sgRNA selection and cloning
Target site selection
A sgRNA target site is 20 nucleotides long, and the only restriction on target site selection is that the three nucleotides following the target site need to adhere to the PAM sequence NGG. In addition, for plasmid-based delivery using a U6 promoter, the sgRNA should start with an A or G. If the target sequence does not start with A or G, simply add a G to the 5'-end (the sequence to be inserted into the vector is then 21 nt long). The following guidelines should be be followed to design an efficient sgRNA:
- Choose a target cut site close to the insertion or mutation site: <30 bp for repair templates with short homologous arms (oligo or PCR templates with 35 bp homology arms), <100 bp for selection based homologous repair templates with longer homologous arms (>500 bp).
- If no appropriate site can be found, consider choosing 2 cut sites further apart, replacing the entire intervening sequence.
- Choose a guide RNA that ends on G or GG, and not on C. High scores in the prediction algorithm published by Doench et al. also seems to correlate with good success rates.
Cloning the sgRNA
We currently clone our sgRNAs into vector pJJR50, which contains the A-U flipped and hairpin extended sgRNA sequence described in Chen et al., under control of the R07E5.16 U6 promoter. Target sequences are cloned into BbsI digested vector as pairs of annealed oligonucleotides with a 5'-TCTT overhang added to the forward oligo, and a 5'-AAAC overhang added to the reverse oligo. The following images show the pJJR50 sequence:
Follow these steps to clone a sgRNA sequence into pJJR50:
- Digest vector pJJR50 with BbsI. Do NOT dephosphorylate the vector.
- Order a pair of complimentary oligo's, consisting of the targets site sequence (adding an extra G to the 5'-end if necessary), and a 5'-TCTT overhang added to the forward oligo, and a 5'-AAAC overhang added to the reverse oligo (see example below). There is no need to phosphorylate the oligos.
- Anneal the oligos as described here.
- Ligate into the digested pJJR50.