For the large-scale CRISPR-Cas9 protocol, we used the 4D Nucleofector from Lonza. The program that was adopted was EO-115 and the buffer used was P3 buffer for primary cells. As in the small-scale CRISPR-Cas9 protocol, we first prepared the crRNA plus tracrRNA duplex for each crRNA by incubating them at 95°C for 5 minutes in a thermocycler at equimolar concentrations. Cas9 protein (IDT) and gRNA (crRNA plus tracrRNA combination) were then incubated at room temperature for 15 minutes. To electroporate 5 x 10^6 cells, we used 2.2 µM Cas9 and 2.4 µM gRNA. For the larger cell doses, reagents were then scaled up by dividing the cell dose of interest by 5 x 10^6 and then multiplying this number by the final amount of RNP complex used to electroporate 5 x 10^6 cells.
Off-target identification: The GUIDE-seq method was used for unbiased discovery of off-target editing events.8 In this study, HEK293 cells that constitutively express the S.p. Cas9 nuclease (“HEK293-Cas9” cells) were used as the source of Cas9. Alt-R gRNA complexes were formed by combining Alt-R tracrRNA and Alt-R crRNA XT at a 1:1 molar ratio. gRNA complexes were delivered by nucleofection using the Amaxa Nucleofector 96-well Shuttle System (Lonza). For each nucleofection, 3.5 x 10^5 HEK293-Cas9 cells were washed with 13 phosphate-buffered saline, resuspended in 20 µL of solution SF (Lonza), and combined with 10 µM gRNA together with 0.5 µM GUIDE-seq double-stranded DNA donor fragment. This mixture was transferred into 1 well of a Nucleocuvette plate (Lonza) and electroporated using protocol 96-DS-150.