CRISPR Display: A modular method for locus-specific targeting of long noncoding RNAs and synthetic RNA devices in vivo [RNA-Seq]

Noncoding RNAs (ncRNAs) comprise an important class of natural regulators that mediate a vast array of biological processes, including the modulation of chromatin architecture. Moreover, artificial ncRNAs have revealed that the functional capabilities of RNA are extremely broad. To further investigate and harness these capabilities, we developed CRISPR-Display ("CRISP-Disp"), a targeted localization strategy that uses Cas9 to deploy large RNA cargos to specific DNA loci. We demonstrate that exogenous RNA domains can be functionally appended onto the CRISPR scaffold at multiple insertion points, allowing the construction of Cas9 complexes with RNAs nearing one kilobase in length, with structured RNAs, protein-binding cassettes, artificial aptamers and pools of random sequences. CRISP-Disp also allows the simultaneous multiplexing of disparate functions at multiple targets. We anticipate that this technology will provide a powerful method with which to ectopically localize functional RNAs and ribonuceloprotein complexes at specified genomic loci. Whole cell poly(A) selected RNA seq, from HEK293FT cells bearing lentivirally-integrated Gaussia and Cypridina luciferase reporter loci. Cells were transiently transfected with dCas9~VP64 alone, or with dCas9~VP and one of several modified sgRNAs,each targeting the Gaussia reporter.

非编码RNA（noncoding RNAs, ncRNAs）是一类重要的天然调控因子，介导包括染色质结构调控在内的大量生物学过程。此外，对人工合成非编码RNA的研究表明，RNA的功能潜能极为广泛。为进一步探究并利用这些潜能，我们开发了CRISPR-Display（简称CRISP-Disp），这是一种利用Cas9蛋白将大型RNA载荷递送至特定DNA位点的靶向定位策略。本研究证实，外源性RNA结构域可通过多个插入位点功能性连接至CRISPR骨架上，由此可构建长度接近1千碱基的Cas9复合物，其搭载结构化RNA、蛋白结合盒、人工适配体以及随机序列库。CRISP-Disp还可实现在多个靶点上同时搭载多种不同功能。我们预计该技术将为在指定基因组位点异位定位功能性RNA及核糖核蛋白复合物提供一种强有力的手段。本数据集的测序样本来自整合了慢病毒介导的高斯荧光素酶（Gaussia luciferase）与西里迪纳荧光素酶（Cypridina luciferase）报告基因位点的HEK293FT细胞，采用全细胞poly(A)富集RNA测序技术。实验中，细胞仅被瞬时转染dCas9~VP64融合蛋白，或同时转染dCas9~VP融合蛋白与数条靶向高斯荧光素酶报告基因的修饰单向导RNA（single guide RNA, sgRNA）之一。