Domingo Júlia, Minaeva Mariia, Morris John A, Ghatan Sam, Ziosi Marcello, Sanjana Neville E, Lappalainen Tuuli
New York Genome Center, New York, NY 10013, USA.
Science for Life Laboratory, Department of Gene Technology, KTH Royal Institute of Technology, Stockholm, Sweden.
bioRxiv. 2024 Aug 6:2024.03.01.582837. doi: 10.1101/2024.03.01.582837.
Genomic loci associated with common traits and diseases are typically non-coding and likely impact gene expression, sometimes coinciding with rare loss-of-function variants in the target gene. However, our understanding of how gradual changes in gene dosage affect molecular, cellular, and organismal traits is currently limited. To address this gap, we induced gradual changes in gene expression of four genes using CRISPR activation and inactivation. Downstream transcriptional consequences of dosage modulation of three master trans-regulators associated with blood cell traits (GFI1B, NFE2, and MYB) were examined using targeted single-cell multimodal sequencing. We showed that guide tiling around the TSS is the most effective way to modulate gene expression across a wide range of fold-changes, with further effects from chromatin accessibility and histone marks that differ between the inhibition and activation systems. Our single-cell data allowed us to precisely detect subtle to large gene expression changes in dozens of genes, revealing that many responses to dosage changes of these three TFs are non-linear, including non-monotonic behaviours, even when constraining the fold-changes of the master regulators to a copy number gain or loss. We found that the dosage properties are linked to gene constraint and that some of these non-linear responses are enriched for disease and GWAS genes. Overall, our study provides a straightforward and scalable method to precisely modulate gene expression and gain insights into its downstream consequences at high resolution.
与常见性状和疾病相关的基因组位点通常是非编码的，可能会影响基因表达，有时与靶基因中罕见的功能丧失变异同时出现。然而，我们目前对基因剂量的逐渐变化如何影响分子、细胞和生物体性状的理解有限。为了填补这一空白，我们使用CRISPR激活和失活诱导了四个基因的基因表达逐渐变化。使用靶向单细胞多模态测序研究了与血细胞性状相关的三个主要转录调节因子（GFI1B、NFE2和MYB）剂量调节的下游转录后果。我们表明，围绕转录起始位点（TSS）的引导序列平铺是在广泛的倍数变化范围内调节基因表达的最有效方法，染色质可及性和组蛋白标记在抑制和激活系统之间存在差异，会产生进一步影响。我们的单细胞数据使我们能够精确检测数十个基因中从细微到巨大的基因表达变化，揭示出即使将主要调节因子的倍数变化限制为拷贝数增加或减少，对这三个转录因子剂量变化的许多反应也是非线性的，包括非单调行为。我们发现剂量特性与基因约束有关，并且这些非线性反应中的一些在疾病和全基因组关联研究（GWAS）基因中富集。总体而言，我们的研究提供了一种直接且可扩展的方法，以精确调节基因表达并在高分辨率下深入了解其下游后果。
