Genome-scale CRISPR screening for modifiers of cellular LDL uptake.
Genome-scale CRISPR screening for modifiers of cellular LDL uptake.
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DOI：
10.1371/journal.pgen.1009285
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发表时间：
2021-01
期刊：
影响因子：
4.5
通讯作者：
Ginsburg D
中科院分区：
文献类型：
--
作者：
Emmer BT;Sherman EJ;Lascuna PJ;Graham SE;Willer CJ;Ginsburg D
高胆固醇血症是动脉粥样硬化性心血管疾病的一个致病和可改变的危险因素。调节胆固醇稳态的关键途径涉及LDL受体介导的低密度脂蛋白进入肝细胞的受体介导的内吞作用。我们应用基因组规模的CRISPR筛选来查询在富含脂蛋白或脂蛋白饥饿条件下培养的HuH7细胞中细胞LDL摄取的遗传决定因素。候选LDL摄取调节剂通过以更大覆盖深度合成和二次筛选定制的gRNA文库来验证。相对于初级全基因组筛选，该次级筛选产生了显著改善的性能，具有更好的内部阳性对照的区分，没有阴性对照的鉴定，并且在基因和gRNA水平上的筛选命中之间的一致性改善。然后，我们将我们定制的gRNA文库应用于正交筛选，该正交筛选测试了LDL与转铁蛋白内吞作用的每个候选调节剂的特异性、LDLR缺失的遗传上位性的存在或不存在、每个扰动对LDLR表达和运输的影响以及LDL摄取调节剂在多种细胞类型中的普遍性。这些发现确定了几个以前未被认识的基因与LDL摄取的假定作用，并提出了其与LDLR的功能相互作用机制。低密度脂蛋白（LDL）中血液循环的胆固醇水平是心血管疾病（包括心脏病发作和中风）总体风险的重要决定因素。这一水平是通过从循环中清除LDL进入肝细胞来调节的。虽然参与LDL摄取的许多分子已被表征，但我们假设其他目前未被认识到的遗传相互作用也参与了这一过程。因此，我们应用CRISPR介导的基因组编辑来系统地测试人类基因组中每个基因对肝源性细胞系摄取LDL的贡献。我们合成了一个二级CRISPR文库，靶向来自这个初始全基因组筛选的顶级候选基因，以确认它们在LDL摄取中的作用，并测试它们对其他细胞功能的影响。我们的研究结果证实了先前已知参与LDL摄取的基因的作用，并为这一过程的整体调控提供了新的见解。
Hypercholesterolemia is a causal and modifiable risk factor for atherosclerotic cardiovascular disease. A critical pathway regulating cholesterol homeostasis involves the receptor-mediated endocytosis of low-density lipoproteins into hepatocytes, mediated by the LDL receptor. We applied genome-scale CRISPR screening to query the genetic determinants of cellular LDL uptake in HuH7 cells cultured under either lipoprotein-rich or lipoprotein-starved conditions. Candidate LDL uptake regulators were validated through the synthesis and secondary screening of a customized library of gRNA at greater depth of coverage. This secondary screen yielded significantly improved performance relative to the primary genome-wide screen, with better discrimination of internal positive controls, no identification of negative controls, and improved concordance between screen hits at both the gene and gRNA level. We then applied our customized gRNA library to orthogonal screens that tested for the specificity of each candidate regulator for LDL versus transferrin endocytosis, the presence or absence of genetic epistasis with LDLR deletion, the impact of each perturbation on LDLR expression and trafficking, and the generalizability of LDL uptake modifiers across multiple cell types. These findings identified several previously unrecognized genes with putative roles in LDL uptake and suggest mechanisms for their functional interaction with LDLR. The level of cholesterol circulating in the blood in low-density lipoproteins (LDL) is an important determinant of overall risk for cardiovascular diseases, including heart attack and stroke. This level is regulated by the removal of LDL from circulation into liver cells. While many molecules involved in LDL uptake have been characterized, we hypothesized that other currently unrecognized genetic interactions are also involved in this process. We therefore applied CRISPR-mediated genome editing to systematically test the contribution of every gene in the human genome to the uptake of LDL by a liver-derived cell line. We synthesized a secondary CRISPR library targeting the top candidate genes from this initial genome-wide screen to confirm their role in LDL uptake and to test their influence on other cellular functions. Our findings confirm the role of genes previously known to participate in LDL uptake and also provide novel insight into the overall regulation of this process.
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影响因子：
21.3
作者：
McNally KE;Faulkner R;Steinberg F;Gallon M;Ghai R;Pim D;Langton P;Pearson N;Danson CM;Nägele H;Morris LL;Singla A;Overlee BL;Heesom KJ;Sessions R;Banks L;Collins BM;Berger I;Billadeau DD;Burstein E;Cullen PJ
通讯作者：
Cullen PJ
影响因子：
9.3
作者：
Blattmann, Peter;Henriques, David;Aebersold, Ruedi
通讯作者：
Aebersold, Ruedi
影响因子：
64.5
作者：
NOVICK, P;FIELD, C;SCHEKMAN, R
通讯作者：
SCHEKMAN, R
影响因子：
14.9
作者：
Brinkman EK;Chen T;Amendola M;van Steensel B
通讯作者：
van Steensel B
影响因子：
7.7
作者：
Emmer, Brian T.;Hesketh, Geoffrey G.;Ginsburg, David
通讯作者：
Ginsburg, David