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.

中文翻译：

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基于慢病毒的 Cas9/gRNA 表达系统，并随后通过 Cre 介导的重组去除


CRISPR 和相关基因组工程技术的一个主要问题是长期暴露于 Cas9 和相关编辑酶导致的脱靶诱变。为了帮助减轻这种担忧，我们在基于 HIV 的慢病毒载体的 3'-LTR 中添加了一个 loxP 位点，该载体能够在多种哺乳动物细胞类型中表达 Cas9/gRNA 复合物。易感靶细胞的转导产生了表达所需 Cas9/gRNA 复合物的整合原病毒。逆转录过程还会导致 3'-LTR 复制，从而使整合的原病毒两侧出现 loxP 位点（floxed）。 Cre 重组酶的后续表达会导致 loxP 到 loxP 位点特异性重组，从而删除 Cas9/gRNA 有效负载并有效防止额外的 Cas9 介导的突变。该构建体还表达具有单个转录终止序列的 gRNA，这导致更高的表达水平和更有效的基因组工程，如 SAMHD1 基因的破坏所证明的那样。这种打了就跑的 CRISPR 方法通过重建自然的 APOBEC3B 缺失和破坏错配修复基因 MSH2 得到了验证。这种打了就跑的策略可能在许多领域具有广泛的用途，特别是那些难以通过瞬时递送核糖核蛋白复合物来改造细胞类型的领域。