We combined 560 ng of Cas9(D10A) or base editor expression plasmid with 240 ng of sgRNA-expression plasmid in a volume that did not exceed 1.5 μl. Detailed plasmid maps for plasmids ABEmax and ABEmaxAW are in fig. S10. This combined plasmid mixture was nucleofected in a final volume of 20 μl per sample in a 16-well Nucleocuvette strip (Lonza). K562 cells were nucleofected using the SF Cell Line 4D-Nucleofector X Kit (Lonza) with 5 × 105 cells per sample (program FF-120), according to the manufacturer’s protocol. U2OS cells were nucleofected using the SE Cell Line 4D-Nucleofector X Kit (Lonza) with 3 × 105 to 4 × 105 cells per sample (program DN-100), according to the manufacturer’s protocol. RNA and DNA were isolated 48 hours after nucleofection. U2OS cells were trypsinized and resuspended in phosphate-buffered saline (PBS), and K562 cells were directly resuspended in PBS before being spun down by centrifugation (800g for 2 min) to isolate cell pellets. Cell pellets were resuspended in PBS (20 μl), and 3 μl was placed in 50 μl of DNA lysis buffer [10 mM tris-HCl (pH 7.0), 0.05% SDS, and proteinase K (25 μg/ml; Sigma-Aldrich)], which was incubated on a heat block at 37°C for 60 min and then 80°C for 20 min. The remaining 17 μl of cells suspended in PBS was pelleted again by centrifugation (800g for 2 min), and RNA extraction was begun on these pellets with the addition of RLT Plus Lysis Buffer (QIAGEN) to the cell pellet. RNA isolation proceeded with the RNeasy Plus Mini Kit (QIAGEN), as described below.

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