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

Publication ,  Journal Article
Kabadi, AM; Ousterout, DG; Hilton, IB; Gersbach, CA
Published in: Nucleic acids research
October 2014

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.

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Published In

Nucleic acids research

DOI

EISSN

1362-4962

ISSN

0305-1048

Publication Date

October 2014

Volume

42

Issue

19

Start / End Page

e147

Related Subject Headings

  • Transcriptional Activation
  • Trans-Activators
  • RNA, Guide, CRISPR-Cas Systems
  • Lentivirus
  • Humans
  • HEK293 Cells
  • Genome
  • Genetic Vectors
  • Fibroblasts
  • Developmental Biology
 

Citation

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Kabadi, A. M., Ousterout, D. G., Hilton, I. B., & Gersbach, C. A. (2014). Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector. Nucleic Acids Research, 42(19), e147. https://doi.org/10.1093/nar/gku749
Kabadi, Ami M., David G. Ousterout, Isaac B. Hilton, and Charles A. Gersbach. “Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector.Nucleic Acids Research 42, no. 19 (October 2014): e147. https://doi.org/10.1093/nar/gku749.
Kabadi AM, Ousterout DG, Hilton IB, Gersbach CA. Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector. Nucleic acids research. 2014 Oct;42(19):e147.
Kabadi, Ami M., et al. “Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector.Nucleic Acids Research, vol. 42, no. 19, Oct. 2014, p. e147. Epmc, doi:10.1093/nar/gku749.
Kabadi AM, Ousterout DG, Hilton IB, Gersbach CA. Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector. Nucleic acids research. 2014 Oct;42(19):e147.
Journal cover image

Published In

Nucleic acids research

DOI

EISSN

1362-4962

ISSN

0305-1048

Publication Date

October 2014

Volume

42

Issue

19

Start / End Page

e147

Related Subject Headings

  • Transcriptional Activation
  • Trans-Activators
  • RNA, Guide, CRISPR-Cas Systems
  • Lentivirus
  • Humans
  • HEK293 Cells
  • Genome
  • Genetic Vectors
  • Fibroblasts
  • Developmental Biology