Considerations and practical implications of performing a phenotypic CRISPR/Cas survival screen.
Considerations and practical implications of performing a phenotypic CRISPR/Cas survival screen.
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DOI：
10.1371/journal.pone.0263262
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发表时间：
2022
期刊：
影响因子：
3.7
通讯作者：
Geijsen N
中科院分区：
文献类型：
--
作者：
Ashoti A;Limone F;van Kranenburg M;Alemany A;Baak M;Vivié J;Piccioni F;Dijkers PF;Creyghton M;Eggan K;Geijsen N
具有活力作为读数的全基因组筛选有助于识别必需基因。使用CRISPR-Cas进行基因敲除筛选的发展为鉴定这些基因提供了更灵敏的方法。在这里，我们进行了详尽的全基因组CRISPR/Cas9表型拯救筛选，以鉴定由先锋转录因子DUX 4诱导的细胞毒性调节剂。由于表观遗传抑制机制的失败导致的DUX 4的错误表达是面肩肱型肌营养不良症（FHSD）的基础，这是一种迄今为止仍然无法治疗的复杂肌肉疾病。顾名思义，FSHD通常始于面部和肩带的肌肉。我们的CRISPR/Cas9筛选显示，除了DUX 4本身之外，没有关键的效应物可以调节DUX 4的细胞毒性，这表明FSHD的治疗工作应该针对DUX 4本身的直接调节。然而，我们的筛选确实揭示了一些罕见和意想不到的基因组事件，这对我们数据的解释产生了重要影响。我们的研究结果可能为规划未来的CRISPR/Cas9表型生存筛选提供重要考虑因素。
Genome-wide screens that have viability as a readout have been instrumental to identify essential genes. The development of gene knockout screens with the use of CRISPR-Cas has provided a more sensitive method to identify these genes. Here, we performed an exhaustive genome-wide CRISPR/Cas9 phenotypic rescue screen to identify modulators of cytotoxicity induced by the pioneer transcription factor, DUX4. Misexpression of DUX4 due to a failure in epigenetic repressive mechanisms underlies facioscapulohumeral muscular dystrophy (FHSD), a complex muscle disorder that thus far remains untreatable. As the name implies, FSHD generally starts in the muscles of the face and shoulder girdle. Our CRISPR/Cas9 screen revealed no key effectors other than DUX4 itself that could modulate DUX4 cytotoxicity, suggesting that treatment efforts in FSHD should be directed towards direct modulation of DUX4 itself. Our screen did however reveal some rare and unexpected genomic events, that had an important impact on the interpretation of our data. Our findings may provide important considerations for planning future CRISPR/Cas9 phenotypic survival screens.
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