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

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

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • CRISPR-Associated Protein 9
  • Cell Line
  • Chromosome Mapping / methods*
  • Cloning, Molecular
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • DNA / genetics
  • DNA / metabolism
  • DNA Restriction Enzymes / genetics
  • DNA Restriction Enzymes / metabolism
  • Endonucleases / genetics
  • Endonucleases / metabolism
  • Gene Library
  • Genome*
  • Humans
  • Mice
  • MicroRNAs / genetics*
  • MicroRNAs / metabolism
  • Oligonucleotide Array Sequence Analysis
  • RNA, Guide / genetics*
  • RNA, Guide / metabolism
  • Untranslated Regions*

Substances

  • Bacterial Proteins
  • MIRN125 microRNA, human
  • MIRN142 microRNA, human
  • MicroRNAs
  • RNA, Guide
  • Untranslated Regions
  • DNA
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Endonucleases
  • DNA Restriction Enzymes