Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector

Nucleic Acids Res. 2014 Oct 29;42(19):e147. doi: 10.1093/nar/gku749. Epub 2014 Aug 13.

Abstract

Engineered DNA-binding proteins that manipulate the human genome and transcriptome have enabled rapid advances in biomedical research. In particular, the RNA-guided CRISPR/Cas9 system has recently been engineered to create site-specific double-strand breaks for genome editing or to direct targeted transcriptional regulation. A unique capability of the CRISPR/Cas9 system is multiplex genome engineering by delivering a single Cas9 enzyme and two or more single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can be used to simultaneously create multiple DNA breaks or to target multiple transcriptional activators to a single promoter for synergistic enhancement of gene induction. To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells. This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • CRISPR-Associated Proteins / genetics*
  • CRISPR-Associated Proteins / metabolism
  • CRISPR-Cas Systems*
  • Cell Engineering*
  • Cells, Cultured
  • DNA-Binding Proteins / genetics*
  • DNA-Binding Proteins / metabolism
  • Fibroblasts / metabolism
  • Genetic Vectors*
  • Genome
  • HEK293 Cells
  • Humans
  • Lentivirus / genetics*
  • RNA, Guide / metabolism
  • Trans-Activators / genetics
  • Transcriptional Activation

Substances

  • CRISPR-Associated Proteins
  • DNA-Binding Proteins
  • RNA, Guide
  • Trans-Activators