Death receptor-based enrichment of Cas9-expressing cells.
Death receptor-based enrichment of Cas9-expressing cells.
复制标题
DOI：
10.1186/s12896-016-0250-4
复制
发表时间：
2016-02-16
影响因子：
3.5
通讯作者：
Beaudouin J
中科院分区：
文献类型：
--
作者：
Liesche C;Venkatraman L;Aschenbrenner S;Grosse S;Grimm D;Eils R;Beaudouin J
CRISPR/Cas9 基因组编辑系统极大地促进和扩展了我们设计哺乳动物基因组的能力，包括靶向基因敲除。然而，敲除效应的表型分析需要较高的DNA编辑效率。在这里，我们报告了一种基于外在细胞凋亡途径的用户友好策略，通过选择共表达死亡受体显性失活突变体的 Cas9 转染细胞，可以富集多克隆基因编辑的细胞群。外源性细胞凋亡途径可以在许多哺乳动物细胞类型中触发，配体易于产生，不需要纯化，并且比最先进的选择药物嘌呤霉素更快地杀死细胞。通过桑格测序、T7 核酸内切酶 I (T7E1) 测定和直接表型分析对我们的先进选择策略进行严格评估，证实了 Cas9 表达细胞群的强大而快速的富集，在某些情况下在一小时内达到 100%。值得注意的是，这些富集细胞中靶标 DNA 切割的效率达到了高水平，超出了 T7E1 测定的可靠范围，这一结论可以推广到编辑效率超过 30%。此外，我们的数据强调，特定 gRNA 诱导的插入和缺失模式在不同细胞系中是可重复的。本文报告的工作流程和研究结果应该会简化未来一系列低通量或高通量基因敲除筛选，并应在很大程度上改善 CRISPR 实验的数据解释。本文的在线版本 (doi:10.1186/s12896-016-0250-4) 包含补充材料，可供授权用户使用。
The CRISPR/Cas9 genome editing system has greatly facilitated and expanded our capacity to engineer mammalian genomes, including targeted gene knock-outs. However, the phenotyping of the knock-out effect requires a high DNA editing efficiency. Here, we report a user-friendly strategy based on the extrinsic apoptosis pathway that allows enrichment of a polyclonal gene-edited cell population, by selecting Cas9-transfected cells that co-express dominant-negative mutants of death receptors. The extrinsic apoptosis pathway can be triggered in many mammalian cell types, and ligands are easy to produce, do not require purification and kill much faster than the state-of-the-art selection drug puromycin. Stringent assessment of our advanced selection strategy via Sanger sequencing, T7 endonuclease I (T7E1) assay and direct phenotyping confirmed a strong and rapid enrichment of Cas9-expressing cell populations, in some cases reaching up to 100 % within one hour. Notably, the efficiency of target DNA cleavage in these enriched cells reached high levels that exceeded the reliable range of the T7E1 assay, a conclusion that can be generalized for editing efficiencies above 30 %. Moreover, our data emphasize that the insertion and deletion pattern induced by a specific gRNA is reproducible across different cell lines. The workflow and the findings reported here should streamline a wide array of future low- or high-throughput gene knock-out screens, and should largely improve data interpretation from CRISPR experiments. The online version of this article (doi:10.1186/s12896-016-0250-4) contains supplementary material, which is available to authorized users.