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体外研究 用于靶向表观遗传沉默的工程化转录抑制因子的选择

JoVE Journal
Bioengineering

This content is Free Access.

JoVE Journal Bioengineering

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Please note that all translations are automatically generated. Click here for the English version.

体外研究用于靶向表观遗传沉默的工程化转录抑制因子的选择

DOI:

10.3791/64403-v

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10:44 min

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May 05, 2023

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Alessandro Migliara, Martino Alfredo Cappelluti, Francesca Giannese, Sara Valsoni, Alberto Coglot, Ivan Merelli³, Davide Cittaro, Angelo Lombardo⁴

¹San Raffaele Telethon Institute for Gene Therapy (SR-Tiget),IRCCS San Raffaele Scientific Institute, ²Center for Omics Sciences,IRCCS San Raffaele Institute, ³Institute for Biomedical Technologies,National Research Council, ⁴Vita-Salute San Raffaele University

Chapters

00:05Introduction
01:34Engineering a Fluorescence-Based Reporter Cell Line
03:10Designing gRNAs for CRISPR/dCas9-Based Epigenetic Silencing of Target Gene
04:13Arrayed Transient Delivery of CRISPR/dCas9-Based ETRs in Reporter Cell Line
07:19Analyzing Transcriptional Activity of Target Gene Over Time
08:11Results: dCas9 ETR-Mediated Epigenetic Silencing of Human B2M Gene by gRNAs
09:23Conclusion

Summary

Automatic Translation

在这里，我们提出了一种用于体外选择工程转录抑制因子（ETR）的方案，该方案具有高、长期、稳定、靶向沉默效率和低全基因组脱靶活性。该工作流程允许将初始的、复杂的候选 ETR 库减少到一个简短的列表，适合在治疗相关环境中进行进一步评估。

Tags

Keywords: Epigenetic Silencing Gene Silencing Artificial Nucleases Engineered Transcriptional Repressors Chromatin Compaction Transcriptional Inhibition Gene Disruption RNA Interference B2M Gene CRISPR Cas9 Nucleofection

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