A CRISPR Competition Assay to Identify Cancer Genetic Dependencies
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Author:
Date:
2020-07-20
[Abstract] The CRISPR/Cas9 system is a powerful tool for genome editing, wherein the RNA-guided nuclease Cas9 can be directed to introduce double-stranded breaks (DSBs) at a targeted locus. In mammalian cells, these DSBs are typically repaired through error-prone processes, resulting in insertions or deletions (indels) at the targeted locus. Researchers can use these Cas9-mediated lesions to probe the consequences of loss-of-function perturbations in genes of interest. Here, we describe an optimized protocol to identify specific genes required for cancer cell fitness through a CRISPR-mediated cellular competition assay. Identifying these genetic dependencies is of utmost importance, as they provide potential targets for anti-cancer drug development. This protocol provides researchers with a robust ...
[摘要] [摘要] CRISPR / Cas9系统是用于基因组编辑的强大工具,其中RNA引导的核酸酶Cas9可以直接在目标基因座处引入双链断裂(DSB)。在哺乳动物细胞中,这些DSB通常通过容易出错的过程进行修复,从而导致在目标基因座处插入或缺失(indel)。研究人员可以使用这些Cas9介导的病变来探究结果目标基因的功能丧失扰动。在这里,我们描述了一种优化的协议,可通过CRISPR介导的细胞竞争测定法来鉴定癌细胞适应性所需的特定基因。鉴定这些遗传依赖性至关重要,因为它们为抗癌药物的开发提供了潜在的靶标。该协议为研究人员提供了一种强大且可扩展的方法,以研究多种细胞系和癌症类型中的基因依赖性,并验证高通量或全基因组筛选的结果。
[背景] CRISPR / Cas9系统被认为已发展成为一种适应性的原核病毒防御系统(Mojica 等,2005; Makarova 等,2006)。它被发现后不久,就被研究人员选中,并进行了基因组编辑以供实验室使用(Doudna和Charpentier,2014年; Hsu 等人,2014年)。通过转基因表达Cas9核酸酶以及与靶序列互补的短链RNA(sgRNA),可以将双链断裂(DSB)引入各种细胞和生物体的目标位点(Cong 等,2013)。 ...
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Flow Cytometric Analysis of HIV-1 Transcriptional Activity in Response to shRNA Knockdown in A2 and A72 J-Lat Cell Lines
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Author:
Date:
2017-06-05
[Abstract] The main obstacle to eradicating HIV-1 from patients is post-integration latency (Finzi et al., 1999). Antiretroviral treatments target only actively replicating virus, while latent infections that have low or no transcriptional activity remain untreated (Sedaghat et al., 2007). To eliminate viral reservoirs, one strategy focuses on reversing HIV-1 latency via ‘shock and kill’ (Deeks, 2012). The basis of this strategy is to overcome the molecular mechanisms of HIV-1 latency by therapeutically inducing viral gene and protein expression under antiretroviral therapy and to cause selective cell death via the lytic properties of the virus, or the immune system now recognizing the infected cells. Recently, a number of studies have described the therapeutic potential of ...
[摘要] 消除HIV-1患者的主要障碍是后整合延迟(Finzi等人,1999)。抗逆转录病毒治疗仅针对主动复制病毒,而具有低转录活性或无转录活性的潜伏感染仍未得到治疗(Sedaghat等人,2007)。为了消除病毒性水库,一项战略重点是通过“休克和杀死”来逆转HIV-1潜伏期(Deeks,2012)。该策略的基础是通过在抗逆转录病毒治疗下通过治疗性诱导病毒基因和蛋白质表达来克服HIV-1潜伏期的分子机制,并通过病毒的溶解性质或现在识别感染细胞的免疫系统引起选择性细胞死亡。最近,许多研究已经描述了药物抑制人类溴结构域蛋白质的溴结构域和末端(BET)家族的成员的治疗潜力(Filippakopoulos等人,2010; Dawson等人& / em>,2011; Delmore等人,2011),其包括BRD2,BRB3,BRD4和BRDT。小分子BET抑制剂,例如JQ1(Filippakopoulos et al。,2010; Delmore等人,2011),I-BET(Nicodeme等人< / ...
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