Abstract
Gene targeting by homologous recombination at chromosomal endogenous loci has traditionally been considered a low-efficiency process. However, the effectiveness of such so-called genome surgery or genome editing has recently been drastically improved through technical developments, chiefly the use of designer nucleases like zinc-finger nucleases (ZFNs), meganucleases, transcription activator-like effector nucleases (TALENs) and CRISPR/Cas nucleases. These enzymes are custom designed to recognize long target sites and introduce double-strand 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 blot-based 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 integration-deficient lentiviral vectors (IDLVs) coding for ZFNs and a suitable DNA repair template.
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Acknowledgments
The authors acknowledge financial support from the 7th EU Framework Programme (PERSIST project, grant agreement no. 222878) and the Primary Immunodeficiency Association.
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Rocca, C.J., Abdul-Razak, H.H., Holmes, M.C., Gregory, P.D., Yáñez-Muñoz, R.J. (2014). A Southern Blot Protocol to Detect Chimeric Nuclease-Mediated Gene Repair. In: Storici, F. (eds) Gene Correction. Methods in Molecular Biology, vol 1114. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-761-7_21
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DOI: https://doi.org/10.1007/978-1-62703-761-7_21
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Publisher Name: Humana Press, Totowa, NJ
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