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[DESCRIPTION]
This protocols is from:<br />Alexandre Paix, et al. (2014) <a href="http://www.genetics.org/content/198/4/1347.full" target="_blank">Scalable and Versatile Genome Editing Using Linear DNAs with Microhomology to Cas9 Sites in Caenorhabditis elegans</a>. <em>Genetics <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">1347</span><span class="cit-sep">-</span><span class="cit-last-page">1356</span></span><span class="cit-sep cit-sep-after-article-pages">;</span><span class="cit-doi"><span class="cit-sep cit-sep-before-article-doi">doi:</span>10.1534/genetics.114.170423</span></em><br /><br />Please see the <a href="http://www.genetics.org/content/198/4/1347.full" target="_blank">full manuscript</a> or additional details<a href="http://www.genetics.org/content/198/4/1347.full" target="_blank"><br /><br /><br /></a>

[GUIDELINES]
<strong>Reagents</strong><br />
<ul>
<li>QIAprep Spin Miniprep Kit: Qiagen, 27104</li>
<li>MinElute PCR Purification Kit: Qiagen, 28004</li>
<li>Phusion High‐Fidelity PCR Master Mix with HF Buffer: NEB, M0531L</li>
<li>GoTaq Green Master Mix: Promega, M7122</li>
<li>Taq DNA Polymerase: Invitrogen, 10342‐046</li>
<li>Recovery buffer: 5mm HEPES pH 7.2, 3mM CaCl2, 3mM MgCl2, 66mM NaCl, 2.4mM KCl, 4% Glucose (w/v)</li>
<li>10X M9: 420mM Na2HPO4, 220mM KH2PO4, 860mM NaCl, 10mM MgSO4</li>
<li>Q5 Site‐Directed Mutagenesis Kit: NEB, E0554S</li>
<li>Lysis buffer:50mM KCl, 10mM Tris pH8.3, 2.5mM MgCl2, 0.45% NP40, 0.45% Tween20. Before worm lysis, add proteinase K to 0.1 ug/ul.</li>
</ul>
------------------------<br /><strong>Design and cloning of the sgRNAs  <br /></strong>‐Use this website <a href="http://crispr.mit.edu/" target="_blank">http://crispr.mit.edu/</a> to select sgRNAs.  <strong><br /></strong><br />Choose sgRNAs that are 1) as close as possible to the modification site and 2) have few off‐target sites. If there are off‐targets sites, they should have 3 or more mismatches, preferentially near the PAM. If possible, choose more than one sgRNAs near each other. Not every sgRNA works, so it’s best to use more than one, until you have one that you know works.   <br /><br />‐The website shows sgRNAs with 20 bases, but we only use the last 19 bases (skip the first base).<strong><br /></strong><br />‐Order the following primers:<strong><br />Forward Q5</strong>: (N19)gttttagagctagaaatagcaagt<br /><strong>Reverse Q5</strong>: caagacatctcgcaatagg<br /><strong>Forward sequencing</strong> (for sequencing sgRNA insertion only): tatgaaatgcctacaccctctc<br />--------------------------<br /><strong>Design of repair ssODNs</strong>   <br /><br />The repair oligo should contain flanking bases at both the 5’ and 3’ ends for homologous recombination (sequences identical to genomic DNA). Ideally, flanking sequences should terminate with a C or G and contain good sequence diversity at their extremities (no hairpins). 43nt is the shortest flanking sequence we have tested and 100 is the longest. We do not know the optimal homology arm length for ssODNs. Make sure the homology arms extend as close to the sgRNA cut(s) as possible.   <br /><br />The repair oligo should contain mutations that make it resistant to cutting by Cas9/sgRNA.  You can mutate the PAM, insert new bases between the sgRNA sequence and the PAM, or mutate the sgRNA sequence near the PAM (we typically create 4 mismatches).  <br /><br />If the sgRNA targets a coding region, be careful to make only silent changes using codons that are used at similar frequency as<br />original codon (<a href="http://www.genscript.com/cgi-bin/tools/codon_freq_table" target="_blank">This site</a> for codon usage in C. elegans may be useful). If possible, avoid sgRNAs that target non‐coding sequences since mutations in these sequences could possibly affect regulatory (splicing, promoters) motifs.   <br /><br />‐If possible, engineer a restriction site (RE) in your oligo to facilitate screening. Make sure that the RE site is either directly in<br />your edit, or on the distal side of your edit relative to the cut, to ensure that both are incorporated.  <br /><br />‐If you want to insert a premature stop, insert a NheI site between the PAM and sgRNA sequence. Use the TAG codon inside the<br />NheI site. For maximal gene disruption, we recommend also adding a base to create a frameshift after the TAG codon.<br /><br />‐Suggestions for protein tags (capital letters represent bases modified to create a RE site):<br /><br />V5: ggtaagcctatccctaaccctctcctcggtctAgatAGTacT (contains XbaI and ScaI sites)<br /><br />HA: tacccataTgatgtCccGgattacgct (contains NdeI and NciI sites)<br /><br />TetraCys: tgctgcccaggatgctgc (contains BstNI site)<br /><br />3xFLAG: gactacaaagaccatgacggtgattataaagatcatgaTatcgaTtacaaggatgacgatgacaag (contains EcoRV and ClaI sites)<br /><br />Myc: gaacaaaaactGatAtcagaagaggatctg (contains EcoRV site)<br /><br />OLLAS: tccggattcgccaacGAGCTCggaccacgtctcatgggaaag (contains SacI site)<br /><br />‐Order single stranded oligo from Operon (125nt max, 10nmol, salt free) or from IDT (200nt max, 4nM ultramer, salt free). Reconstitute oligo at 1ug/ul according to the amount provided by the manufacturer.<br />--------------------------<br /><strong>Construction of PCR donor templates for GFP insertion<br /></strong><br />Optional Nested PCR step: The nested PCR step is included to amplify your PCR further and to eliminate any long primers remaining from the first PCR. This step may be omitted if your PCR is already &gt;500ng/ul and if you used relatively short primers (&lt;60 bases) that are efficiently removed by the minelute PCR purification kit.  Perform three nested PCR on this purification using 22‐25nt Fw/Rev primers complementary to the 5’/3’ ends of the template (45s elongation step, 30 cycles, 3*50ul reaction, 60°C annealing). Check the PCR products on agarose gel.<br /><br />Optional Dpn1 digestion step: This step may not be necessary but is included to eliminate any remaining GFP plasmid template that could form an extrachromosomal array and give you a false GFP+.  Add to each 50ul PCR reactions 30ul of H2O, 10ul of Dpn1 (200 units, NEB), 10ul of 10X cutsmart buffer, and incubate at 37°C for 12h and next at 80°C for 20min.<br />--------------------------<br /><strong>PCR Screening<br /><br />We recommend testing each gene‐specific PCR assay before starting the injections.  <br /></strong><br />We recommend the following Taq polymerases:<br /><br />If the PCR product will be processed enzymatically (restriction enzyme): Invitrogen recombinant taq (follow the<br />manufacturer recommendation for a 50ul reaction, do 30 cycles, 1min or more by kb). Most of restriction enzymes work with<br />the Invitrogen recombinant Taq buffer.<br /><br />If the PCR product does not need processing (only looking for a size shift or a positive PCR using GFP internal primer):<br />Promega Go Taq 2x Master mix.  <br /><br />For PCR products &gt;1.5kb: NEB Phusion 2X Master mix, 30s by kb, 30 cycles. Note that, although regular Taq could<br />amplify larger fragment, the full length DNA will compete with the edited one, and for amplification of both fragments in the<br />same mixture, the Phusion Taq is a better choice. Most of the restriction enzymes work with the Phusion buffer.<br /><br />‐PCR strategies (Figure 1B):  <br /><br />For large insertions, use a primer specific to the insert (“internal primer”): This is best for detecting a large insert (such<br />as GFP) in a large pool of F1s (8). Use your gene‐specific forward external primer and an internal reverse primer (for GFP use<br />the GFP reverse primer: cattaacatcaccatctaattc). Do the PCR using GoTaq, 50ul reaction, 30 cycles, 45s‐1min elongation. Note<br />that the GFP reverse primer works well with annealing temperatures from 55 to 60 °C using a GoTaq.<br /><br />For small insertions and modifications, design forward and reverse external primers around the edited site (but<br />OUTSIDE the sequences in the repair template). Ideally, the PCR product should be about 500 bp centered around the edited site. Test your primers on N2 lysis with the appropriate Taq, using a gradient from 55 to 65 °C for regular Taq or 60 to 72 °C for Phusion Taq.<br /><br />PCR size shift: use a 2.5% gel for insertion/deletion &gt;18bp. For deletion &gt; to 200bp, 1.25% agarose gel is the preferred.<br />A large number of F1s can be pooled together when looking for a deletion, but we generally pool 8 F1s (makes it easier to<br />recover edits among F2/3s).  <br /><br />Restriction Enzyme (RE) site insertion: This is the preferred method for a small modification/insertion. 2 F1s can be<br />pooled. Use 5ul of the PCR product and add RE/buffer/H2O to 10ul. Do the reaction overnight. Most of the REs work with this<br />approach (we prefer NheI because it works very well at 37 °C, is available in master mix package from NEB, and contains a<br />putative STOP codon). Run the digestion product on 2.5% gel, short run.<br /><br />‐Controls:  <br /><br />When screening F1s, we make a reaction master mix (PCR reagents and primers) that is added to each of the F1 lysis<br />samples. We also prepare several N2 worm lysis samples with master mix only (negative control) and a few with master mix + a<br />positive control (see below). Be sure to add this positive control PCR after all the other tubes have been closed to avoid<br />contamination.  <br /><br />Positive control for RE digestion: use a PCR fragment containing the selected restriction sites. This control could be<br />amplified from a plasmid using the same polymerase than the one used for the screen. Use 5ul of the unpurified PCR.<br /><br />Positive control for GFP screen: we recommend making a synthetic template containing both your gene‐specific<br />external forward primer and the GFP internal reverse primer. Using 0.1ul of the repair GFP template, do a PCR using GoTaq, the<br />GFP internal reverse primer, and a new forward primer containing the sequence of your forward external primer fused to 25‐<br />28nt complementary to the 5’ end of your repair GFP template. Do the PCR at 60 °C annealing, 50ul reaction, 30 cycles, 45s<br />elongation. Purify the PCR product and use it as a positive control (100pg).  <br /><br />Another control that can be used when screening for GFP are lysed N2s that receives the PCR master mix as well as the external reverse primer (positive control).

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