Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA.
DST-INSPIRE Faculty, Department of Microbiology, Ramanarain Ruia Autonomous College, Matunga, Mumbai, India.
Nat Methods. 2019 Jul;16(7):633-639. doi: 10.1038/s41592-019-0436-5. Epub 2019 Jun 24.
Mammalian genomes are folded into tens of thousands of long-range looping interactions. The cause-and-effect relationship between looping and genome function is poorly understood, and the extent to which loops are dynamic on short time scales remains an unanswered question. Here, we engineer a new class of synthetic architectural proteins for directed rearrangement of the three-dimensional genome using blue light. We target our light-activated-dynamic-looping (LADL) system to two genomic anchors with CRISPR guide RNAs and induce their spatial colocalization via light-induced heterodimerization of cryptochrome 2 and a dCas9-CIBN fusion protein. We apply LADL to redirect a stretch enhancer (SE) away from its endogenous Klf4 target gene and to the Zfp462 promoter. Using single-molecule RNA-FISH, we demonstrate that de novo formation of the Zfp462-SE loop correlates with a modest increase in Zfp462 expression. LADL facilitates colocalization of genomic loci without exogenous chemical cofactors and will enable future efforts to engineer reversible and oscillatory loops on short time scales.
哺乳动物基因组折叠成数以万计的长距离环互作用。环与基因组功能之间的因果关系还了解甚少，在短时间尺度上环的动态程度仍然是一个未解决的问题。在这里，我们使用蓝光工程设计了一类新的合成结构蛋白，用于定向重排三维基因组。我们将我们的光激活动态环（LADL）系统靶向两个基因组锚点，使用 CRISPR 指导 RNA，并通过蓝光诱导隐花色素 2 和 dCas9-CIBN 融合蛋白的异二聚化诱导它们的空间共定位。我们将 LADL 应用于重新引导一段延伸增强子（SE）远离其内源 Klf4 靶基因，并使其与 Zfp462 启动子共定位。使用单分子 RNA-FISH，我们证明了 Zfp462-SE 环的新形成与 Zfp462 表达的适度增加相关。LADL 促进基因组位点的共定位，而无需外源化学共因子，并且将能够实现未来在短时间尺度上设计可逆和振荡环的努力。
