene deletion mutant. The antibiotic cassette can be designed to include flippase recognition sites that allow the excision of the cassette by a site-specific recombinase to produce a clean knock-out with only a ~100-base-pair-long scar sequence in the genome. We demonstrate the protocol by knocking out the tamA gene encoding an assembly factor involved in autotransporter biogenesis and test the effect of this knock-out on the biogenesis and function of two trimeric autotransporter adhesins. Though gene deletion by P1 transduction has its limitations, the ease and speed of its implementation make it an attractive alternative to other methods of gene deletion.

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

Here we present a protocol for the use of pre-existing antibiotic resistance-cassette deletion constructs as a basis for making deletion mutants in other E. coli strains. Such deletion mutations can be mobilized and inserted into the corresponding locus of a recipient strain using P1 bacteriophage transduction.

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol ...

Direct Liquid-Culture Screening for Evaluating the Production of Heterologous Proteins Using an Auxotrophic Mutant of Aspergillus Oryzae

Aspergillus oryzae, a filamentous fungus, is one of the most widely used hosts for industrial applications including large-scale production of proteins. A polyethylene glycol (PEG)-mediated protoplast transformation method is generally used for the introduction of heterologous genes into A. oryzae. The conventional method typically requires three weeks for the screening of favorable transformants. Here, a new technique, the direct liquid-culture (DLC) screening method, is introduced which reduces the screening time to six days in a 200 mL flask format or to 10 days in a 24 well microplate format. The DLC screening method ensures the acquisition of positive transformants and evaluation of the secretory production of heterologous proteins in a single step, unlike the conventional screening method where two separate steps are required for the same. The protocol for PEG-mediated protoplast transformation of A. oryzae is described, which consists of five steps: preparation of fresh spore suspension, preculture, preparation of protoplasts, introduction of DNA, and DLC screening. For successful results in DLC screening, it is critical to use a nutrient-rich medium with optimized osmotic pressure. The protocol should further popularize the use of A. oryzae as a host of choice in the industrial production of proteins.

Direct Liquid-Culture Screening for Evaluating the Production of Heterologous Proteins Using an Auxotrophic Mutant of Aspergillus Oryzae

A direct liquid-culture (DLC) screening method has been developed which significantly reduces the time required for polyethylene glycol (PEG)-mediated protoplast transformation of the filamentous fungus, Aspergillus oryzae, when employed for evaluation of the secretory production of heterologous proteins. This method dramatically increases the throughput of the evaluation protocol.

Aspergillus oryzae, a filamentous fungus, is one of the most widely used hosts for industrial applications including large-scale production of proteins. A ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

Biology

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) and Wuschel2 (Wus2). Bbm is regulated by the maize phospholipid transferase gene (Pltp) promoter, and Wus2 is under the control of a maize auxin-inducible (Axig1) promoter. An Agrobacterium strain carrying these morphogenic genes on transfer DNA (T-DNA) and extra copies of Agrobacterium virulence (vir) genes are used to infect maize immature embryo explants. Somatic embryos form on the scutella of infected embryos and can be selected by herbicide resistance and germinated into plants. A heat-activated cre/loxP recombination system built into the DNA construct allows for removal of morphogenic genes from the maize genome during an early stage of the transformation process. Transformation frequencies of approximately 14%, 4%, and 4% (numbers of independent transgenic events per 100 infected embryos) can be achieved for W22, B73, and Mo17, respectively, using this protocol.

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Plant morphogenic genes can be used to improve genetic transformation of recalcitrant genotypes. Described here is an Agrobacterium-mediated genetic transformation (QuickCorn) protocol for three important public maize inbred lines.

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) ...

Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs

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