Figure 3.

Cas9-mediated editing of Trp53 in Arf^−/−Eμ-myc lymphomas is positively selected for following DXR treatment in vivo. (A) Schematic diagram of in vivo fitness assay. (B) Kaplan-Meier analysis of tumor-free survival of mice injected with Rosa26 or Trp53 Cas9 targeted Arf^−/−Eμ-myc and p53^−/−Eμ-myc lymphomas following treatment with DXR. (C) Detection of GFP in tumors arising from QCiG-p53-infected Arf^−/−Eμ-myc lymphomas following exposure to DXR and analyzed 3 d later. White arrows denote GFP fluorescence in lymph nodes originating from the presence of QCiG-p53 in the resulting tumors. (D) FACS analysis of the indicated Cas9 targeted Eμ-myc lymphomas analyzed before injection into mice (input), from tumors arising in vivo (pre-DXR), and from tumors for which the host had received DXR treatment (post-DXR). (E) SURVEYOR assay of DNA from QCiG-p53- and QCiG-Rosa-infected Arf^−/−Eμ-myc lymphomas isolated from mice prior to DXR treatment. (F) Immunoblot showing long-term Cas9, p53, and GFP expression in QCiG-Rosa and QCiG-p53 Arf^−/−Eμ-Myc lymphomas in vivo. Samples are from three separate tumors isolated prior to (pre-DXR) or following (post-DXR) DXR treatment. In the case of post-DXR samples for QCiG-Rosa Arf^−/−Eμ-myc lymphomas, tumors were harvested after relapse (∼10 d after post-DXR treatment). The asterisk highlights a truncated p53 protein arising in the Cas9-edited samples.