Methods for Scarless, Selection-Free Generation of Human Cells and Allele-Specific Functional Analysis of Disease-Associated SNPs and Variants of Uncertain Significance

Sci Rep. 2017 Nov 8;7(1):15044. doi: 10.1038/s41598-017-15407-4.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Base Sequence
  • CRISPR-Cas Systems
  • Colorectal Neoplasms / genetics
  • Colorectal Neoplasms / metabolism
  • Colorectal Neoplasms / pathology
  • Fanconi Anemia Complementation Group N Protein / genetics
  • Fanconi Anemia Complementation Group N Protein / metabolism
  • Gene Editing / methods*
  • Genetic Predisposition to Disease / genetics*
  • Genome-Wide Association Study / methods*
  • Genotype
  • HCT116 Cells
  • HEK293 Cells
  • Humans
  • Polymerase Chain Reaction / methods*
  • Polymorphism, Single Nucleotide*
  • Sequence Homology, Nucleic Acid
  • Transcription Factor 7-Like 2 Protein / genetics
  • Transcription Factor 7-Like 2 Protein / metabolism


  • Fanconi Anemia Complementation Group N Protein
  • PALB2 protein, human
  • TCF7L2 protein, human
  • Transcription Factor 7-Like 2 Protein