▲ CRITICAL STEP For HDR applications, we recommend cloning sgRNA guides into one of the sgRNA expression plasmids described in Step 5B, rather than using the PCR-based expression approach. (A) For the cotransfection of the HDR-targeting plasmid with the Cas9 plasmid: (i) Pre-mix the following DNA in PCR tubes: Cas9 plasmid (pSpCas9(sgRNA))500 ng Linearized targeting plasmid500 ng(B) For the cotransfection of ss0DN and with the Cas9 plasmid: (i) Pre-mix the following DNA in PCR tubes: Cas9 plasmid (pSpCas9(sgRNA))500 ng ssODN template (10 μM)1 μl 20Dissociation of cells for transfection. Remove the medium and rinse the cells once gently with DPBS, taking care not to dislodge cells. Add 2 ml of TrypLE to a T75 flask and incubate it for 5 min at 37 °C, and then add 10 ml of warm D10 medium and triturate gently in a 50-ml Falcon tube.
