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URL: https://dx.doi.org/DOI:10.17504/protocols.io.mmic44e

Authors: Janga H, Aznaourova M, Boldt F, Damm K, Grünweller A, Schulte LN

Summary: This protocol describes how to knockout mammalian non-coding RNA genes through excision of an epigenetic transcriptional start site (TSS) signature. To this end two guideRNAs are cloned onto the pX458 CRISPR vector to induce DNA cleavage right up- and downstream of the TSS element.The protocol describes how to generate homozygous knockout cell clones. Optionally, successful target DNA excision by the dual guideRNA pX458 construct may be pre-validated by transfection into an easy-to-transfect cell line (e.g. Hek293) and genomic PCR without prior single cell clonal expansion.

Associated Publications: Janga H, Aznaourova M, Boldt F, Damm K, Grünweller A, Schulte LN (2018) Cas9-mediated excision of proximal DNaseI/H3K4me3 signatures confers robust silencing of microRNA and long non-coding RNA genes. PLoS ONE 13(2): e0193066. doi: 10.1371/journal.pone.0193066

Affiliations: Institute for Lung Research, Philipps University, Marburg, Germany, Institute for Lung Research, Philipps University, Marburg, Germany, Institute for Lung Research, Philipps University, Marburg, Germany, Institute for Pharmaceutical Chemistry, Philipps University, Marburg, Germany, Institute for Pharmaceutical Chemistry, Philipps University, Marburg, Germany, Institute for Lung Research, Philipps University, Marburg, Germany

External URL: https://doi.org/10.1371/journal.pone.0193066

Version: 2

Publication Date: 2018

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