A lentivirus-based system for Cas9/gRNA expression and subsequent removal by Cre-mediated recombination

Michael A. Carpenter, Emily K. Law, Artur Serebrenik, William L. Brown, Reuben S. Harris

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

A major concern of CRISPR and related genome engineering technologies is off-target mutagenesis from prolonged exposure to Cas9 and related editing enzymes. To help mitigate this concern we added a loxP site to the 3′-LTR of an HIV-based lentiviral vector capable of expressing Cas9/gRNA complexes in a wide variety of mammalian cell types. Transduction of susceptible target cells yields an integrated provirus that expresses the desired Cas9/gRNA complex. The reverse transcription process also results in duplication of the 3′-LTR such that the integrated provirus becomes flanked by loxP sites (floxed). Subsequent expression of Cre recombinase results in loxP-to-loxP site-specific recombination that deletes the Cas9/gRNA payload and effectively prevents additional Cas9-mediated mutations. This construct also expresses a gRNA with a single transcription termination sequence, which results in higher expression levels and more efficient genome engineering as evidenced by disruption of the SAMHD1 gene. This hit-and-run CRISPR approach was validated by recreating a natural APOBEC3B deletion and by disrupting the mismatch repair gene MSH2. This hit-and-run strategy may have broad utility in many areas and especially those where cell types are difficult to engineer by transient delivery of ribonucleoprotein complexes.

Original languageEnglish (US)
Pages (from-to)79-84
Number of pages6
JournalMethods
Volume156
DOIs
StatePublished - Mar 1 2019

Keywords

  • CRISPR
  • Cas9
  • Cre recombinase
  • gRNA
  • loxP
  • pLentiCRISPR1000

PubMed: MeSH publication types

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

Fingerprint Dive into the research topics of 'A lentivirus-based system for Cas9/gRNA expression and subsequent removal by Cre-mediated recombination'. Together they form a unique fingerprint.

  • Cite this