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[DESCRIPTION]
This is a positive control experiment (to test protocol in your hands and/or activity of your home‐made Cas9): tag <em>gtbp‐1</em> with <em>eGFP</em> or <em>mCherry</em> using <em>dpy‐10</em> co‐CRISPR.. The protocol is from:<br /><br /><span class="cit-title"><span class="cit-auth cit-auth-type-author">Paix A</span><span class="cit-sep cit-sep-separator">,</span><span class="cit-auth cit-auth-type-author"> Folkmann A</span><span class="cit-sep cit-sep-separator">,</span><span class="cit-auth cit-auth-type-author"> Rasoloson D</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"> Seydoux G (2015) <a href="http://www.genetics.org/content/early/2015/07/17/genetics.115.179382.abstract" target="_blank">High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR/Cas9 Ribonucleoprotein Complexes</a></span>.</span>  Genetics <span class="cit-elocation">genetics.115.179382</span><span class="cit-sep cit-sep-after-article-pages">; </span><span class="cit-ahead-of-print-date"><span class="cit-sep cit-sep-before-article-ahead-of-print-date">Early online </span>July 17, 2015. <span class="cit-doi"><span class="cit-sep cit-sep-before-article-doi">doi:</span>10.1534/genetics.115.179382<br /><br /></span></span>Please see the <a href="http://www.genetics.org/content/early/2015/07/17/genetics.115.179382.abstract" target="_blank">full manuscript</a> for detail.

[GUIDELINES]
Main protocol: <a href="https://www.protocols.io/view/Direct-delivery-CRISPR-HDR-editing-protocol-for-C-dri54d" target="_blank">Direct delivery CRISPR-HDR editing protocol for <em>C. elegans</em></a><br /><br /><strong>Reagents for gtbp‐1 eGFP and mCherry tagging<br /></strong> <br />crRNA gtbp‐1 Ct (#728 in Table S3): CCACGAGGUGGUAUGCGCAG + GUUUUAGAGCUAUGCUGUUUUG<br /><br />Primers pairs to generate template (from eGFP containing‐plasmid pAP682‐1):<br /><br />Forward primer (5’ to 3’, lower case indicating the homology arm sequence) for eGFP insertion:<br />ggttcgggtggtgctccacgaggtggtatgcgcGTGAGTAAAGGAGAAGAAC<br /><br />Reverse primer (5’ to 3’, lower case indicating the homology arm sequence) for eGFP insertion:<br />cttctaattttgtcccgcattttggaaaccgctCTTGTACAGCTCGTCCATGCC<br /><br />Primers pairs to generate template (from mCherry containing‐plasmid pAP582‐1):<br /><br />Forward primer (5’ to 3’, lower case indicating the homology arm sequence) for mCherry insertion:<br />ggttcgggtggtgctccacgaggtggtatgcgcGTCTCAAAGGGTGAAGAAGATAAC<br /><br />Reverse primer (5’ to 3’ lower case indicating the homology arm sequence) for mCherry insertion:<br />cttctaattttgtcccgcattttggaaaccgctCTTATACAATTCATCCATGCC<br /><br />PCR condition: use annealing temperature of 63 <span class="st">°</span>C, elongation step of 45s, pAP682‐1 (eGFP) or pAP582‐1<br />(mCherry) as a plasmid template. Do 8 PCR reactions and pool them (400µl total), purify on minelute<br />column (see <a href="https://www.protocols.io/view/Direct-delivery-CRISPR-HDR-editing-protocol-for-C-dri54d?step=6" target="_blank">Repair template synthesis</a>).

[STEPS]
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