medium warm.19Use option A in the table below for preparing the co-transfection of the HDR targeting plasmid with the Cas9 plasmid or option B for the co-transfection of ssODN with the Cas9 plasmid. To prepare transfection controls, see Step 9. If an sgRNA is cloned into pSpCas9(BB)-2A-GFP, cells may also be sorted by fluorescence. If you are using Cas9 nickase to mediate HDR, substitute pSpCas9(sgRNA) with pSpCas9n(sgRNA) from Step 5B(v).