DECKO: Single-oligo, dual-CRISPR deletion of genomic elements including long non-coding RNAs.
DECKO: Single-oligo, dual-CRISPR deletion of genomic elements including long non-coding RNAs.
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DOI：
10.1186/s12864-015-2086-z
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发表时间：
2015-10-23
期刊：
影响因子：
4.4
通讯作者：
Johnson R
中科院分区：
文献类型：
--
作者：
Aparicio-Prat E;Arnan C;Sala I;Bosch N;Guigó R;Johnson R
CRISPR基因组编辑技术使得快速、廉价地删除非蛋白质编码调控元件成为可能。我们提出了一种适合于此目的的载体系统，称为DECKO（双切除CRISPR敲除），它采用简单的两步克隆来生成同时表达两种引导rna （gRNAs）的慢病毒载体。DECKO的主要特点是它使用了一个165 bp的起始寡核苷酸，携带两个grna的可变序列，使其从单位点研究到复杂文库克隆完全可扩展。我们使用DECKO来删除一个蛋白质编码基因和两个致癌lncrna的启动子，UCA1和高表达的MALAT1，这是许多先前使用RNA干扰方法研究的重点。DECKO成功地在四种人类细胞系中删除了大小从100到3000 bp不等的基因组片段。使用持续约20天的克隆衍生工作流程，我们在3个人类细胞系中获得了9个纯合子和17个杂合子启动子敲除。观察到频繁的靶区反转。这些克隆的稳态MALAT1 RNA水平降低高达98%，并显示增殖率降低。我们提出了一种双CRISPR工具DECKO，它使用单个起始寡核苷酸克隆，从而为非编码基因组元件（包括长链非编码rna）的CRISPR敲除研究提供了简单性和可扩展性。本文的在线版本（doi:10.1186/s12864-015-2086-z）包含补充材料，可供授权用户使用。
CRISPR genome-editing technology makes it possible to quickly and cheaply delete non-protein-coding regulatory elements. We present a vector system adapted for this purpose called DECKO (Double Excision CRISPR Knockout), which applies a simple two-step cloning to generate lentiviral vectors expressing two guide RNAs (gRNAs) simultaneously. The key feature of DECKO is its use of a single 165 bp starting oligonucleotide carrying the variable sequences of both gRNAs, making it fully scalable from single-locus studies to complex library cloning. We apply DECKO to deleting the promoters of one protein-coding gene and two oncogenic lncRNAs, UCA1 and the highly-expressed MALAT1, focus of many previous studies employing RNA interference approaches. DECKO successfully deleted genomic fragments ranging in size from 100 to 3000 bp in four human cell lines. Using a clone-derivation workflow lasting approximately 20 days, we obtained 9 homozygous and 17 heterozygous promoter knockouts in three human cell lines. Frequent target region inversions were observed. These clones have reductions in steady-state MALAT1 RNA levels of up to 98 % and display reduced proliferation rates. We present a dual CRISPR tool, DECKO, which is cloned using a single starting oligonucleotide, thereby affording simplicity and scalability to CRISPR knockout studies of non-coding genomic elements, including long non-coding RNAs. The online version of this article (doi:10.1186/s12864-015-2086-z) contains supplementary material, which is available to authorized users.