To nick or not to nick: comparison of I-SceI single- and double-strand break-induced recombination in yeast and human cells.

Genetic modification of a chromosomal locus to replace an existing dysfunctional allele with a corrected sequence can be accomplished through targeted gene correction using the cell's homologous recombination (HR) machinery. Gene targeting is stimulated by generation of a DNA double-strand brea...

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Autores principales:	Samantha S Katz, Frederick S Gimble, Francesca Storici
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/3ce394158ba24f668fcf0cb80d0a81d1
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spelling	oai:doaj.org-article:3ce394158ba24f668fcf0cb80d0a81d12021-11-18T08:32:14ZTo nick or not to nick: comparison of I-SceI single- and double-strand break-induced recombination in yeast and human cells.1932-620310.1371/journal.pone.0088840https://doaj.org/article/3ce394158ba24f668fcf0cb80d0a81d12014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24558436/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Genetic modification of a chromosomal locus to replace an existing dysfunctional allele with a corrected sequence can be accomplished through targeted gene correction using the cell's homologous recombination (HR) machinery. Gene targeting is stimulated by generation of a DNA double-strand break (DSB) at or near the site of correction, but repair of the break via non-homologous end-joining without using the homologous template can lead to deleterious genomic changes such as in/del mutations, or chromosomal rearrangements. By contrast, generation of a DNA single-strand break (SSB), or nick, can stimulate gene correction without the problems of DSB repair because the uncut DNA strand acts as a template to permit healing without alteration of genetic material. Here, we examine the ability of a nicking variant of the I-SceI endonuclease (K223I I-SceI) to stimulate gene targeting in yeast Saccharomyces cerevisiae and in human embryonic kidney (HEK-293) cells. K223I I-SceI is proficient in both yeast and human cells and promotes gene correction up to 12-fold. We show that K223I I-SceI-driven recombination follows a different mechanism than wild-type I-SceI-driven recombination, thus indicating that the initial DNA break that stimulates recombination is not a low-level DSB but a nick. We also demonstrate that K223I I-SceI efficiently elevates gene targeting at loci distant from the break site in yeast cells. These findings establish the capability of the I-SceI nickase to enhance recombination in yeast and human cells, strengthening the notion that nicking enzymes could be effective tools in gene correction strategies for applications in molecular biology, biotechnology, and gene therapy.Samantha S KatzFrederick S GimbleFrancesca StoriciPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 2, p e88840 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Samantha S Katz Frederick S Gimble Francesca Storici To nick or not to nick: comparison of I-SceI single- and double-strand break-induced recombination in yeast and human cells.
description	Genetic modification of a chromosomal locus to replace an existing dysfunctional allele with a corrected sequence can be accomplished through targeted gene correction using the cell's homologous recombination (HR) machinery. Gene targeting is stimulated by generation of a DNA double-strand break (DSB) at or near the site of correction, but repair of the break via non-homologous end-joining without using the homologous template can lead to deleterious genomic changes such as in/del mutations, or chromosomal rearrangements. By contrast, generation of a DNA single-strand break (SSB), or nick, can stimulate gene correction without the problems of DSB repair because the uncut DNA strand acts as a template to permit healing without alteration of genetic material. Here, we examine the ability of a nicking variant of the I-SceI endonuclease (K223I I-SceI) to stimulate gene targeting in yeast Saccharomyces cerevisiae and in human embryonic kidney (HEK-293) cells. K223I I-SceI is proficient in both yeast and human cells and promotes gene correction up to 12-fold. We show that K223I I-SceI-driven recombination follows a different mechanism than wild-type I-SceI-driven recombination, thus indicating that the initial DNA break that stimulates recombination is not a low-level DSB but a nick. We also demonstrate that K223I I-SceI efficiently elevates gene targeting at loci distant from the break site in yeast cells. These findings establish the capability of the I-SceI nickase to enhance recombination in yeast and human cells, strengthening the notion that nicking enzymes could be effective tools in gene correction strategies for applications in molecular biology, biotechnology, and gene therapy.
format	article
author	Samantha S Katz Frederick S Gimble Francesca Storici
author_facet	Samantha S Katz Frederick S Gimble Francesca Storici
author_sort	Samantha S Katz
title	To nick or not to nick: comparison of I-SceI single- and double-strand break-induced recombination in yeast and human cells.
title_short	To nick or not to nick: comparison of I-SceI single- and double-strand break-induced recombination in yeast and human cells.
title_full	To nick or not to nick: comparison of I-SceI single- and double-strand break-induced recombination in yeast and human cells.
title_fullStr	To nick or not to nick: comparison of I-SceI single- and double-strand break-induced recombination in yeast and human cells.
title_full_unstemmed	To nick or not to nick: comparison of I-SceI single- and double-strand break-induced recombination in yeast and human cells.
title_sort	to nick or not to nick: comparison of i-scei single- and double-strand break-induced recombination in yeast and human cells.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/3ce394158ba24f668fcf0cb80d0a81d1
work_keys_str_mv	AT samanthaskatz tonickornottonickcomparisonofisceisingleanddoublestrandbreakinducedrecombinationinyeastandhumancells AT fredericksgimble tonickornottonickcomparisonofisceisingleanddoublestrandbreakinducedrecombinationinyeastandhumancells AT francescastorici tonickornottonickcomparisonofisceisingleanddoublestrandbreakinducedrecombinationinyeastandhumancells
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To nick or not to nick: comparison of I-SceI single- and double-strand break-induced recombination in yeast and human cells.

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