Journal: Scientific Reports

Article Title: Selective targeting of KRAS oncogenic alleles by CRISPR/Cas9 inhibits proliferation of cancer cells

doi: 10.1038/s41598-018-30205-2

Figure Lengend Snippet: CRISPR/Cas9 targeting of KRAS c.35 G > T in SW620 and SW480 cells. ( a ) Indel frequencies in target KRAS region of SW620 and SW480 cells with sgKRAS-G12V, and in HEK293T cells with sgKRAS-WT. ( b ) Major indel patterns detected in the KRAS target region of SW620 cells with sgKRAS-G12V. Blue: sgRNA target sequence; bold blue: c.35 G > T single-nucleotide missense substitution; orange: PAM sequence; red: mismatched nucleotides. ( c ) Cell proliferation assays of indicated cell lines following transduction with lentiviral vector encoding Cas9, Cas9 and sgKRAS-G12V, or Cas9 and sgKRAS-WT. Red: sgRNA sample relevant to tested cell line. Bars represent the mean ± S.E.M. **** (P < 0.0001), ns: not significant.

Article Snippet: One day after seeding, the cells were transfected in GibcoTM Opti-MEMTM I Reduced Serum Medium (Life Technologies, USA) using 1 μg of target lentiviral vector (empty lentiCRISPR v2 or lentiCRISPR v2 containing sgKRAS-WT, sgKRAS-G12V, or sgKRAS-G12D), 0.25 μg of pMD2.G (Addgene, USA), 0.75 μg of psPAX2 (Addgene, USA) and 7 μl of LipofectamineTM 3000 (Life Technologies, USA).

Techniques: CRISPR, Sequencing, Transduction, Plasmid Preparation

Journal: Scientific Reports

Article Title: Selective targeting of KRAS oncogenic alleles by CRISPR/Cas9 inhibits proliferation of cancer cells

doi: 10.1038/s41598-018-30205-2

Figure Lengend Snippet: Indel ratios and transduced cell viability before and after puromycin selection. Indel frequencies in target KRAS region of AsPC-1 and SNU407 cells with sgKRAS-G12D were determined before ( a ) and after ( c ) antibiotic selection. Transduced cell viability was determined using the MTT assay before ( b ) and after ( d ) puromycin selection. For cell proliferation assays, AsPC-1 and SNU407 cells were transfected with lentiviral vectors encoding Cas9, Cas9 and sgKRAS-G12D, or Cas9 and sgKRAS-WT. Red: sgRNA sample relevant to tested cell line. Bars represent the mean ± S.E.M. **** (P < 0.0001), ns: not significant.

Article Snippet: One day after seeding, the cells were transfected in GibcoTM Opti-MEMTM I Reduced Serum Medium (Life Technologies, USA) using 1 μg of target lentiviral vector (empty lentiCRISPR v2 or lentiCRISPR v2 containing sgKRAS-WT, sgKRAS-G12V, or sgKRAS-G12D), 0.25 μg of pMD2.G (Addgene, USA), 0.75 μg of psPAX2 (Addgene, USA) and 7 μl of LipofectamineTM 3000 (Life Technologies, USA).

Techniques: Selection, MTT Assay, Transfection