Children's Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
Department of Pediatrics, Harold C. Simmons Comprehensive Cancer Center, and Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
Nat Commun. 2020 Jan 24;11(1):485. doi: 10.1038/s41467-020-14362-5.
Tissue-specific gene expression requires coordinated control of gene-proximal and -distal cis-regulatory elements (CREs), yet functional analysis of gene-distal CREs such as enhancers remains challenging. Here we describe CRISPR/dCas9-based enhancer-targeting epigenetic editing systems, enCRISPRa and enCRISPRi, for efficient analysis of enhancer function in situ and in vivo. Using dual effectors capable of re-writing enhancer-associated chromatin modifications, we show that enCRISPRa and enCRISPRi modulate gene transcription by remodeling local epigenetic landscapes at sgRNA-targeted enhancers and associated genes. Comparing with existing methods, the improved systems display more robust perturbations of enhancer activity and gene transcription with minimal off-targets. Allele-specific targeting of enCRISPRa to oncogenic TAL1 super-enhancer modulates TAL1 expression and cancer progression in xenotransplants. Single or multi-loci perturbations of lineage-specific enhancers using an enCRISPRi knock-in mouse establish in vivo evidence for lineage-restricted essentiality of developmental enhancers during hematopoiesis. Hence, enhancer-targeting CRISPR epigenetic editing provides opportunities for interrogating enhancer function in native biological contexts.
组织特异性基因表达需要协调控制基因近端和远端顺式调控元件（CREs），但功能分析基因远端 CREs 如增强子仍然具有挑战性。在这里，我们描述了基于 CRISPR/dCas9 的增强子靶向表观遗传编辑系统 enCRISPRa 和 enCRISPRi，用于在原位和体内高效分析增强子功能。使用能够重新编写增强子相关染色质修饰的双重效应物，我们表明 enCRISPRa 和 enCRISPRi 通过重塑 sgRNA 靶向增强子和相关基因的局部表观遗传景观来调节基因转录。与现有方法相比，改进的系统显示出更强的增强子活性和基因转录的扰动，并且最小化了脱靶效应。enCRISPRa 对致癌性 TAL1 超级增强子的等位基因特异性靶向调节了 TAL1 的表达和异种移植中的癌症进展。使用 enCRISPRi 敲入小鼠对谱系特异性增强子进行单或多位点扰动，在造血过程中为发育增强子在谱系限制中的必要性提供了体内证据。因此，靶向增强子的 CRISPR 表观遗传编辑为在天然生物背景下研究增强子功能提供了机会。
