CRISPR-Cas genome editing technologies have revolutionized the fields of functional genetics and genome engineering, but with the recent discovery and optimization of RNA-targeting Cas ribonucleases, we may soon see a similar revolution in the study of RNA function and transcriptome engineering. However, to date, successful proof of principle for Cas ribonuclease RNA targeting in eukaryotic systems has been limited. Only recently has successful modification of RNA expression by a Cas ribonuclease been demonstrated in animal embryos. This previous work, however, did not evaluate endogenous expression of Cas ribonucleases and only focused on function in early developmental stages. A more comprehensive evaluation of this technology is needed to assess its potential impact in the field. Here we report on our efforts to develop a programmable platform for RNA targeting using a Cas ribonuclease, CasRx, in the model organism Drosophila melanogaster. By genetically encoding CasRx in flies, we demonstrate moderate transcript targeting of known phenotypic genes in addition to unexpected toxicity and lethality. We also report on the off-target effects following on-target transcript cleavage by CasRx. Taken together, our results present the current state and limitations of a genetically encoded programmable RNA-targeting Cas system in Drosophila melanogaster, paving the way for future optimization of the system.

中文翻译：

---

在果蝇中使用 CasRx 进行可编程 RNA 靶向。


CRISPR-Cas基因组编辑技术彻底改变了功能遗传学和基因组工程领域，但随着最近RNA靶向Cas核糖核酸酶的发现和优化，我们可能很快就会在RNA功能和转录组工程研究中看到类似的革命。然而，迄今为止，Cas 核糖核酸酶 RNA 靶向真核系统的成功原理证明还很有限。直到最近，Cas 核糖核酸酶对 RNA 表达的成功修饰才在动物胚胎中得到证实。然而，之前的工作并未评估 Cas 核糖核酸酶的内源表达，仅关注早期发育阶段的功能。需要对该技术进行更全面的评估，以评估其在该领域的潜在影响。在这里，我们报告了我们在模式生物果蝇中使用 Cas 核糖核酸酶 CasRx 开发用于 RNA 靶向的可编程平台的努力。通过在果蝇中对 CasRx 进行基因编码，我们证明了除了意想不到的毒性和致死性之外，对已知表型基因的中等转录靶向性。我们还报告了 CasRx 进行靶向转录本切割后的脱靶效应。总而言之，我们的结果展示了果蝇中基因编码的可编程 RNA 靶向 Cas 系统的当前状态和局限性，为未来系统的优化铺平了道路。