(B) Cloning sgRNA into the pSpCas9(BB) vector for co-expression with Cas9 ● TIMING 3 d Preparation of the sgRNA oligos inserts. Resuspend the top and bottom strands of oligos for each sgRNA design (Step 1) to a final concentration of 100 μM. Prepare the following mixture for phosphorylating and annealing the sgRNA oligos (top and bottom strands): ComponentAmount (μl)sgRNA top (100 μM)1sgRNA bottom (100 μM)1T4 ligation buffer, 10×1T4 PNK1ddH2O6Total10Phosphorylate and anneal the oligos in a thermocycler by using the following parameters: 37 °C for 30 min; 95 °C for 5 min; ramp down to 25 °C at 5 °C min–1.Dilute phosphorylated and annealed oligos 1:200 by adding 1 μl of oligo to 199 μl of room temperature ddH2O.Cloning the sgRNA oligos into pSpCas9(BB). Set up a ligation reaction for each sgRNA, as described below. We recommend also setting up a no-insert, pSpCas9(BB)-only negative control for ligation. Note: if you are using the Cas9 D10A nickase mutant for subsequent applications, substitute pSpCas9(BB) with pSpCas9n(BB). Alternatively, if fluorescence-based screening or selection is needed, substitute with pSpCas9(BB)-2A-GFP, pSpCas9(BB)-2A-Puro, pSpCas9n(BB)-2A-GFP or pSpCas9n(BB)-2A-Puro. The following
