"Zinc fingers" for gene modification – fast and inexpensive
A new method of assembling promising gene modification tools – "zinc fingers"
NanoNewsNet based on the materials of Massachusetts General Hospital:
MGH researchers develop faster method of engineering zinc-finger nucleasesA group of scientists led by specialists from Massachusetts General Hospital (MGH) has developed a way to quickly obtain targeted synthetic enzymes that allow inactivating, restoring or changing certain DNA sequences.
An article about this success, Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA), published online in Nature Methods by the MGH Molecular Pathology group, describes a highly efficient and less time-consuming way to assemble a powerful tool – zinc-containing nuclease proteins called "zinc fingers" (zinc-finger nucleases, ZFNs).
"Using our approach, called context-dependent assembly, any scientist can use either standard methods of molecular biology or commercial DNA synthesis to create ZFNs for the desired target genes," says J. Keith Joung, MD and PhD, senior author of the article. "ZFNs is a widely used, powerful tool for manipulating the genome of cells of various organisms, including humans. They can provide effective correction of gene mutations responsible for human health, avoiding problems arising from the inaccuracy of today's gene therapy approaches using viral vectors."
Most human transcription factors that regulate the translation of a genetic signal into a protein interact with certain DNA sequences using peptides called "zinc fingers". Zinc finger nucleases are synthetic, "designer" proteins consisting of a protein Zn domain designed to bind to a specific DNA sequence and an enzyme that cleaves both DNA strands at the site of the target site. Despite the huge potential of ZFNs, the creation of the required proteins has remained a difficult task until now.
In the simplest approach, known as modular assembly, individual peptides are connected to each other like beads on a string, creating a multi-finger protein theoretically capable of recognizing long stretches of DNA. Zhong and others have shown that in practice, modular assembly of multi-finger proteins does not bring success. Most of the failures are probably due to the "context-dependent" effects that individual zinc fingers can have on the DNA binding activity of neighboring fingers. Assembling peptides that are unable to work together adequately can be compared to trying to assemble a puzzle from parts that do not fit into it.
In 2008, Zhong and his colleagues from the University of Minnesota, members of the Zinc Finger Consortium, reported on the development of a method called OPEN (Oligomerized Pool Engineering), which takes into account such context-dependent effects. But, despite the fact that OPEN gives good results, it is very laborious and requires an extremely long time – up to a year for the laboratory to install the technology, and two months to obtain the necessary ZFNs. To solve these problems, scientists from MGH have collected an extensive archive of zinc fingers, which are known to work well when they are next to each other – essentially already assembled pieces of the puzzle. Using this context-sensitive method, they were able to collect dozens of ZFNs in just four days.
"With such an archive, any scientist can easily get his own ZFNs in less than a week, and for this he does not need any special knowledge," explains Zhong. "Using a context-sensitive assembly, you can create a large number of ZFNs at the same time, which is difficult to do using the OPEN method, since it is more time-consuming."
As in the case of OPEN, the Zhong Laboratory and Zinc Finger Consortium will make the software and reagents necessary for the practical application of context-sensitive assembly available to all academic laboratories.
One example of the work of zinc finger nucleases (ZFNs).
ZFNs is a tool for inducing the breaking of a double DNA chain.
ZFNs are chimeric genes consisting of a synthetic DNA–recognizing domain of three C2H2 zinc fingers associated with a non-specific restriction enzyme (usually FokI endonuclease) that cleaves DNA (A).
ZFN genes, in which each finger recognizes a three-nucleotide sequence, can potentially be developed to recognize any combination of nine nucleotides, for example, such as is shown in Figure (B).
Since FokI functions as a dimer, two sets of ZFNs with a unique combination of 18 nucleotides are used to bind to the target DNA.
After binding to the DNA of the cell (C), the FokI endonuclease domain cleaves the DNA and creates a double break (D), which in many cases leads to deletion (deletion) of the mutation and subsequent restoration of the site.
(Picture: mcdb.lsa.umich.edu )
"One of the Holy Grails of genetics is the ability to modify individual target genes," says Laurie Tompkins, who oversees grants in the field of genetics at the National Institute of General Medical Sciences (National Institute of General Medical Sciences), one of the institutes of the National Institutes of Health (National Institutes of Health) USA, an ardent supporter of the study Jun. "Dr. Zhong and his colleagues have developed an extremely simple and effective strategy for using zinc finger technology to replace altered gene variants with normal ones, or vice versa, providing both scientists and clinicians with a research tool with a wide range of possibilities."
"I believe that at the moment context-dependent assembly will have the greatest impact on research using ZFNs for genetic manipulation of model organisms, perhaps even models derived from pluripotent stem cells. It will also have a great impact on the ability to create knockout mutations in large series of genes involved in the development or associated with certain diseases. ZFNs can also be used to create complete collections of mutants for each gene of any organism," adds Zhong.
Dr. Zhong is also an associate professor of Pathology at Harvard Medical School and a member of the Center for Computational and Integrative Biology and the Center for Cancer Research at Massachusetts General Hospital.
Abstract to the article: Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA)
Portal "Eternal youth" http://vechnayamolodost.ru21.12.2010