Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors.
Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors.
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DOI：
10.7554/elife.81856
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发表时间：
2022-12-28
期刊：
影响因子：
7.7
通讯作者：
Jost, Marco
中科院分区：
文献类型：
--
作者：
Replogle, Joseph M.;Bonnar, Jessica L.;Pogson, Angela N.;Liem, Christina R.;Maier, Nolan K.;Ding, Yufang;Russell, Baylee J.;Wang, Xingren;Leng, Kun;Guna, Alina;Norman, Thomas M.;Pak, Ryan A.;Ramos, Daniel M.;Ward, Michael E.;Gilbert, Luke A.;Kampmann, Martin;Weissman, Jonathan S.;Jost, Marco
CRISPR干扰（CRISPRi）能够在哺乳动物细胞中对基因表达进行可编程的、可逆的和可滴定的抑制（敲低）。最初的CRISPRi介导的遗传筛选已经展示了解决细胞生物学、遗传学和生物技术中基本问题的潜力，但CRISPRi筛选的更广泛部署受到大尺寸的单向导RNA（sgRNA）文库和生成具有一致CRISPRi介导的敲低的细胞模型的挑战的限制。在这里，我们提出了下一代CRISPRi sgRNA文库和效应子表达构建体，它们能够在哺乳动物细胞模型中实现强而一致的敲低。首先，我们将联合收割机经验sgRNA选择与双sgRNA文库设计相结合，以产生超紧凑（每个基因1-3个元件）、高活性CRISPRi sgRNA文库。接下来，我们比较了CRISPRi效应子，以表明最近发表的Zim 3-dCas 9在强烈的靶向敲减和对细胞生长或转录组的最小非特异性影响之间提供了极好的平衡。最后，我们设计了一套具有Zim 3-dCas 9稳定表达和稳健的靶向敲减的细胞系。我们的结果和公开可用的试剂为CRISPRi基因筛查建立了最佳实践。
CRISPR interference (CRISPRi) enables programmable, reversible, and titratable repression of gene expression (knockdown) in mammalian cells. Initial CRISPRi-mediated genetic screens have showcased the potential to address basic questions in cell biology, genetics, and biotechnology, but wider deployment of CRISPRi screening has been constrained by the large size of single guide RNA (sgRNA) libraries and challenges in generating cell models with consistent CRISPRi-mediated knockdown. Here, we present next-generation CRISPRi sgRNA libraries and effector expression constructs that enable strong and consistent knockdown across mammalian cell models. First, we combine empirical sgRNA selection with a dual-sgRNA library design to generate an ultra-compact (1–3 elements per gene), highly active CRISPRi sgRNA library. Next, we compare CRISPRi effectors to show that the recently published Zim3-dCas9 provides an excellent balance between strong on-target knockdown and minimal non-specific effects on cell growth or the transcriptome. Finally, we engineer a suite of cell lines with stable expression of Zim3-dCas9 and robust on-target knockdown. Our results and publicly available reagents establish best practices for CRISPRi genetic screening.