A Southern Blot Protocol to Detect Chimeric Nuclea

Authors: Céline J Rocca Hayder H AbdulRazak Michael C Holmes Philip D Gregory Rafael J YáñezMuñoz

Publish Date: 2014

Volume: , Issue: , Pages: 325-338

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Abstract

Gene targeting by homologous recombination at chromosomal endogenous loci has traditionally been considered a lowefficiency process However the effectiveness of such socalled genome surgery or genome editing has recently been drastically improved through technical developments chiefly the use of designer nucleases like zincfinger nucleases ZFNs meganucleases transcription activatorlike effector nucleases TALENs and CRISPR/Cas nucleases These enzymes are custom designed to recognize long target sites and introduce doublestrand breaks DSBs at specific target loci in the genome which in turn mediate significant improvements in the frequency of homologous recombination Here we describe a Southern blotbased assay that allows detection of gene repair and estimation of repair frequencies in a cell population useful in cases where the targeted modification itself cannot be detected by restriction digest This is achieved through detection of a silent restriction site introduced alongside the desired mutation in our particular example using integrationdeficient lentiviral vectors IDLVs coding for ZFNs and a suitable DNA repair template

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