Li Shen, Garay Joseph P, Tubbs Colby A, Franco Hector L
The Lineberger Comprehensive Cancer Center, Department of Genetics, University of North Carolina at Chapel Hill, NC, USA.
FEBS Open Bio. 2021 Jun;11(6):1537-1551. doi: 10.1002/2211-5463.13139. Epub 2021 Mar 20.
Precise genome engineering of living cells has been revolutionized by the introduction of the highly specific and easily programmable properties of the clustered regularly interspaced short palindromic repeats (CRISPR) 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 knock-in 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 knock-in 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 mutant FOXA1 protein expression and intended amino acid substitutions via western blotting and mass spectrometry. In addition, we show that mutant allele frequency estimation is easily achieved by topoisomerase-based 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 wild-type FOXA1 with precise knock-in mutants to enable the systematic analysis of its molecular mechanisms within the appropriate physiological context.
成簇规律间隔短回文重复序列（CRISPR）技术具有高度特异性和易于编程的特性，其引入彻底改变了活细胞的精确基因组工程。这极大地加速了人类健康研究，并推动了新型疗法的发现。CRISPR-Cas9因其能够使特定基因失活或敲除而被最广泛应用，但它也可用于引入DNA序列的细微位点特异性替换，从而导致蛋白质氨基酸组成的变化。尽管基于CRISPR的基因敲入策略在典型的二倍体细胞（即含有两组染色体的细胞）中已被证明是成功的，但对通常具有不稳定基因组和多拷贝染色体的癌细胞进行精确编辑更具挑战性，且文献中对此并未充分探讨。在此，我们详细介绍了在多倍体癌细胞中用预期的敲入突变体替换内源性蛋白质的方法，并讨论了我们的实验设计、筛选策略和简便的等位基因频率估计方法。作为原理验证，我们在MCF-7乳腺癌细胞中对先驱转录因子FOXA1（雌激素和雄激素受体信号传导的关键成分）内的特定氨基酸进行了基因组编辑。我们通过蛋白质印迹法和质谱法确认了突变型FOXA1蛋白的表达和预期的氨基酸替换。此外，我们表明，通过基于拓扑异构酶的克隆与等位基因特异性PCR相结合，可以轻松实现突变等位基因频率的估计，我们随后通过下一代RNA测序对其进行了确认。通常，乳腺癌细胞中有4至5个FOXA1拷贝（等位基因），因此对该蛋白质进行编辑本身就具有挑战性。因此，大多数专注于FOXA1突变体的研究都依赖于从质粒中异位过表达FOXA1。因此，我们提供了一种优化方法，用精确的敲入突变体替换内源性野生型FOXA1，以便在适当的生理背景下对其分子机制进行系统分析。
