Successful Transient Expression of Cas9 and Single Guide RNA Genes in Chlamydomonas reinhardtii
Successful Transient Expression of Cas9 and Single Guide RNA Genes in Chlamydomonas reinhardtii
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DOI：
10.1128/ec.00213-14
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发表时间：
2014-11-01
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影响因子：
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通讯作者：
Weeks, Donald P.
中科院分区：
文献类型：
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作者：
Jiang, Wenzhi;Brueggeman, Andrew J.;Weeks, Donald P.
成簇的规则间隔短回文重复序列（CRISPR）/Cas9系统已经成为用于靶向基因修饰（例如，基因敲除和基因置换）。最初尝试将这项技术应用于单细胞衣原体模型，莱茵衣藻，未能产生含有编辑基因的细胞。为了确定Cas9和单向导RNA（sgRNA）基因在C.在reinhardtii中，我们测试了密码子优化的Cas9基因沿着四种不同sgRNA之一在转化后立即24小时期间引起靶向基因破坏的能力。所有三个外源基因靶标以及C. reinhardtii显示出不同的Cas9/sgRNA介导的靶位点修饰，如通过靶位点区域的克隆PCR扩增子的DNA测序所确定的。Cas9和sgRNA基因的瞬时表达的成功与在涉及>10（9）个细胞的16个独立转化实验中仅回收携带适当修饰的FKB 12靶位点的单个雷帕霉素抗性集落形成对比。在用单独的Cas9基因（或甚至用编码缺乏核酸酶活性的Cas9的基因）转化后，未能回收具有完整或表达的Cas9基因的转化体提供了当在C中组成型产生Cas9时Cas9毒性的强有力的暗示性证据。莱因哈德氏菌目前的结果提供了令人信服的证据，表明Cas9和sgRNA基因在C. Reinhardtii引起靶向基因修饰，并指出需要专注于开发适当阻止基因编辑后Cas9产生和/或活性的方法。
The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has become a powerful and precise tool for targeted gene modification (e.g., gene knockout and gene replacement) in numerous eukaryotic organisms. Initial attempts to apply this technology to a model, the single-cell alga, Chlamydomonas reinhardtii, failed to yield cells containing edited genes. To determine if the Cas9 and single guide RNA (sgRNA) genes were functional in C. reinhardtii, we tested the ability of a codon-optimized Cas9 gene along with one of four different sgRNAs to cause targeted gene disruption during a 24-h period immediately following transformation. All three exogenously supplied gene targets as well as the endogenous FKB12 (rapamycin sensitivity) gene of C. reinhardtii displayed distinct Cas9/sgRNA-mediated target site modifications as determined by DNA sequencing of cloned PCR amplicons of the target site region. Success in transient expression of Cas9 and sgRNA genes contrasted with the recovery of only a single rapamycin-resistant colony bearing an appropriately modified FKB12 target site in 16 independent transformation experiments involving >10(9) cells. Failure to recover transformants with intact or expressed Cas9 genes following transformation with the Cas9 gene alone (or even with a gene encoding a Cas9 lacking nuclease activity) provided strong suggestive evidence for Cas9 toxicity when Cas9 is produced constitutively in C. reinhardtii. The present results provide compelling evidence that Cas9 and sgRNA genes function properly in C. reinhardtii to cause targeted gene modifications and point to the need for a focus on development of methods to properly stem Cas9 production and/or activity following gene editing.