Fig. S5.

CRISPR-mediated gene knockout in primary T cells and BMDMs. (A) Cas9-expressing T cells were mixed with wild-type T cells. The cells were activated with anti-CD3 and anti-CD28 antibodies for 2 d. The activated T cells were transduced with retroviral particles expressing sgRNAs. The transduced T cells were selected with puromycin for 4 d before assessment of knockout efficiencies. (B) Scheme of CRISPR-mediated gene knockout in primary BMDMs. BM cells from R26-Cas9iGFP/+ mice and wild-type mice were differentiated for 2 wk in the presence of M-CSF. BMDMs were transduced with lentiviruses expressing sgRNAs targeting mouse CD64 and CD14 genes. The transduced cells were selected with puromycin for 4 d and the gene knockout efficiency was analyzed by flow cytometry. (C) FACS plots of the transduced BMDMs derived from control animals (Top) and R26-Cas9iGFP/+ animals (Bottom) stained with antibodies against the targeted surface markers. The summary of the experiments is shown at Right. The data are representative of three individual Cas9 transgenic mice (each representing a data point in the summary).