Howard Hughes Medical Institute and Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.
Elife. 2020 Jun 4;9:e56193. doi: 10.7554/eLife.56193.
In a previous study, we established a forward genetic screen to identify genes required for multicellular development in the choanoflagellate, (Levin et al., 2014). Yet, the paucity of reverse genetic tools for choanoflagellates has hampered direct tests of gene function and impeded the establishment of choanoflagellates as a model for reconstructing the origin of their closest living relatives, the animals. Here we establish CRISPR/Cas9-mediated genome editing in by engineering a selectable marker to enrich for edited cells. We then use genome editing to disrupt the coding sequence of a C-type lectin gene, , and thereby demonstrate its necessity for multicellular rosette development. This work advances as a model system in which to investigate how genes identified from genetic screens and genomic surveys function in choanoflagellates and evolved as critical regulators of animal biology.
在之前的一项研究中，我们建立了一个正向遗传学筛选方法，以鉴定领鞭毛虫（ ）多细胞发育所必需的基因。（Levin 等人，2014 年）。然而，领鞭毛虫缺乏反向遗传学工具，这阻碍了对基因功能的直接测试，并妨碍了将领鞭毛虫作为重建其最亲近的动物亲属起源的模型的建立。在这里，我们通过工程设计一个可选择的标记来富集编辑细胞，从而在 中建立了 CRISPR/Cas9 介导的基因组编辑。然后，我们使用基因组编辑来破坏一个 C 型凝集素基因的编码序列 ，从而证明了它对多细胞玫瑰花结发育的必要性。这项工作推进了 作为一个模型系统，在这个系统中可以研究从遗传筛选和基因组调查中鉴定的基因在领鞭毛虫中是如何发挥作用的，并进化为动物生物学的关键调控因子。
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