Hepatocytes were isolated from 6 −12-week-old female mTmG mice on a C57BL/6 background by collagenase perfusion as previously described(10 (link)). A chemically modified synthetic single guide RNA (sequence: 5’-UCGUGGGGGUCCUGACCUAC-3’) targeting the mouse Cypor gene was obtained (Synthego). Along with spCas9 protein (Integrated DNA Technologies), sgRNA was delivered to primary hepatocytes using the CRISPRMAX reagent (Thermo Fisher Scientific) with modifications to manufacturer protocol. Briefly, the assembled ribonucleoprotein complexes were added to non-attached primary hepatocytes in suspension at 1×106 cells / mL in HCM media (Lonza). Cells were incubated with rocking at 37°C for 2 hours before transplantation. Hepatocyte transplantation was conducted by injecting 5×105 hepatocytes into the spleen. An aliquot of transfected cells was plated on a collagen-coated plate in HCM media and harvested at day 5 post-transfection for gDNA extraction and indel assessment.