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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 S.
Methods 2019 v.156 pp. 79-84
DNA repair, base pair mismatch, enzymes, gene editing, genes, mammals, mutagenesis, proviruses, reverse transcription, ribonucleoproteins, site-specific recombination, transcription termination
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.