Exploiting the use of DNA single- and double-strand breaking forms of the I-SceI endonuclease to stimulate homologous recombination and gene targeting in budding yeast and in human cells, the research of Samantha S. Katz in Francesca Storici’ lab provides new mechanistic insights into the process of nick-induced DNA recombination and on the function of nicking enzymes in genetic engineering.

Enzymes generating a site-specific double-strand break (DSB) in DNA, including homing endonucleases, such as I-SceI, are widely utilized to promote strand exchange between homologous sequences for purposes of characterizing mechanisms of DNA recombination and repair, and to facilitate targeted gene correction in many cellular systems from bacteria to human cells. However, in the most recent years, enzymes capable of making single-strand breaks (SSBs), nickases, have attracted a lot of attention. While a DSB can efficiently stimulate recombination, the competing non-homologous end-joining pathway for DSB repair is often favored, especially in human cells, and poses a major safety problem for gene targeting strategies, in particular for gene therapy applications, because it frequently leads to in/dels or chromosomal rearrangements. Recent work has shown that an SSB not only facilitates gene targeting, but importantly also leads to less off-site targeting damage than a DSB.

Despite the relevance of nicking enzymes, there are only very few available nicking systems, and still a lot remains to be understood about how a nick stimulates recombination and gene targeting in cells. The work conducted by Samantha S. Katz, recent PhD recipient in Francesca Storici lab at the School of Biology of Georgia Tech, in collaboration with Dr. Frederick Gimble from Purdue University, pioneers the in vivo function of the first available I-SceI nicking variant (K223I I-SceI). The team demonstrates that K223I I-SceI nickase efficiently stimulates gene correction in both yeast and human cells, and that such stimulation can occur even at loci 10 kb distant from the break site. Moreover, said Dr. Storici: <<we prove that the K223I I-SceI nickase stimulates recombination via a mechanism that is different from that by which the wild-type I-SceI double-strand nuclease works>>. The authors propose two models for nick-induce gene correction, either by simple unwinding of the broken strand at the nick site, or as a consequence of replication fork collapse and strand resection.
    
This study provides robust support to the fact that SSB-driven gene editing is a valuable mechanism for applications in molecular biology and biotechnology. The study is just published as an article in the journal PLoS One (Tuesday February 18, 2014):

Katz, S. S., Gimble, F. S. and Storici, F. To nick or not to nick: comparison of I-SceI single- and double-strand break-induced recombination in yeast and human cells
PLoS One, Vol 9, Issue 2, e88840, 2014 http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0088840


This project was supported by the Georgia Cancer Coalition grant (award R9028), the National Science Foundation grant MCB-1021763, and the Graduate Assistance in Areas of National Need (GAANN) fellowship.