PMID: 35177623
开头用的是CRISPR screen，engineered HEK293A cells stably expressing Cas9 and GTIIC-GFP reporter, a transcription reporter of YAP/TEAD activity.Since cells have decreased YAP activity when Hippo signaling is activated at high cell density, regular GFP cannot be used to track YAP activity due to its low turnover rate. (因为传统上非磷酸化的YAP才会入核，与TEAD结合调控转录，这里的系统就是通过检测下游基因GTIIC-GFP的荧光值来代表YAP活性) We therefore fused a PEST sequence, which is found in many short-lived proteins, to the C-terminal of GFP to increase its turnover rate.（PEST sequence，蛋白质上一段特征序列，主要与蛋白质的泛素化降解有关；被认为是泛素化识别序列之一）
通过流式分选出荧光强度在20%的细胞，再送去测序；检验一个体系的准确性，有时看结果的阳性对照是否存在，如NF2,SAV1已被报道与YAP信号通路相关。
the Cancer Dependency Map project (DepMap portal—https://depmap.org/portal](https://depmap.org/portal)).The DepMap is an ongoing project to systematically assess the effect of single-gene inactivation on cell proliferation by genome-wide RNAi or CRISPR screens across a large panel of well-characterized human cancer cell lines. The essentiality of the gene for cell proliferation is represented as a dependency score—denoted as CERES score for CRISPR screens (Project Achilles)30,31.
Transmembrane protein KIRREL1 regulates Hippo signaling via a feedback loop and represents a therapeutic target in YAP/TAZ-active cancers
与上篇文章发现的是同一基因，但出发点不一样，在癌细胞和HEK293A筛选发现
1）the Hippo pathway status in cancer specimens by analyzing the expression of CTGF and CYR61, two canonical YAP/TAZ target genes.nrichment analysis combining CTGF and CYR61 levels was used to define the status (ON or OFF) of YAP/TAZ in stomach cancer specimens, and we found that patients with active YAP/TAZ (ON) had a significantly worse prognosis.
2）using proximity-dependent biotin identification (BioID) coupled with mass spectrometry (MS)。邻近标记技术也可以用于研究蛋白-RNA以及蛋白-DNA间的相互作用。例如，通过融合dCas9和邻近标记工具酶，我们可以分析基因组特定位置的结合蛋白。https://www.nature.com/articles/s41592-020-01010-5 该技术依赖于具有邻近标记功能的工具酶，包括过氧化氢酶(e.g. APEX, HRP)和生物素连接酶(e.g. BioID, TurboID)等，在活细胞水平对邻近生物分子进行生物素标记。https://www.tinglab.org/proximitylabeling-workshop-2020
在后续研究如何影响方面，很相似。
