Dlk1 KO using the Alt-R CRISPR-Cas9 system

To KO Dlk1, validated mouse Dlk1 Alt-R CRISPR-Cas9 gRNA (crRNA) #1 (Design ID: Mm.Cas9.DLK1.1. AA), Dlk1 crRNA #2 (Design ID: Mm.Cas9.DLK1.1. AB), Dlk1 crRNA #3 (Design ID: Mm.Cas9.DLK1.1. AC) and Alt-R S.p. Cas9 nuclease V3 (catalog number: #1081058) as the negative control were obtained from the Alt-R CRISPR-Cas9 System (Integrated DNA Technologies Inc., Coralville, IA, USA). According to the manufacturer’s instructions, crRNAs at a final concentration of 10 nM were premixed with the same amount of tracrRNA (#1072532, Integrated DNA Technologies Inc.) in 1× nuclease-free duplex buffer (Integrated DNA Technologies Inc.). Then, the mixture was mixed with Alt-R S.p. Cas9 nuclease (#1081058, Integrated DNA Technologies Inc.) in Opti-MEM (Thermo Fisher Scientific Inc.) to form the RNP complex. The RNP complex was mixed with RNAiMAX transfection reagent (Thermo Fisher Scientific Inc.) in Opti-MEM. Then, the final mixture was added to wells containing 3T3L1 preadipocytes at 1 × 10⁵ per well in six-well plates in 2 ml of high-glucose DMEM with 10% FBS without penicillin/streptomycin. Eighteen hours after transfection, the medium containing the RNP complex was replaced with fresh medium with adipogenic differentiation cocktail to induce differentiation. The efficiency of Dlk1 gene disruption was confirmed by T7 endonuclease I (T7EI) assay.