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. 2019 Feb 15;10(14):1440–1457. doi: 10.18632/oncotarget.26677

Figure 3. Generation of CRISPR-mediated NF1 and RASA1 knock out in patient-derived CRC organoids.

Figure 3

(A) Selection strategy to generate NF1 and RASA1 knock out organoids after CRISPR-mediated homologous recombination. (B, D) Genetic strategy to target the NF1 and RASA1 locus for homologous directed repair via the CRISPR/Cas9. The structure of the NF1 and RASA1 gene and the targeted exon is depicted at the top. Black boxes illustrate exons, separated by introns. Red scissors show sgRNA-generated double stranded breaks. Blue arrows illustrate PCR primer pairs. The agarose electrophoresis gel shows the ~1kb PCR product of the allele that was repaired by NHEJ of NF1 (Clone # 6 and 12) and RASA1 (Clone # 1 allele 1 and 2, Clone # 2 allele 1 and 2) in selected clones. Sanger sequencing indicate the introduced small indels per clone. Nonmatching bases are shown in orange. Regions of the sgRNA complementary to the protospacer (underlined) are shown in blue. Red arrow heads indicate cleavage sites. (C, E) Western blot analysis for NF1 and RASA1 presence in the indicated organoid lines.