Gene editing with CRISPR/Cas9
We employed the CRISPR/Cas9 genome editing platform for the generation of the desired targeted genomic rearrangement in the A549
lung cancer cell line. Single guide RNAs (sgRNAs) designed and built to guide Cas9 to bind and cut desired intronic regions in the EML4 and
ALK gene targets, trigger the paracentric genomic rearrangement event upon co-transfection (Figure 1).
Figure 1. Identification of sgRNA target
sites at EML4 and ALK genomic loci.
SgRNAs designed and built to guide
Cas9 to bind and cut desired intronic
regions in the EML4 and ALK gene
targets can trigger the paracentric
genomic rearrangement event upon cotransfection.
‘A’, ‘B’, ‘C’ and ‘D’ denote
primer locations. Transfected cells are
sorted into single cells and expanded for
screening of gene translocation events
by junction PCR across the 5 prime (5p)
and 3 prime (3p) genomic breakpoint
junctions as shown. The occurrence of a
positive ~500bp PCR band for any
isolated single cell clone using the
primers ‘A’ and ‘C’ as well as ‘B’ and ‘D’
suggests the successful generation of
an EML4-ALK fusion product.
Genotype of EML4-ALK mutated cell line
The introduction of the EML4-ALK mutation in the cell line was confirmed via Sanger sequencing as shown in Figure 2 (A, B) for the expected
5p and 3p genomic breakpoints. Sanger sequencing of prepared EML4-ALK cDNA from mRNA of the mutated cell line was carried out to
confirm the expression of the EML4-ALK fusion transcript (Figure 2C). We subsequently confirmed expression of the EML4-ALK fusion protein
to be an 86 kDa fragment as expected by western blotting (data not shown).
Figure 2. (A, B) Sanger sequencing results for the 5p and 3p genomic
junction PCR confirmed the accuracy of the generated EML4-ALK
fusion product. Shown below each chromatogram is a cartoon
representation of the edited genomic locus. The red dashed line
indicates the genomic fusion junction for the ALK and EML4 genes.
(C) Sequence of EML4-ALK fusion transcript across cDNA breakpoint
for the isolated clone. The cyan dashed line is the EML4-ALK cDNA
Functional Characterization of EML4-ALK mutated cell lines
Functional testing of the isogenic A549 EML4-ALK cell line gave a favorable drug response in comparison to its parental A549 cell line (Figure
3). Dose response curves for cells treated with ALK inhibitors crizotinib and ceritinib (Figure 3 A, B) showed that the isogenic A549 EML4-ALK
cell line has selective drug sensitivity to ALK inhibitors crizotinib and ceritinib relative to the parental A549 cell line. Furthermore, this trend is
consistent irrespective of whether it is a dose-response based assay monitored via IncuCyte FLR® live cell imaging system (Essen BioScience;
Figure 3 A, B) or the CellTiter-Glo® Lumniscent Cell Viability Assay (Promega; Figure 3 C, D).
Figure 3. CCL-185IG™ is sensitive to ALK inhibitor drugs. (A, B) A549 and CCL-185IG™ cells were treated with the indicated concentrations of ALK
inhibitors crizotinib and ceritinib and cell survival was determined via live cell analysis. (C, D) A549 and CCL-185IG™ cells were treated with 1 µM
of same compounds and cell survival was confirmed by CellTiter-Glo® Luminescent Cell Viability Assay.