Abstract
Precise genome engineering of living cells has been revolutionized by the introduction of the highly specific and easily programmable properties of CRISPR-Cas9 technology. This has greatly accelerated research into human health and has facilitated the discovery of novel therapeutics. CRISPR-Cas9 is most widely employed for its ability to inactivate, or knockout, specific genes, but can be also used to introduce subtle site-specific substitutions of DNA sequences that can lead to changes in the amino acid composition of proteins. Despite the proven success of CRISPR-based knockin strategies of genes in typical diploid cells (i.e. cells containing two sets of chromosomes), precise editing of cancer cells, that typically have unstable genomes and multiple copies of chromosomes, is more challenging and not adequately addressed in the literature. Herein we detail our methodology for replacing endogenous proteins with intended knockin mutants in polyploid cancer cells and discuss our experimental design, screening strategy, and facile allele-frequency estimation methodology. As proof of principle, we performed genome editing of specific amino acids within the pioneer transcription factor FOXA1, a critical component of estrogen and androgen receptor signaling, in MCF-7 breast cancer cells. We confirm proper levels of mutant FOXA1 protein expression and intended amino acids substitutions via western blotting and mass spectrometry. In addition, we show that mutant allele-frequency estimation is easily achieved by TOPO cloning combined with allele-specific PCR, which we later confirmed by next-generation RNA-sequencing. Typically, there are 4 - 5 copies (alleles) of FOXA1 in breast cancer cells making the editing of this protein inherently challenging. As a result, most studies that focus on FOXA1 mutants rely on ectopic overexpression of FOXA1 from a plasmid. Therefore, we provide an optimized methodology for replacing endogenous wildtype FOXA1 with precise knockin mutants to enable the systematic analysis of its molecular mechanisms within the appropriate physiological context.
