Reliable CRISPR/Cas9 Genome Engineering in Caenorhabditis elegans Using a Single Efficient sgRNA and an Easily Recognizable Phenotype

CRISPR/Cas9 genome engineering strategies allow the directed modification of the Caenorhabditis elegans genome to introduce point mutations, generate knock-out mutants, and insert coding sequences for epitope or fluorescent tags. Three practical aspects, however, complicate such experiments. First, the efficiency and specificity of single-guide RNAs (sgRNA) cannot be reliably predicted. Second, the detection of animals carrying genome edits can be challenging in the absence of clearly visible or selectable phenotypes. Third, the sgRNA target site must be inactivated after editing to avoid further double-strand break events. We describe here a strategy that addresses these complications by transplanting the protospacer of a highly efficient sgRNA into a gene of interest to render it amenable to genome engineering. This sgRNA targeting the dpy-10 gene generates genome edits at comparatively high frequency. We demonstrate that the transplanted protospacer is cleaved at the same time as the dpy-10 gene. Our strategy generates scarless genome edits because it no longer requires the introduction of mutations in endogenous sgRNA target sites. Modified progeny can be easily identified in the F1 generation, which drastically reduces the number of animals to be tested by PCR or phenotypic analysis. Using this strategy, we reliably generated precise deletion mutants, transcriptional reporters, and translational fusions with epitope tags and fluorescent reporter genes. In particular, we report here the first use of the new red fluorescent protein mScarlet in a multicellular organism. wrmScarlet, a C. elegans-optimized version, dramatically surpassed TagRFP-T by showing an eightfold increase in fluorescence in a direct comparison.


Supplementary Tables
Supplementary Table 1: Prevalence of GNGG and GGNGG protospacers in and close to exons of two-pore domain potassium channel genes.

Building sgRNA expression vectors using pPT2
This protocol describes the steps and tools used to generate sgRNA expression vectors using the pPT2 vector backbone. See the materials and methods section for the required reagents (e.g. Gibson assembly reagents, sequencing primers). We recommend the online service benchling.com for sgRNA, oligo and vector design.

I. Identifying a suitable protospacer motif
• A protospacer is a 19-20 bp sequence flanked at its 3' end by an NGG PAM (protospacer adjacent motif). Different online tools are available to identify possible protospacers in a region of interest (crispr.mit.edu ; tefor.net/crispor/crispor.cgi ; benchling.com).
• When multiple protospacer sequences are possible, select the closest (to the site to engineer) and/or the most specific sequence (use the off-target prediction tool provided by benchling for example). In general, four non-matching bases should be enough to significantly reduce off-target cutting, especially if the mismatches are located in the 3' region of the protospacer (Hsu et al. 2013).

II. Building the sgRNA vector sequence in silico
• The pPT2 vector contains the U6 promoter and 3' UTR of K09B11.12 (Friedland et al. 2013) and two restriction sites (PmeI and SexAI) to linearize the vector, followed by the invariant sgRNA scaffold sequence (see Figure 1A).
• To generate the sgRNA expression vector sequence, insert the protospacer sequence (without the PAM, i.e. NGG) between the U6 promoter and the sgRNA scaffold as shown in figure 1B.
• If the selected protospacer sequence does not begin with a guanine residue, add this nucleotide manually to the 5' of the protospacer (i.e. resulting in a "19+1" bp insertion in pPT2, see figure 1B).
• Name this vector pXYn where XY are the initials of the person building the vector and n the number of the vector. Accordingly, the protospacer sequence is then labeled CRpXYn (generate a "feature" with the sequence, excluding the added G, to identify it easily in the genomic sequence).
• Generate one 60 bp oligonucleotide centered on the protospacer sequence as shown in figure 1C (forward or reverse). Gibson assembly can be performed with a single primer (see IIIa). Alternatively, generate two complementary 60 bp oligonucleotides centered on the protospacer sequence as shown in figure 1C (see IIIb). IIIa. Building the sgRNA vector using a single oligonucleotide The protospacer sequence is incorporated into the pPT2 vector as follows: 1 | Linearize pPT2 using the PmeI and SexAI restriction enzymes.
2 | Purify the linearized pPT2 vector using gel extraction. IIIb. Building the sgRNA vector using oligonucleotide dimers

| Gibson assembly
• The protospacer sequence is incorporated into the pPT2 vector as follows.
• Run the program below on a thermal cycler to anneal primers.
• Add 30 µL of water to the resulting sample. • Separate into 50 µL aliquots. Store at -20°C. The enzymes remain active after 10 cycles of freeze-thaw.