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In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing
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
Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.
