A split-Cas9 architecture for inducible genome editing and transcription modulation.
A split-Cas9 architecture for inducible genome editing and transcription modulation.
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DOI：
10.1038/nbt.3149
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发表时间：
2015-02
影响因子：
46.9
通讯作者：
Zhang F
中科院分区：
文献类型：
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作者：
Zetsche B;Volz SE;Zhang F
转导后 4 周，通过深度测序分析来自野生型 (wt)-Cas9 转导的 HEK293FT 细胞的 DNA，而来自 LSC-5 转导细胞的 DNA 在 6 周后进行分析，以允许用 200 nM 雷帕霉素连续处理 12 天（图 1g）。在用携带 wt-Cas9 和 EMX1 靶向 sgRNA 的慢病毒转导的细胞中，我们检测到目标位点的插入缺失频率约为 95%，以及四个已验证的脱靶位点（OT-1、2、3 和 4）的突变（OT-1= 2%、OT-2= 2%、OT-3 0.7% 和 OT-4= 38%）。相比之下，雷帕霉素处理 12 天后，用 LSC-5 转导的细胞中的目标插入缺失频率约为 43%。在未经处理的细胞中，LSC-5 和对照样本之间未检测到 EMX1 靶向插入缺失的显着差异。值得注意的是，无论雷帕霉素处理如何，在用 LSC-5 转导的细胞中均未检测到脱靶位点的显着修饰（这是不太可能与核定位的 Cas9 (C)-FKBP 片段二聚化的一种方式，我们用单个核输出序列 (NES)(Cas9 (N)-FRB-NES) 替换了 Cas9 (N)-FRB 上的两个核定位序列 (NLS)。雷帕霉素，Cas9 (N)-FRB-NES 与 Cas9 (C)-FKBP-2× NLS 二聚化，重建完整的 Cas9 蛋白，从而将核运输的平衡转向核输入并允许 DNA 靶向（图 1c、d）。我们使用 split-4 和 split-5 测试了我们的策略（图 1a），发现单个 NES 足以将背景活性降低到 SURVEYOR 测定的检测限以下（图 1a）。 1e) 我们的数据表明，Cas9 (N)-FRB 和 Cas9 (C)-FKBP 分裂片段在细胞内的空间隔离，与雷帕霉素激活的二聚化相结合，可以诱导激活 Cas9 核酸酶。高剂量的 Cas9 会加剧脱靶序列的 indel 频率。我们推测，与组成型活性相比，诱导低水平的 Cas9 活性可减少脱靶 indel。 Cas9，可能表现出较高的靶向活性，但也表现出较高水平的脱靶活性。
DNA from wild-type (wt)-Cas9–transduced HEK293FT cells was analyzed by deep sequencing 4 weeks after transduction, whereas DNA from LSC-5–transduced cells was analyzed after 6 weeks, to allow for 12 consecutive days of treatment with 200 nM rapamycin (Fig. 1g). In cells transduced with a lentivirus carrying both wt-Cas9 and an EMX1-targeting sgRNA, we detected~ 95% indel frequency at the on-target site as well as mutations at four validated off-target sites (OT-1, 2, 3 and 4) OT-1= 2%, OT-2= 2%, OT-3 0.7% and OT-4= 38%. In comparison, on-target indel frequency in cells transduced with LSC-5 was~ 43% after 12 days of rapamycin treatment. In untreated cells, no significant difference in EMX1 on-target indels could be detected between LSC-5 and control samples. Notably, no significant modification of off-target sites could be detected in cells transduced with LSC-5, regardless of rapamycin treatment (one-way where it is less likely to dimerize with the nuclear-localized Cas9 (C)-FKBP fragment, we replaced the two nuclear localization sequences (NLSs) on Cas9 (N)-FRB with a single nuclear export sequence (NES)(Cas9 (N)-FRB-NES). In the presence of rapamycin, Cas9 (N)-FRB-NES dimerizes with Cas9 (C)-FKBP-2× NLS to reconstitute a complete Cas9 protein, which shifts the balance of nuclear trafficking toward nuclear import and allows DNA targeting (Fig. 1c, d). We tested our strategy with split-4 and split-5 (Fig. 1a) and found that a single NES is sufficient to reduce background activity below the detection limit of the SURVEYOR assay (Fig. 1e). Our data show that spatial sequestration of Cas9 (N)-FRB and Cas9 (C)-FKBP split fragments inside the cell, combined with rapamycin-activated dimerization, allows inducible activation of the Cas9 nuclease.High dosage of Cas9 can exacerbate indel frequencies at off-target sequences9. We speculated that induction of low levels of Cas9 activity could be used to reduce off-target indels compared to constitutively active Cas9, which may exhibit high on-target activity but also elevated levels of off-target activity.