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

Methods. 2019 Mar 1:156:79-84. doi: 10.1016/j.ymeth.2018.12.006. Epub 2018 Dec 19.


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.

Keywords: CRISPR; Cas9; Cre recombinase; gRNA; loxP; pLentiCRISPR1000.

Publication types

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

MeSH terms

  • Base Pairing
  • Base Sequence
  • CRISPR-Associated Protein 9 / genetics*
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems*
  • Cytidine Deaminase / deficiency
  • Cytidine Deaminase / genetics
  • Exons
  • Gene Deletion
  • Gene Editing / methods*
  • Genetic Vectors / chemistry
  • Genetic Vectors / metabolism
  • HEK293 Cells
  • Humans
  • Integrases / genetics*
  • Integrases / metabolism
  • Introns
  • Lentivirus / genetics*
  • Lentivirus / metabolism
  • MCF-7 Cells
  • Minor Histocompatibility Antigens / genetics
  • MutS Homolog 2 Protein / deficiency
  • MutS Homolog 2 Protein / genetics
  • RNA, Guide, CRISPR-Cas Systems / genetics*
  • RNA, Guide, CRISPR-Cas Systems / metabolism
  • Recombination, Genetic*
  • SAM Domain and HD Domain-Containing Protein 1 / deficiency
  • SAM Domain and HD Domain-Containing Protein 1 / genetics


  • Minor Histocompatibility Antigens
  • RNA, Guide, CRISPR-Cas Systems
  • Cre recombinase
  • Integrases
  • CRISPR-Associated Protein 9
  • SAM Domain and HD Domain-Containing Protein 1
  • SAMHD1 protein, human
  • APOBEC3B protein, human
  • Cytidine Deaminase
  • MSH2 protein, human
  • MutS Homolog 2 Protein