Fig 1.
Construction of pLentiCrispr-V2-mOrange (V2mO), its lentiviral production in HEK293T and transduced mOrange expression in GC cell lines AGS and GT5.
A. V2mO plasmid construct, with mOrange cistronically inserted between Cas9 and puromycin cDNAs. Between the original pLentiCrispr-Cas9-V2 plasmid (top) and the V2mO plasmid (bottom) are shown the overlapping sequences and restriction enzymes for the construction. B. HEK293T cells expressed mOrange in lentiviral production, which enables estimation of transformation efficiency and viral titer. C & D. Target cell lines AGS (C) and GT5 (D) were transduced with lentiviral V2mO-RhoA.g5 after puromycin selection and mOrange sorting. E. Cell line GT5 was transduced with lentiviral V2mO-Gli1.g4 after puromycin selection and mOrange sorting. F & G. Cell lines AGS (F) and GT5 (G) were transduced with lentiviral V2mO-Gal3.g1 after puromycin selection and mOrange sorting.
Table 1.
Primers used in this study.
Fig 2.
Western blots showing expressions of RhoA, Gli1 and Gal3 in GC cells, their transduced pools and respective single clones isolated from potential pools.
A. Western blot showed elevated expression of endogenous RhoA in all tested gastric cancer cell lines. B. Western blots showed levels of RhoA knockout in cell lines AGS and GT5 transduced with lentiviral V2mO-RhoA.g5 after puromycin selection and mOrange sorting. C & D. In the AGS and GT5 cell lines, respectively, Western blots of RhoA showed single clones from pool 1 that was transduced with V2mO-RhoA.g5. E. Western blots showed Gli1 pools transduced with lentiviral V2mO-Gli1.g2 and g4. F & G. Western blots of Gal3 in AGS and GT5 cell lines, respectively, and their single clones after transduction with lentiviral V2mO-Gal3.g1.
Fig 3.
Sequencing electropherograms of PCR products and direct estimation of editing efficiencies around indicated binding sites.
A. RhoA gRNA5 binding site in AGS cells. Editing efficiency ratio = 12.5%. B. RhoA gRNA5 binding site in GT5 cells. Editing efficiency ratio = 28.5%. C. Gli1 gRNA4 binding site in GT5 cells. Editing efficiency ratio = 36.1%. D. Gal3 gRNA1 binding site in AGS cells. Editing efficiency ratio = 26.7%. E. Around the Gal3 gRNA1 binding site in GT5 cells. Editing efficiency ratio = 50%.
Fig 4.
TIDE (tracking of indels by decomposition) analyses showing the indel spectra and aberrant sequences of CRISPR/Cas9-gRNA-transduced cell populations versus untreated parental cell populations.
The graphs on the left analyzed indel frequencies within ±10 bp from theoretical gRNA breakpoints. The graphs on the right depicted PCR sequence aberrations; theoretical gRNA cuts were indicated by blue lines. A1 & A2. The RhoA gRNA5 region in AGS cells. Total editing efficiency = 15.6%. B1 & B2. The RhoA gRNA5 region in GT5 cells. Total editing efficiency = 35.7%. C1 & C2. The Gli1 gRNA4 region in GT5 cells. Total editing efficiency = 21.1%. D1 & D2. The Gal3 gRNA1 region in AGS cells. Total editing efficiency = 27.0%. E1 & E2. The Gal3 gRNA1 region in GT5 cells. Total editing efficiency = 41.5%. Eff, efficiency.
Table 2.
Comparison of the editing efficiencies by direct electropherogram estimation, and TIDE and ICE analyses.
Fig 5.
ICE (Inference of CRISPR edits) analyses showing indel and discordance plots.
A. The RhoA gRNA5 region in AGS cells showing a knockout (KO) score of 16. B. The RhoA gRNA5 region in GT5 cells showing a KO score of 40. C. The Gli1 gRNA4 region in GT5 cells showing a KO score of 17. D. The Gal3 gRNA1 region in AGS cells showing a KO score of 25. E. The Gal3 gRNA1 region in GT5 cells showing a KO score of 42.
Fig 6.
RhoA wildtype, RhoA.Y42C and Gal3 gene rescue and cDNA modifications.
A & B. Western blots of RhoA, Flag, and β-actin showed before (A) and after (B) three nonconsecutive nucleotide modifications in the gRNA5 binding site. C. Western blots of RhoA, Flag, and β-actin showing RhoA rescue after DNA modifications in AGS and GT5 cells with Flag being at C-terminal. D. Alignment of rescue plasmids p3xFlag-RhoA.dgWt and p3xFlag-RhoA.dgY42C at the gRNA5 binding site was shown aligned alongside the original RhoA genomic DNA sequence. RhoA gRNA5 was shown at the top, and the amino acid translation was shown at the bottom. Note that wildtype is highlighted in pink for codon Y(TAT), while mutant is Y42C is highlighted in green for codon C(TGT). Three nonconsecutive nucleotides modified are in blue. E. Western blot of Gal3 in AGS cells showed the effect of overexpressing p3xFlag-Gal3.Wt and nucleotide modified p3xFlag-Gal3.dg1, dg2 and dg3. F. Alignment of the rescue plasmids p3xFlag-Gal3.Wt and p3xFlag-Gal3.dg1, dg2, and dg3 with, respectively, 1, 2, and 3 nonconsecutive nucleotides modified from the gRNA5 binding site. The amino acid translations are shown at the bottom. Note that the changed nucleotides are in green. KO, knockout.