RNA interference is a powerful experimental tool for RNA knockdown, but not all organisms are amenable. Here, we provide a proof of principle demonstration that a type III Csm effector complex can be used for programmable mRNA transcript degradation in eukaryotes. In zebrafish, Streptococcus thermophilus Csm complex (StCsm) proved effective for knockdown of maternally expressed EGFP in germ cells of Tg(ddx4:ddx4-EGFP) fish. It also led to significant, albeit less drastic, fluorescence reduction at one day postfertilization in Tg(myl7:GFP) and Tg(fli1:EGFP) fish that express EGFP zygotically. StCsm targeted against the endogenous tdgf1 elicited the characteristic one-eyed phenotype with greater than 50% penetrance, and hence with similar efficiency to morpholino-mediated knockdown. We conclude that Csm-mediated knockdown is very efficient for maternal transcripts and can also be used for mixed maternal/early zygotic and early zygotic transcripts, in some cases reaching comparable efficiency to morpholino-based knockdown without significant off-target effects.

中文翻译：

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通过 III 型 CRISPR-Cas 复合物对斑马鱼进行靶向 RNA 敲除。

RNA 干扰是 RNA 敲除的强大实验工具，但并非所有生物体都适用。在这里，我们提供了原理证明，证明 III 型 Csm 效应复合物可用于真核生物中可编程 mRNA 转录物降解。在斑马鱼中，嗜热链球菌Csm 复合物 (StCsm) 被证明可有效敲低Tg(ddx4:ddx4-EGFP)鱼生殖细胞中母源表达的EGFP。它还导致在受精后一天，合子表达 EGFP 的Tg (myl7:GFP)和Tg ( fli1:EGFP)鱼的荧光显着降低，尽管不太剧烈。针对内源性tdgf1 的StCsm引发了具有大于 50% 外显率的特征性单眼表型，因此与吗啉代介导的敲低具有相似的效率。我们得出的结论是，Csm 介导的敲低对于母体转录本非常有效，也可用于混合母体/早期合子和早期合子转录本，在某些情况下达到与基于吗啉基的敲低相当的效率，而没有明显的脱靶效应。