Cryopreserved C57BL/6J plateable mouse hepatocytes (product code: MBCP01; Lonza). Cryopreserved human hepatocytes were obtained from Lonza.

The NIH 3T3, Hepa 1-6, HEK293 cells, and human hepatocytes were electroporated with a 4D-Nucleofector X Unit (Lonza). NIH 3T3 cells were electroporated using program EN-158, SG Cell Line 4D-Nucleofector solution (Lonza), and the following conditions: 20µL SG nucleofection buffer, 2.2×105 cells, 0.5µL of 20µg/µL sgRNA (TriLink Biotechnologies), and 1.7µL of 61µM 3NLS SpCas9 (product code: 1074182; Integrated DNA Technologies). For reactions with Cas9 RNPs, the SpCas9 RNP was incubated with the Hpd targeting sgRNA for 20min at room temperature. Hepa 1-6 and HEK293 cells were electroporated using the SF Cell Line 4D-Nucleofector solution (Lonza) along with the 4D-Nucleofector programs CM-138 and CM-130, respectively.

The Hepa1-6 and HEK293 cell electroporation conditions were as follows: 20?µL SF nucleofection buffer; 1.2×105 cells; 0.5µL of 20µg/µL sgRNA; and 0.76µL of 1.32µg/µL Cas9 pDNA, 1µL of 1µg/µL CleanCap Cas9 mRNA (product code: L-7206-1000; TriLink Biotechnologies), 1.7µL of 61µM 3NLS SpCas9, or 1.7 uL of 61µM 3NLS HiFi SpCas9 (product code: 1078728; Integrated DNA Technologies). For cell lines, delivery efficiency was determined by separately electroporating cells with 0.4µL of 1µg/µL pmaxGFP (Lonza) and analyzing cells using a Guava Flow Cytometer at 24h after electroporation (Supplementary Table S4).

Primary human hepatocytes were electroporated using modified procedures described in Zabulica et al.39 Briefly, hepatocytes were electroporated with P3 Primary Cell 4D-Nucleofector solution (Lonza), program CA-137, and the following electroporation conditions: 100µL P3 nucleofection buffer, 1.8µL of 20µg/µL sgRNA, 5µL of 61µM V3 SpCas9 (product code: 1081059; Integrated DNA Technologies) or 5µL of 61µM V3 HiFi SpCas9 (product code: 1081061; Integrated DNA Technologies), and 3µL of 100µM Electroporation Enhancer (product code: 1075916; Integrated DNA Technologies).

For primary mouse hepatocytes, we first compared different electroporation programs using Lonza 4D and 2b Nucleofectors and observed the highest transfection efficiency using program T-028 on the 2b Nucleofector system (Supplementary Fig. S2). In subsequent experiments, primary mouse hepatocytes were electroporated using Lonza Nucleofector 2b (program T-028), Mouse/Rat Hepatocyte Nucleofector solution (Lonza), and the following electroporation conditions: 100?µL Mouse/Rat Hepatocyte Nucleofector solution, 1.2×106 cells, 1.5µL of 20µg/µL sgRNA, and 4µL of 1µg/µL CleanCap Cas9 mRNA, 4.9µL of 61µM 3NLS SpCas9, and 4.9µL of 61µM 3NLS HiFi SpCas9.