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[DESCRIPTION]
This protocols is from:<br />Daniel J. Dickinson, et al. (2015) <a href="http://www.genetics.org/content/early/2015/06/03/genetics.115.178335" target="_blank">Streamlined genome engineering with a self-excising drug selection cassette</a>. <em>Genetics</em><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>June 3, 2015<span class="cit-sep cit-sep-after-article-ahead-of-print-date">,</span></span><span class="cit-doi"><span class="cit-sep cit-sep-before-article-doi">doi:</span>10.1534/genetics.115.178335</span><br /><br />Please see the <a href="http://www.genetics.org/content/early/2015/06/03/genetics.115.178335" target="_blank">full manuscript</a> or additional details. Also, updates are available on the <a href="http://wormcas9hr.weebly.com/" target="_blank">author's website</a><a href="http://www.genetics.org/content/198/4/1347.full" target="_blank">.<br /></a>

[GUIDELINES]
Please see the <a href="http://wormcas9hr.weebly.com/" target="_blank">author's website</a> for updates and additional details.<br /><br /><strong>Before the Experiment</strong><br /><br /><span style="text-decoration: underline;">Choose the Cas9 target site<br /></span>1) Identify a 100-200 bp region in which the Cas9 target site should be located. We generally use a 200 bp window centered on the start codon (for N-terminal tags) or stop codon (for Cterminal tags. <br /><br />2) Submit this genomic sequence to the Zhang lab’s CRISPR design tool at <a href="http://crispr.mit.edu/" target="_blank">http://crispr.mit.edu</a>. Make sure you have selected <em>C. elegans</em> as the genome for checking specificity. <br /><br />3) The design tool returns a list of potential targeting sequences, ranked in order of predicted specificity. We always try to choose target sites with a specificity score &gt;95, and in most cases find a site that scores 98 or 99 (100 indicates perfect specificity). If there are several candidate sites with high specificity, choose the site that is closest to the desired insertion site (start or stop codon). The best case scenario is to have the insertion site within the guide sequence and within 10 bp of the PAM (NGG motif), so that insertion of mNG^SEC^3xFlag will disrupt the target site (Figure P1A). If this is not possible, then choose a Cas9 target sequence that is within the coding region of your gene. For the targeting sequence you choose, copy and paste the list of potential off-target sites into a Word document or Excel sheet and save this list for future reference. <br /><br /><span style="text-decoration: underline;">Add the target sequence to the Cas9–sgRNA construct </span><br /><br />1) The design tool returns target sites of the form 5’N20-NGG-3’, where N is any base. You need to insert the N20 sequence into the Cas9–sgRNA construct (pDD162, Addgene #47549). We use NEB’s Q5 Site-Directed Mutagenesis Kit to do this. Use forward primer 5’-N20GTTTTAGAGCTAGAAATAGCAAGT-3’, where N20 is your 20 bp targeting sequence from the design tool, and reverse primer 5’-CAAGACATCTCGCAATAGG-3’. <br /><br />2) IMPORTANT: Do not include the PAM (NGG motif) in your primers for the Cas9-sgRNA construct. The NGG motif must be present in the target DNA, but it is not part of the sgRNA.<br /><br />3) We use sequencing primer <span class="s1">5’-GGTGTGAAATACCGCACAGA-3’ </span>to verify correct insertion of the targeting sequence. <br /><br /><span style="text-decoration: underline;">Design primers to add homology arms to an FP–SEC vector</span> <br /><br />Figure 1C shows our strategy for cloning homology arms into FP–SEC vectors. Homology arms are generated by PCR and inserted in place of the ccdB negative selection markers, which are flanked by restriction sites. We chose these particular restriction sites so that no residual sequence is left behind after addition of the homology arms. ccdB negative selection makes this cloning strategy exceptionally robust and efficient: in a pilot experiment, we generated 8 repair templates in parallel in a single afternoon. 7/8 of these reactions yielded &gt;80% correct clones; the remaining reaction also yielded correct clones, albeit at a lower frequency. <br /><br />You need to design four primers: two for each homology arm. These primers will amplify the homology arms and add sequence overlaps for Gibson assembly to the ends of each arm. If FP::SEC insertion will not disrupt the Cas9 target site, your primers will also need to introduce silent mutations to prevent Cas9 from cutting the repair template. <br /><br />You have a choice of two possible pairs of restriction enzymes to digest the FP–SEC vector: AvrII+SpeI or ClaI+SpeI. If AvrII and SpeI are used, the repair template will include a flexible linker between the 5’ homology arm and FP (this is useful for generating C-terminal tags). If ClaI and SpeI are used, the 5’ homology arm will be fused directly to the FP, with no added sequence (this is useful for N-terminal tags, or when no flexible linker is desired). Figure P1 shows sample primer designs for each situation. <br /><br />Detailed primer design instructions: <br /><br />1) First, decide whether additional mutations are needed to prevent Cas9 from cutting the repair template. We make additional mutations whenever the insertion site is not within the 10 bp of the target sequence closest to the PAM. Additional mutations are made using synonymous codons so that the amino acid sequence is not altered (See Figure P1B for an example). If possible, the simplest and most effective approach is to mutate the PAM (NGG motif), since this motif is absolutely required for cleavage of a substrate by Cas9. If a PAM mutation is not feasible, introduce as many mutations as possible (at least 5-6) in the target sequence.<br /><br />2) Each homology arm should be 500-700 bp long. The positions of the two primers most proximal to the FP^SEC^3xFlag module (i.e., the reverse primer for the 5’ homology arm and the forward primer for the 3’ homology arm) are fixed by the need to insert FP^SEC^3xFlag at a specific location. The positions of the distal primers are more flexible. We design the proximal primers first based on our desired insertion site, and then use Primer-BLAST to pick the best possible distal primers. <br /><br />3) Decide with FP you want to insert, and consult Table P1 for the sequence that needs to be added to the end of each primer to allow Gibson assembly. <br /><br />4) Ideally, the primer length should be less than 60 bp, because longer primers are much more expensive and fail more often. If you find you need a longer primer because your Cas9 target site is far away from the insertion site, it might be more cost effective to purchase a synthetic DNA fragment (we like IDT’s gBlocks) containing the homology arm instead of using PCR. <br /><br />5) Before ordering primers, double check that the mNG::3xFlag will be in frame with your gene of interest.<br /><br /><strong>Injections to Generate Knock-ins</strong><br /><span style="text-decoration: underline;">Day 9 or 10: Look for homozygous plates<br /></span><br />Look for plates where 100% of L4s and adults are Rollers. These are homozygous knock-in animals. They can be maintained indefinitely, outcrossed if desired, or mated to another genetic background. The strong Rol phenotype makes it very easy to follow the knock-in in crosses (but note that Rol males mate poorly). You can also take L1s from these plates and proceed directly to heat shock to remove the selectable markers. <br /><br />It is straightforward to generate lethal mutations with our strategy, because knock-in alleles can be isolated and maintained as heterozygotes. You will know that your knock-in is lethal if you see only heterozygous plates (i.e., plates with ~1/4 wild-type worms and 1/4 dead embryos). You should expect your initial knock-in to be lethal if you are making an N-terminal tag on an essential gene, because the initial knock-in is a transcriptional null mutation.<br /><br />Note: It is impossible to tell whether two strains that originated from the same injection plate derive from independent insertion events or a single insertion event. Therefore, although we single 5-10 worms from each plate in the previous step, we keep only one line from each plate.<br /><br /><strong>Selectable marker removal<br /></strong><br />Figure P2 shows the overall scheme for selectable marker removal. In most cases, the initial knock-in is homozygous viable and marker removal is extremely simple (Figure P2A). <br /><br />If the initial knock-in is lethal, marker removal is slightly more complicated because heterozygous knock-in animals segregate wild-type animals at each generation, which makes it impossible to identify animals that have excised the marker based on wild-type phenotype alone (Figure P2). In this situation, there are two choices. If your knock-in strain is visibly fluorescent, you can simply heat shock heterozygotes and identify animals that have excised the marker based on a wild-type phenotype plus visible fluorescence (Figure P2B). If fluorescence in your knock-in strain is too dim to see by eye, you need to mate in a GFP-marked balancer chromosome first (Figure P2C). Mate males carrying an appropriate GFP-marked balancer to Rol knock-in hermaphrodites. Pick GFP-positive, Rol animals from the F1 progeny. These animals should now no longer segregate wild-type progeny in the absence of heat shock (Figure P2C). Use these balanced knock-in worms for subsequent steps.<br /><br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/baicz36.png" alt="" data-src="https://s3.amazonaws.com/pr-journal/bahcz36.png" data-ofn="figureP1.png" /><br /><strong>Figure P1</strong>: Primer design examples. (A) Primers for an N-terminal tag. In this example, mNG^SEC^3xFlag insertion disrupts the Cas9 target site, so no mutations are required. (B) Primers for a C-terminal tag, including the optional flexible linker that is built into the FP–SEC vectors. In this example, a silent PAM mutation is introduced to block Cas9 cleavage.<br /><br /> <br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/bakcz36.png" alt="" data-src="https://s3.amazonaws.com/pr-journal/bajcz36.png" data-ofn="tablep1.png" /><br /><strong>Table P1</strong>: Primers for insertion of homology arms into FP::SEC vectors.<br /><br /><br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/bancz36.png" alt="" data-src="https://s3.amazonaws.com/pr-journal/bamcz36.png" data-ofn="figureP2.png" /><br /><strong>Figure P2</strong>: Genetic schemes for marker self-excision. (A) For homozygous viable knock-ins, the situation is simple: after heat shock, any wild-type worms will have lost both copies of SEC. (B) If the knock-in is homozygous lethal, the strain produces 1/4 wild-type progeny at each generation. This makes it impossible to unambiguously identify worms that have lost SEC based on wild-type phenotype alone, although knock-ins may be identifiable if they show visible fluorescence. (C) A simple, 1-step cross to introduce a GFP balancer chromosome results in a strain that does not segregate any wild-type progeny. Heat shock-induced marker excision in this background generates wild-type animals that can be easily and unambiguously identified. <br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />

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