Fluorescence Amplification Method for Forward Genetic Discovery of Factors in Human mRNA Degradation

Mol Cell. 2017 Jan 5;65(1):191-201. doi: 10.1016/j.molcel.2016.11.032. Epub 2016 Dec 22.

Abstract

Nonsense-mediated decay (NMD) degrades mRNAs containing a premature termination codon (PTC). PTCs are a frequent cause of human genetic diseases, and the NMD pathway is known to modulate disease severity. Since partial NMD attenuation can potentially enhance nonsense suppression therapies, better definition of human-specific NMD is required. However, the majority of NMD factors were first discovered in model organisms and then subsequently identified by homology in human. Sensitivity and throughput limitations of existing approaches have hindered systematic forward genetic screening for NMD factors in human cells. We developed a method of in vivo amplification of NMD reporter fluorescence (Fireworks) that enables CRISPR-based forward genetic screening for NMD pathway defects in human cells. The Fireworks genetic screen identifies multiple known NMD factors and numerous human candidate genes, providing a platform for discovery of additional key factors in human mRNA degradation.

MeSH terms

  • CRISPR-Cas Systems
  • Cell Separation / methods*
  • Codon, Nonsense
  • Flow Cytometry*
  • Genotype
  • Green Fluorescent Proteins / biosynthesis*
  • Green Fluorescent Proteins / genetics
  • HeLa Cells
  • High-Throughput Screening Assays
  • Humans
  • Mutation
  • Nonsense Mediated mRNA Decay*
  • Phenotype
  • RNA Stability*
  • RNA, Guide / genetics
  • RNA, Guide / metabolism
  • RNA, Messenger / genetics*
  • RNA, Messenger / metabolism*
  • Time Factors
  • Transfection

Substances

  • Codon, Nonsense
  • RNA, Guide
  • RNA, Messenger
  • Green Fluorescent Proteins