CRISPR-Cas9-mediated base-editing screening in mice identifies DND1 amino acids that are critical for primordial germ cell development

Nat Cell Biol. 2018 Nov;20(11):1315-1325. doi: 10.1038/s41556-018-0202-4. Epub 2018 Oct 1.

Abstract

CRISPR-mediated base editing can introduce single-nucleotide changes in the DNA of living cells. One intriguing application of base editing is to screen pivotal amino acids for protein function in vivo; however, it has not been achieved. Here, we report an enhanced third-generation base-editing system with extra nuclear localization sequences that can efficiently introduce a homozygous base mutation in embryonic stem cells. Meanwhile, we establish a strategy to generate base-mutant mice by injection of haploid embryonic stem cells carrying a constitutively expressed enhanced third-generation base-editing system (4B2N1) and single guide RNA into oocytes. Moreover, transfection of 4B2N1 cells with a single guide RNA library targeting the Dnd1 gene allows one-step generation of mutant mice with a base mutation. This enables the identification of four missense mutations that completely deplete primordial germ cells through disruption of DND1 protein stability and protein-protein interaction. Thus, our strategy provides an effective tool for in vivo screening of amino acids that are crucial for protein function.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids / genetics*
  • Amino Acids / metabolism
  • Animals
  • Base Sequence
  • CRISPR-Cas Systems*
  • Embryo, Mammalian / cytology
  • Embryo, Mammalian / embryology
  • Embryo, Mammalian / metabolism
  • Female
  • Gene Editing / methods*
  • Germ Cells / cytology
  • Germ Cells / growth & development
  • Germ Cells / metabolism*
  • HEK293 Cells
  • Humans
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mice, Inbred DBA
  • Mice, Inbred ICR
  • Neoplasm Proteins / genetics*
  • Neoplasm Proteins / metabolism

Substances

  • Amino Acids
  • Dnd1 protein, mouse
  • Neoplasm Proteins