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 journalArticlepeer-review

6 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

Bibliographical note

Funding Information:
The authors thank Amber St. Martin for comments. LentiCRISPR(v1) was a gift from Feng Zhang (Addgene plasmid #49535), and MCF-7L cells were provided by Douglas Yee (University of Minnesota). This work was supported by PHS grants NIAID R37 AI064046 and NIGMS R01 GM110734. RSH is the Margaret Harvey Schering Land Grant Chair for Cancer Research and a Distinguished University McKnight Professor at the University of Minnesota, and an Investigator of the Howard Hughes Medical Institute.

Funding Information:
The authors thank Amber St. Martin for comments. LentiCRISPR(v1) was a gift from Feng Zhang (Addgene plasmid #49535), and MCF-7L cells were provided by Douglas Yee (University of Minnesota). This work was supported by PHS grants NIAID R37 AI064046 and NIGMS R01 GM110734 . RSH is the Margaret Harvey Schering Land Grant Chair for Cancer Research and a Distinguished University McKnight Professor at the University of Minnesota, and an Investigator of the Howard Hughes Medical Institute.

Publisher Copyright:
© 2018 Elsevier Inc.

Keywords

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

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