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

被引:26
作者
Cheng, Jijun [1 ,2 ]
Roden, Christine A. [1 ,2 ,3 ]
Pan, Wen [1 ,2 ]
Zhu, Shu [4 ]
Baccei, Anna [2 ,5 ]
Pan, Xinghua [1 ]
Jiang, Tingting [6 ,7 ]
Kluger, Yuval [6 ,7 ]
Weissman, Sherman M. [1 ]
Guo, Shangqin [2 ,5 ]
Flavell, Richard A. [4 ]
Ding, Ye [8 ]
Lu, Jun [1 ,2 ,7 ,9 ]
机构
[1] Yale Univ, Sch Med, Dept Genet, New Haven, CT 06510 USA
[2] Yale Canc Ctr, Yale Stem Cell Ctr, New Haven, CT 06520 USA
[3] Yale Univ, Grad Program Biol & Biomed Sci, New Haven, CT 06510 USA
[4] Yale Univ, Sch Med, Dept Immunobiol, 333 Cedar St, New Haven, CT 06520 USA
[5] Yale Univ, Sch Med, Dept Cell Biol, 333 Cedar St, New Haven, CT 06520 USA
[6] Yale Univ, Sch Med, Dept Pathol, 333 Cedar St, New Haven, CT 06520 USA
[7] Yale Univ, Interdept Program Computat Biol & Bioinformat, New Haven, CT 06511 USA
[8] New York State Dept Hlth, Wadsworth Ctr, Albany, NY 12208 USA
[9] Yale Ctr RNA Sci & Med, New Haven, CT 06520 USA
来源
NATURE COMMUNICATIONS | 2016年 / 7卷
关键词
MEDIATED CONTROL; DIFFERENTIATION; MIR-142-3P; MICRORNAS; CELLS; BIOGENESIS; GENOMICS;
D O I
10.1038/ncomms11178
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
引用
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页数:10
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