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CRISPR/Cas9 mutagenesis in Volvox carteri （团藻） Volvox carteri and other volvocine green algae comprise an excellent model for investigating developmental complexity and its origins. Here we describe a method for targeted mutagenesis in V. carteri using CRISPR/Cas9 components expressed from transgenes. We used V. carteri nitrate reductase gene (nitA) regulatory sequences to conditionally express Streptococcus pyogenes Cas9, and V. carteri U6 RNA gene regulatory sequences to constitutively express single‐guide RNA (sgRNA) transcripts. V. carteri was bombarded with both the Cas9 vector and with one of several sgRNA vectors programmed to target different test genes (glsA, regA, and invA), and transformants were selected for expression of a hygromycin‐resistance marker present on the sgRNA vector. Hygromycin‐resistant transformants grown with nitrate as sole nitrogen source (inducing for nitA) were tested for Cas9 and sgRNA expression, and for ability to generate progeny with expected mutant phenotypes. Some transformants of a Somatic Regenerator (Reg) mutant strain receiving sgRNA plasmid with glsA protospacer sequence yielded progeny (at a rate of ~.01%) with a Gonidialess (Gls) phenotype similar to that observed for previously described glsA mutants, and sequencing of the glsA gene in independent mutants revealed short deletions within the targeted region of glsA, indicative of Cas9‐directed non‐homologous end joining. Similarly, bombardment of a morphologically wild type strain with the Cas9 plasmid and sgRNA plasmids targeting regA orinvA yielded regA and invA mutant transformants/progeny, respectively (at rates of 0.1%‐100%). The capacity to make precisely directed frameshift mutations should greatly accelerate the molecular genetic analysis of development in V. carteri, and of developmental novelty in the volvocine algae. AI译文（仅供参考）：Volvox carteri（团藻）和其他volvocine绿藻是研究发育复杂性及其起源的优秀模型。 在这里，我们描述了使用由转基因表达的CRISPR / Cas9组分在V. carteri中进行定向诱变的方法。 我们使用V. carteri硝酸盐还原酶基因（ nitA ）调节序列有条件地表达化脓性链球菌 Cas9和V. carteri U6 RNA基因调控序列以组成型表达单向RNA（sgRNA）转录物。 用Cas9载体和编程以靶向不同测试基因（ glsA ， regA和invA ）的几种sgRNA载体之一轰击V. carteri ，并选择转化体用于表达sgRNA载体上存在的潮霉素抗性标记。 测试用硝酸盐作为唯一氮源（诱导nitA ）生长的潮霉素抗性转化体的Cas9和sgRNA表达，以及产生具有预期突变表型的后代的能力。 接受具有glsA原型间隔区序列的sgRNA质粒的体细胞再生器（Reg）突变体菌株的一些转化体产生后代（以~0.01％的比率）具有类似于先前描述的glsA突变体所观察到的Gonidialess（Gls）表型，并且测序独立突变体中的glsA基因揭示了glsA靶向区域内的短缺失，表明Cas9指导的非同源末端连接。 类似地，用Cas9质粒和靶向regA或invA的sgRNA质粒轰击形态学野生型菌株分别产生regA和invA突变体转化体/后代（以0.1％-100％的比率）。 精确定向移码突变的能力应该大大加速V. carteri发育的分子遗传分析，以及volvocine藻类的发育新颖性。 欢迎点击「PaperRss」↑关注我们！  |
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