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Nat Biotechnol. 2016 Dec;34(12):1279-1286. doi: 10.1038/nbt.3715. Epub 2016 Oct 31.

Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR-Cas9 library.

Zhu S1,2, Li W3,4, Liu J1,2, Chen CH3,4, Liao Q5, Xu P1, Xu H6, Xiao T4,7, Cao Z1,8, Peng J1, Yuan P1, Brown M4,7,9, Liu XS3,4, Wei W1.

Author information

1
Biodynamic Optical Imaging Center (BIOPIC), Beijing Advanced Innovation Center for Genomics, Peking-Tsinghua Center for Life Sciences, State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China.
2
Peking University-Tsinghua University-National Institute of Biological Sciences Joint Graduate Program (PTN), Peking University, China.
3
Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA.
4
Center for Functional Cancer Epigenetics, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.
5
Department of Prevention Medicine, School of Medicine, Ningbo University, Ningbo, Zhejiang, China.
6
Broad Institute of MIT and Harvard, Cambridge Center, Cambridge, Massachusetts, USA.
7
Division of Molecular and Cellular Oncology, Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.
8
Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China.
9
Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.

Abstract

CRISPR-Cas9 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 CRISPR-Cas9-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.

PMID:
27798563
PMCID:
PMC5592164
DOI:
10.1038/nbt.3715
[Indexed for MEDLINE]
Free PMC Article

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