title: A Molecular Chipper technology for CRISPR sgRNA library generation and functional mapping of noncoding regions.
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.
authors.affiliation: Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510, USA.|Yale Stem Cell Center, Yale Cancer Center, New Haven, Connecticut 06520, USA.
authors.forename: Jijun
authors.initials: J
authors.lastname: Cheng
authors.affiliation: Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510, USA.|Yale Stem Cell Center, Yale Cancer Center, New Haven, Connecticut 06520, USA.|Graduate Program in Biological and Biomedical Sciences, Yale University, New Haven, Connecticut 06510, USA.
authors.forename: Christine A
authors.initials: CA
authors.lastname: Roden
authors.affiliation: Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510, USA.|Yale Stem Cell Center, Yale Cancer Center, New Haven, Connecticut 06520, USA.
authors.forename: Wen
authors.initials: W
authors.lastname: Pan
authors.affiliation: Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
authors.forename: Shu
authors.initials: S
authors.lastname: Zhu
authors.affiliation: Yale Stem Cell Center, Yale Cancer Center, New Haven, Connecticut 06520, USA.|Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
authors.forename: Anna
authors.initials: A
authors.lastname: Baccei
authors.affiliation: Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
authors.forename: Xinghua
authors.initials: X
authors.lastname: Pan
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.|Interdepartmental Program in Computational Biology and Bioinformatics, Yale University, New Haven, Connecticut 06511, USA.
authors.forename: Tingting
authors.initials: T
authors.lastname: Jiang
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.|Interdepartmental Program in Computational Biology and Bioinformatics, Yale University, New Haven, Connecticut 06511, USA.
authors.forename: Yuval
authors.initials: Y
authors.lastname: Kluger
authors.affiliation: Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
authors.forename: Sherman M
authors.initials: SM
authors.lastname: Weissman
authors.affiliation: Yale Stem Cell Center, Yale Cancer Center, New Haven, Connecticut 06520, USA.|Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
authors.forename: Shangqin
authors.initials: S
authors.lastname: Guo
authors.affiliation: Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Flavell
authors.affiliation: Wadsworth Center, New York State Department of Health, Albany, New York 12208, USA.
authors.forename: Ye
authors.initials: Y
authors.lastname: Ding
authors.affiliation: Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510, USA.|Yale Stem Cell Center, Yale Cancer Center, New Haven, Connecticut 06520, USA.|Interdepartmental Program in Computational Biology and Bioinformatics, Yale University, New Haven, Connecticut 06511, USA.|Yale Center for RNA Science and Medicine, New Haven, Connecticut 06520, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Lu
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D000076987:CRISPR-Associated Protein 9; D002460:Cell Line; D002874:Chromosome Mapping / Q000379:methods*; D003001:Cloning, Molecular; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000235:genetics / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D016678:Genome; D006801:Humans; D051379:Mice; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics* / Q000378:metabolism; D020506:Untranslated Regions
chemical_list: D001426:Bacterial Proteins; C531888:MIRN125 microRNA, human; C555159:MIRN142 microRNA, human; D035683:MicroRNAs; D000094704:RNA, Guide, CRISPR-Cas Systems; D020506:Untranslated Regions; D004247:DNA; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases; D004262:DNA Restriction Enzymes