Figure 1.

DNA constructs and overview of the experimental setup. (A) Schematic map of the plasmid expressing three single-guide RNAs (sgRNAs) simultaneously. The sgRNA is expressed under the control of the U6 promoter, while the expression of spCas9 and enhanced green fluorescent protein is driven by the CBh (chicken beta-actin hybrid) promoter. A third sgRNA was introduced to target Nf1, Nf2, Tsc2, Cdkn2a, or Rb1 along with the first and second sgRNAs to target Tp53 and Pten, respectively. P2A, 2A self-cleaving peptide; pA; poly A signal. (B) Schematic representation of the experimental setup. After the establishment of the mouse tumor model through hydrodynamic tail vein injection of the clustered regularly interspaced short palindromic repeat-caspase 9 (CRISPR-Cas9) constructs, organoids were prepared from the generated mouse liver tumor to analyze their sensitivity to radiation.