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[DESCRIPTION]
This protocol is from:<br /><span class="cit-auth cit-auth-type-author">Alexander WG</span><span class="cit-sep cit-sep-separator">, </span><span class="cit-auth cit-auth-type-author">Doering DT</span><span class="cit-sep cit-sep-separator">,</span><span class="cit-sep cit-sep-last-separator">and</span><span class="cit-auth cit-auth-type-author"> Hittinger CT (2014) <a href="http://www.genetics.org/content/198/3/859.full" target="_blank">High-Efficiency Genome Editing and Allele Replacement in Prototrophic and Wild Strains of Saccharomyces</a>. <em>Genetics</em> <span class="cit-vol">198<span class="cit-sep cit-sep-after-article-vol">:</span></span><span class="cit-pages"><span class="cit-first-page">859</span><span class="cit-sep">-</span><span class="cit-last-page">866; </span></span><span class="cit-doi"><span class="cit-sep cit-sep-before-article-doi">doi:</span>10.1534/genetics.114.170118<br /><br /></span></span>Please see the <a href="http://www.genetics.org/content/198/3/859.full" target="_blank">full manuscript</a> for additional details.<br /><br />The purpose of this document is to provide you with an easy‐to‐follow guide to using the HERP cassettes. We will go through the preparation of the selection and counterselection media, the culturing and transformation for insertion of the HERP cassettes, and counterseletive replacement of the HERP cassettes.

[GUIDELINES]
<strong>C. Inserting the HERP cassettes<br /></strong><br /><strong>1) Design primers with overhangs that target the cassette to your desired locus.    </strong><br /><br />a) The 5' overhangs dictate where the cassette will be integrated, and the length needed depends on the<br />species you're manipulating (40 bp for <em>S. cerevisiae, S. paradoxus, S. uvarum</em>, &amp; <em>S. eubayanus</em>, 50 bp for <em>S. mikatae</em>,<br />and 70 for <em>S. kudriavzevii; S. arboricola</em>'s length requirement is unknown). The longer these overhangs are the more<br />efficient integration will be, although longer overhangs usually mean longer oligonucleotides, which are expensive<br />and sometimes difficult to use.<br /><br />b) The 3' ends amplify the cassette from either the primer or yeast genomic DNA. While we have<br />constructed both plasmids and stably‐integrated yeast strains with all three HERP cassettes, the yeast strains provide<br />an advantage over the plasmid constructs. HERP cassettes with an adjacent I‐SceI recognition site are unstable in<br />bacteria, resulting in the plasmid never being recovered. In yeast, SCE1 is actively repressed while growing on glucose,<br />which prevents leaky nuclease expression. Because of this active repression, the yeast strains tolerate an I‐SceI site<br />adjacent to the HERP cassettes, which in turn reduces the length of oligonucleotide needed to provide both a priming<br />site and a targeting overhang. The authors <span style="text-decoration: underline;"><strong>strongly</strong></span> recommend using the yeast strains as PCR templates for HERP<br />cassette amplification. If the plasmids are used, then the 18‐bp I‐SceI sequence must be included on the oligo<br />between the 3' amplification sequence and the 5' targeting overhang (Table S3).

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