Site-specific randomization of the endogenous genome by a regulatable CRISPR-Cas9 piggyBac system in human cells

Sci Rep. 2018 Jan 10;8(1):310. doi: 10.1038/s41598-017-18568-4.

Abstract

Randomized mutagenesis at an endogenous chromosomal locus is a promising approach for protein engineering, functional assessment of regulatory elements, and modeling genetic variations. In mammalian cells, however, it is challenging to perform site-specific single-nucleotide substitution with single-stranded oligodeoxynucleotide (ssODN) donor templates due to insufficient homologous recombination and the infeasibility of positive selection. Here, we developed a DNA transposon based CRISPR-Cas9 regulated transcription and nuclear shuttling (CRONUS) system that enables the stable transduction of CRISPR-Cas9/sgRNA in broad cell types, but avoids undesired genome cleavage in the absence two chemical inducing molecules. Highly efficient single nucleotide alterations induced randomization of desired codons (up to 4 codons) at a defined genomic locus in various human cell lines, including human iPS cells. Thus, CRONUS provides a novel platform for modeling diseases and genetic variations.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems*
  • Cells, Cultured
  • Codon / genetics
  • DNA Transposable Elements*
  • Female
  • Gene Editing / methods
  • HEK293 Cells
  • Humans
  • Male
  • Mutagenesis, Site-Directed / methods*
  • RNA, Guide / genetics
  • Random Allocation
  • Transduction, Genetic / methods

Substances

  • Codon
  • DNA Transposable Elements
  • RNA, Guide