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Nat Commun. 2016 Mar 30;7:11178. doi: 10.1038/ncomms11178.

A Molecular Chipper technology for CRISPR sgRNA library generation and functional mapping of noncoding regions.

Cheng J1,2, Roden CA1,2,3, Pan W1,2, Zhu S4, Baccei A2,5, Pan X1, Jiang T6,7, Kluger Y6,7, Weissman SM1, Guo S2,5, Flavell RA4, Ding Y8, Lu J1,2,7,9.

Author information

1
Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
2
Yale Stem Cell Center, Yale Cancer Center, New Haven, Connecticut 06520, USA.
3
Graduate Program in Biological and Biomedical Sciences, Yale University, New Haven, Connecticut 06510, USA.
4
Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
5
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
6
Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
7
Interdepartmental Program in Computational Biology and Bioinformatics, Yale University, New Haven, Connecticut 06511, USA.
8
Wadsworth Center, New York State Department of Health, Albany, New York 12208, USA.
9
Yale Center for RNA Science and Medicine, New Haven, Connecticut 06520, USA.

Abstract

Clustered regularly-interspaced palindromic repeats (CRISPR)-based genetic screens using single-guide-RNA (sgRNA) libraries have proven powerful to identify genetic regulators. Applying CRISPR screens to interrogate functional elements in noncoding regions requires generating sgRNA libraries that are densely covering, and ideally inexpensive, easy to implement and flexible for customization. Here we present a Molecular Chipper technology for generating dense sgRNA libraries for genomic regions of interest, and a proof-of-principle screen that identifies novel cis-regulatory domains for miR-142 biogenesis. The Molecular Chipper approach utilizes a combination of random fragmentation and a type III restriction enzyme to derive a densely covering sgRNA library from input DNA. Applying this approach to 17 microRNAs and their flanking regions and with a reporter for miR-142 activity, we identify both the pre-miR-142 region and two previously unrecognized cis-domains important for miR-142 biogenesis, with the latter regulating miR-142 processing. This strategy will be useful for identifying functional noncoding elements in mammalian genomes.

PMID:
27025950
PMCID:
PMC4820989
DOI:
10.1038/ncomms11178
[Indexed for MEDLINE]
Free PMC Article
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