| Literature DB >> 26443227 |
Tobias Schmidt1, Jonathan L Schmid-Burgk1, Thomas S Ebert1, Moritz M Gaidt1, Veit Hornung2.
Abstract
Recent developments in the field of designer nucleases allow the efficient and specific manipulation of genomic architectures in eukaryotic cell lines. To this end, it has become possible to introduce DNA double strand breaks (DSBs) at user-defined genomic loci. If located in critical coding regions of genes, thus induced DSBs can lead to insertions or deletions (indels) that result in frameshift mutations and thereby the knockout of the target gene. In this chapter, we describe a step-by-step workflow for establishing knockout cell clones of the difficult-to-transfect suspension cell line THP1. The here described protocol encompasses electroporation, cell cloning, and a deep sequencing-based genotyping step that allows the in-parallel analysis of 96 cell clones per gene of interest. Furthermore, we describe the use of the analysis tool OutKnocker that allows rapid identification of cell clones with all-allelic frameshift mutations.Entities:
Keywords: Barcoded deep sequencing; CRISPR/Cas9; Genome editing; Genotyping; Knockout cell lines
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Year: 2016 PMID： 26443227 DOI： 10.1007/978-1-4939-2932-0_19
Source DB: PubMed Journal: Methods Mol Biol ISSN： 1064-3745