Integrase-Deficient Lentiviral Vector as an All-in-One Platform for Highly Efficient CRISPR/Cas9-Mediated Gene Editing

Pavel I. Ortinski, Bernadette O'Donovan, Xiaoyu Dong, Boris Kantor

Research output: Contribution to journalArticlepeer-review

79 Scopus citations

Abstract

The CRISPR/Cas9 systems have revolutionized the field of genome editing by providing unprecedented control over gene sequences and gene expression in many species, including humans. Lentiviral vectors (LVs) are one of the primary delivery platforms for the CRISPR/Cas9 system due to their ability to accommodate large DNA payloads and sustain robust expression in a wide range of dividing and non-dividing cells. However, long-term expression of LV-delivered Cas9/guide RNA may lead to undesirable off-target effects characterized by non-specific RNA-DNA interactions and off-target DNA cleavages. Integrase-deficient lentiviral vectors (IDLVs) present an attractive means for delivery of CRISPR/Cas9 components because: (1) they are capable of transducing a broad range of cells and tissues, (2) have superior packaging capacity compared to other vectors (e.g., adeno-associated viral vectors), and (3) they are expressed transiently and demonstrate very weak integration capability. In this manuscript, we aimed to establish IDLVs as a means for safe and efficient delivery of CRISPR/Cas9. To this end, we developed an all-in-one vector cassette with increased production efficacy and demonstrated that CRISPR/Cas9 delivered by the improved IDLV vectors can mediate rapid and robust gene editing in human embryonic kidney (HEK293T) cells and post-mitotic brain neurons in vivo, via transient expression and with higher gene-targeting specificity than the corresponding integrase-competent vectors.

Original languageEnglish
Pages (from-to)153-164
Number of pages12
JournalMolecular Therapy - Methods and Clinical Development
Volume5
DOIs
StatePublished - Jun 16 2017

Bibliographical note

Funding Information:
We thank Duke Center for genomic and computational biology and Sequencing and Genomic Technologies and Genomic Analysis and Bioinformatics Cores for performing and analyzing WES. We thank Udai Singh and the Flow Cytometry Core at the University of South Carolina for assistance. The all-in-one expression plasmid lentiCRISPRv2 (Addgene plasmid #52961) was a kind gift from Feng Zhang (Broad Institute). We thank Sriram Vijayraghavan (Duke University) for his comments and help with manuscript preparation and submission. Support was provided by University of South Carolina-School of Medicine grant RDF18080-E202 (to B.K.) and National Institute on Drug Abuse (NIDA) grant K01 DA031747 (to P.I.O.).

Publisher Copyright:
© 2017 The Author(s)

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics

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