Sue and Bill Gross Stem Cell Research Center, University of California, Irvine, Irvine, CA, 92697, USA.
Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, CA, 92697, USA.
Sci Rep. 2021 Jan 28;11(1):2424. doi: 10.1038/s41598-021-81975-1.
We report that epigenetic silencing causes the loss of function of multi-transcript unit constructs that are integrated using CRISPR-Cas9. Using a modular two color reporter system flanked by selection markers, we demonstrate that expression heterogeneity does not correlate with sequence alteration but instead correlates with chromosomal accessibility. We partially reverse this epigenetic silencing via small-molecule inhibitors of methylation and histone deacetylation. We then correlate each heterogeneously-expressing phenotype with its expected epigenetic state by employing ATAC-seq. The stability of each expression phenotype is reinforced by selective pressure, which indicates that ongoing epigenetic remodeling can occur for over one month after integration. Collectively, our data suggests that epigenetic silencing limits the utility of multi-transcript unit constructs that are integrated via double-strand repair pathways. Our research implies that mammalian synthetic biologists should consider localized epigenetic outcomes when designing complex genetic circuits.
我们报告称，表观遗传沉默导致使用 CRISPR-Cas9 进行整合的多转录单元构建体失去功能。我们使用由选择标记侧翼的模块化双色报告系统，证明表达异质性与序列改变无关，而是与染色质可及性相关。我们通过小分子量的甲基化和组蛋白去乙酰化抑制剂部分逆转这种表观遗传沉默。然后，我们通过 ATAC-seq 将每个异质表达表型与其预期的表观遗传状态相关联。选择压力加强了每种表达表型的稳定性，这表明整合后一个月以上仍可发生持续的表观遗传重塑。总的来说，我们的数据表明，表观遗传沉默限制了通过双链修复途径整合的多转录单元构建体的实用性。我们的研究表明，哺乳动物合成生物学家在设计复杂的遗传电路时应该考虑局部的表观遗传结果。
