The identity of human protein-coding genes is well known, yet our in-depth knowledge of their molecular functions and domain architecture remains limited by shortcomings in homology-based predictions and experimental approaches focused on whole-gene depletion. To bridge this knowledge gap, we developed a method that leverages CRISPR–Cas9-induced mutations across protein-coding genes for the a priori identification of functional regions at the sequence level. As a test case, we applied this method to 48 human mitotic genes, revealing hundreds of regions required for cell proliferation, including domains that were experimentally characterized, ones that were predicted based on homology, and novel ones. We validated screen outcomes for 15 regions, including amino acids 387–402 of Mad1, which were previously uncharacterized but contribute to Mad1 kinetochore localization and chromosome segregation fidelity. Altogether, we demonstrate that CRISPR–Cas9-based tiling mutagenesis identifies key functional domains in protein-coding genes de novo, which elucidates separation of function mutants and allows functional annotation across the human proteome.

中文翻译：

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使用 CRISPR–Cas9 平铺屏幕对人类有丝分裂基因进行功能解剖

人类蛋白质编码基因的身份是众所周知的，但我们对其分子功能和结构域结构的深入了解仍然受到基于同源性的预测和专注于全基因消耗的实验方法的缺陷的限制。为了弥合这一知识鸿沟，我们开发了一种方法，该方法利用 CRISPR-Cas9 诱导的跨蛋白质编码基因突变来先验识别序列水平的功能区域。作为测试案例，我们将该方法应用于 48 个人类有丝分裂基因，揭示了细胞增殖所需的数百个区域，包括实验表征的域、基于同源性预测的域和新的域。我们验证了 15 个区域的筛查结果，包括 Mad1 的氨基酸 387-402，这些以前没有被表征，但有助于 Mad1 动粒定位和染色体分离保真度。总之，我们证明了基于 CRISPR-Cas9 的平铺诱变从头识别蛋白质编码基因中的关键功能域，这阐明了功能突变体的分离并允许跨人类蛋白质组进行功能注释。