(A) Generation of the sgRNA expression construct by PCR amplification ● TIMING 2 h Preparation of diluted U6 PCR template. We recommend using pSpCas9(BB) or pSpCas9n(BB) (supplementary Data 2) as a PCR template, but any U6-containing plasmid can be used. Dilute the template with ddH2O to a concentration of 10 ng μl–1. Note that if a plasmid or cassette already containing a U6-driven sgRNA is used as a template, a gel extraction will need to be performed after PCR (Step 5A(iv)), using the QIAquick gel extraction kit according to the manufacturer's instructions, to ensure that the product contains only the intended sgRNA and no trace of sgRNA carryover from the template.Preparation of diluted PCR primers. Dilute the U6-Fwd and U6-Rev (designed either using the CRISPR Design Tool or by hand and unique for each sgRNA, Step 1) primers (table 1) to a final concentration of 10 μM in ddH2O by adding 10 μl of the 100 μM primer stock to 90 μl of ddH2O.U6-sgRNA PCR. Set up the following reaction for each U6-Rev primer as follows: ComponentAmount (μl)Final concentrationHerculase II
