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 Harris

Research output: Contribution to journalArticle

3 Citations (Scopus)

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

Fingerprint

Guide RNA
Lentivirus
Genetic Recombination
Clustered Regularly Interspaced Short Palindromic Repeats
Genes
Proviruses
Transcription
Genome
Mutagenesis
DNA Mismatch Repair
Ribonucleoproteins
Engineering technology
Reverse Transcription
Repair
Cells
HIV
Technology
Engineers
Mutation
Enzymes

Keywords

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

PubMed: MeSH publication types

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

Cite this

A lentivirus-based system for Cas9/gRNA expression and subsequent removal by Cre-mediated recombination. / Carpenter, Michael A.; Law, Emily K.; Serebrenik, Artur; Brown, William L; Harris, Reuben.

In: Methods, Vol. 156, 01.03.2019, p. 79-84.

Research output: Contribution to journalArticle

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