CRISPR‐based knock‐in mutagenesis of the pioneer transcription factor FOXA1: optimization of strategies for multi‐allelic proteins in cancer cells

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,

New Insights into the Therapeutic Applications of CRISPR/Cas9 Genome Editing in Breast Cancer

“…TOPO cloning allele frequency data and RNA-seq of these cell lines resulted in finding 28-100% mature KI mutation-carrying mRNA. This concludes that CRISPR/Cas9 can facilitate systemic analysis of the molecular mechanisms involved in various breast cancer types, with the utmost precision [174].…”

“…Furthermore, histone deacetylases and cyclin-dependent kinases were recovered using this approach. This provides a broader insight into the druggable targets of TNBC and hints at further studies that can identify multi-target synthetic lethal interactions [174].…”

Functional annotation of breast cancer risk loci: current progress and future directions

“…Reviewed in ref. [ 104 ], with recent additions including CRISPR knock-in [ 106 ] and repression CRISPRoff [ 107 ]. Highly specific assays, multiple target genes can be modulated simultaneously and the introduced genomic changes are potentially reversible.…”

Entering the second decade: FEBS Open Bio in 2022

“…To present articles published in the journal to a lay audience, we started a new series of articles called ‘Insights’ coordinated by our editor Cornelia de Moor. To date, we have published four Insight articles, which cover subjects as diverse as the role of the GAD67 protein in fear in rats [ 12 , 13 ], the use of CRISPR to generate mutations in multiple copies of a gene [ 14 , 15 ], the use of miRNAs as biomarkers for gastric cancer [ 16 , 17 ] and the association between extracellular vesicles and susceptibility to social stress in mice [ 18 , 19 ].…”