CRISPR/Cas9 targeted mutagenesis
Many of the experiments we perform using C. elegans would greatly benefit from the ability to engineer the genome at will – adding tags to genes, deleting genes, or replacing genes with mutant variants. Until recently, however, this ability has eluded C. elegans researchers. In early 2013 several groups reported the use of the Streptococcus pyogenes CRISPR/Cas9 system to generate double strand breaks (DSBs) at specific genomic loci in organisms including yeast, flies, mammalian cells, and zebrafish. DSBs induced by radiation or transposon excision have been used extensively in C. elegans, both for mutagenesis and to induce homologous recombination. The ability to target DSBs to any desired spot in the genome, however, would finally allow true freedom in genome engineering. For this reason, we and several other groups adapted the CRISPR/Cas9 system for use in C. elegans.
The S. pyogenes CRISPR/Cas system effects site-specific cleavage of double stranded DNA through a complex containing the Cas9 endonuclease and two noncoding RNAs (crRNA and tracrRNA). Target site specificity is mediated by a 20 nt spacer region in the crRNA that is complementary to the target DNA, and a 3 nucleotide motif (NGG) following the target site in the DNA (termed PAM, for protospacer adjacent motif). Thus a wide range of target sites can be chosen. Conveniently, a single synthetic guide RNA (sgRNA) that fuses the 3’ end of crRNA to the 5’end of tracrRNA is sufficient to target Cas9 to a specific site and generate DSBs (Figure 1).
Figure 1. Cas9/sgRNA in complex with a target site (click to enlarge). RuvC and HNH endonuclease domains together generate a double-strand break. In the sgRNA sequence, green bases are crRNA derived and red bases tracrRNA derived.
To express Cas9 in the germline, we generated vectors placing the C. elegans optimized Cas9 coding sequence under control of the eft-3 or hsp-16.48 promoters and the tbb-2 3’UTR (Figure 2A). We also generated Cas9::EGFP variants of these vectors in order to follow expression of Cas9. In our hands, expression of Cas9 from the eft-3 promoter was lethal, and we currently exclusively use the hsp-16.48 promoter.
Figure 2. Cas9 and sgRNA vectors we generated for use in C. elegans (click to enlarge). A file with sequence maps can be downloaded here.
To provide the sgRNA, we generated a vector containing a T7 promoter upstream of the sgRNA sequence for in vitro transcription of the sgRNA, and a vector expressing the sgRNA under control of the regulatory sequences of a U6 snRNA. Both vectors contain BsaI restriction sites for simple insertion of the target recognition sequence as an oligomer linker (Figure 2). In our initial experiments, the U6 driven sgRNA was the most efficient. Since it has the additional benefit of not needing to generate RNA in vitro, we currently use this sgRNA expression vector.
To date, we have used these reagents to generated mutations in >6 loci and to add a GFP tag to the 3'-end of a gene.
- Design the sgRNA construct. To identify suitable target sites, we currently use the consensus sequences shown in figure 3.
- Order complementary oligonucleotides that match the desired target site. Oligo's should be ordered phosphorylated, such that after annealing the can be ligated into BsaI digested, dephosphorylated vector.
Figure 3. Cloning of a target sequence into sgRNA vectors linearized by BsaI digestion (click to enlarge). The 20 bp target sequence is outlined in blue, and the PAM in yellow. Two oligonucleotides are ordered that can be annealed to yield the indicated product. In this example the overhangs are designed for insertion into pMB70. For pMB60, use 'tata' instead of 'aatt' for the forward primer.
- Inject animals with an injection mixture containing the following:
- 50 ng/µl Phsp-16.48::Cas9 (pMB67)
- 50 ng/µl U6 driven sgRNA (pMB70)
- 15 ng/µl Pmyo-3::mCherry (pCFJ104 from the Jorgensen lab)
We inject in batches of 20 worms, which takes ~30 min.
- After injection of the last animal, we let the worms recover for 30 min, then subject them to a 1 hr heat shock at 34°C in a water bath. Following the heat shock we maintain the animals at 25°C.
- To identify mutations, we allow transgenic F1 animals (expressing mCherry) to lay eggs, before PCR amplifying and sequencing a region surrounding the targeted site. Mutations are easily identified from heterozygous F1 animals.