Abstract
With the continued emergence of risk loci from Genome-Wide Association studies and variants of uncertain significance identified from patient sequencing, better methods are required to translate these human genetic findings into improvements in public health. Here we combine CRISPR/Cas9 gene editing with an innovative high-throughput genotyping pipeline utilizing KASP (Kompetitive Allele-Specific PCR) genotyping technology to create scarless isogenic cell models of cancer variants in ~1 month. We successfully modeled two novel variants previously identified by our lab in the PALB2 gene in HEK239 cells, resulting in isogenic cells representing all three genotypes for both variants. We also modeled a known functional risk SNP of colorectal cancer, rs6983267, in HCT-116 cells. Cells with extremely low levels of gene editing could still be identified and isolated using this approach. We also introduce a novel molecular assay, ChIPnQASO (Chromatin Immunoprecipitation and Quantitative Allele-Specific Occupation), which uses the same technology to reveal allele-specific function of these variants at the DNA-protein interaction level. We demonstrated preferential binding of the transcription factor TCF7L2 to the rs6983267 risk allele over the non-risk. Our pipeline provides a platform for functional variant discovery and validation that is accessible and broadly applicable for the progression of efforts towards precision medicine.
Highlights
We used HEK293 cells to create isogenic cell models of two VUSs identified from a gastric cancer cohort in the PALB2 locus, a single-base substitution (PALB2-SNV) and a 9-base in-frame deletion (PALB2-DEL)[16]
Plasmid DNA expressing wildtype Cas[9] and guide RNA (gRNA) with on-target activity confirmed by T7 endonuclease I assay were co-transfected with a 127-nt single-stranded oligonucleotide donor for either the single base substitution or the 9-base deletion (Fig. 1b,c; Supplementary Fig. 1a)
We have presented a novel pipeline based on KASP genotyping for generating scarless isogenic cell models of SNPs and VUSs for functional studies
Summary
We demonstrate the power of this novel assay in the detection and quantification of allelic binding preference for transcription factor TCF7L2 and chromatin insulating protein CTCF within edited HCT-116 clones that are heterozygous for the risk SNP rs6983267 produced from our pipeline. By combining genomic DNA of wildtype HCT-116 cells with that of our newly created heterozygous clones (G/T) at varying ratios, we were able to determine the KASP genotyping platform could detect the presence of the mutant (T) allele at concentrations as low as 5% (Supplementary Fig. 5).
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