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Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPRCas9 library
Shiyou Zhu1,2,10, Wei Li3,4,10, Jingze Liu1,2, Chen-Hao Chen3,4, Qi Liao5, Ping Xu1, Han Xu6, Tengfei Xiao4,7, Zhongzheng Cao1,8, Jingyu Peng1, Pengfei Yuan1, Myles Brown4,7,9, Xiaole Shirley Liu3,4,11 & Wensheng Wei1,11
CRISPRCas9 screens have been widely adopted to analyze coding-gene functions, but high-throughput screening of non-coding elements using this method is more challenging because indels caused by a single cut in non-coding regions are unlikely to produce a functional knockout. A high-throughput method to produce deletions of non-coding DNA is needed. We report a high-throughput genomic deletion strategy to screen for functional long non-coding RNAs (lncRNAs) that is based on a lentiviral paired-guide RNA (pgRNA) library. Applying our screening method, we identified 51 lncRNAs that can positively or negatively regulate human cancer cell growth. We validated 9 of 51 lncRNA hits using CRISPRCas9-mediated genomic deletion, functional rescue, CRISPR activation or inhibition and gene-expression profiling. Our high-throughput pgRNA genome deletion method will enable rapid identification of functional mammalian non-coding elements.
2016 Nature America, Inc., part of Springer Nature. All rights reserved.
The CRISPRCas system seen in bacteria and archaea1 has been developed into a genome editing tool with wide-ranging applications24.
Functional screens of coding genes have been widely adopted, in which pooled libraries of single-guide RNAs (sgRNAs) that target the coding regions of genes associated with specific phenotypes can be selected using cell growth or specific markers as a readout512.
Although similar strategies have been used to tile across regulatory elements in order to investigate cis-element function13, such strategies may not work as well for non-coding elements, since indels caused by one gRNA are unlikely to produce loss-of-function phenotypes. Although two gRNAs have been used to generate a large genomic deletion to investigate the function of individual lncRNAs14,15, a high-
throughput screening using such approach has not been reported. We developed a CRISPRCas9 strategy using paired gRNAs (pgRNAs) to produce large-fragment deletions and enable the identification of functional long non-coding RNAs in cancer cells.
RESULTSLentivirally delivered paired-guide RNA system
We constructed a CRISPR pgRNA library such that the genomic sequences between two gRNA-targeting sites could be deleted. First, we tested two approaches to express the pgRNAs...