AsianScientist (Dec. 29, 2015) – Researchers have developed a streamlined protocol for an alternative gene insertion method that does not rely on a DNA repair mechanism known as homologous recombination.
Called PITCh (Precise Integration into Target Chromosome), the protocol was reported in Nature Protocols by Specially Appointed Lecturer Tetsushi Sakuma, Professor Takashi Yamamoto, Specially Appointed Associate Professor Ken-Ichi T Suzuki, and their colleagues at Hiroshima University.
According to the research team, the PITCh system is more convenient and effective than existing methods for inserting foreign DNA into targeted genomic loci by using genome-editing tools. They believe that the new technique could aid the rapid progression of research in fields such as the screening of new drug candidates and creating cell or animal models of human diseases.
Genome editing enables researchers to modify the genome not at random but at a particular target. The current editing method employs engineered nucleases as ‘molecular scissors,’ which create DNA breaks at desired locations in the genome. When DNA breaks are repaired by repair pathways, genetic modifications including the insertion of foreign DNA into the genome (knock-in) and replacement or removal of a targeted genomic locus are induced.
“The PITCh system is an alternative knock-in method that is independent of homologous recombination, one of the DNA-break repair pathways, unlike existing knock-in techniques that use genome editing tools like TALEN or CRISPR-Cas9, which mainly utilize homologous recombination,” said Sakuma.
Sakuma explained that the existing knock-in techniques cannot be applied to every cell type and organism owing to variable HR frequencies. Because of this, the research team aimed at another repair pathway, the microhomology-mediated end-joining (MMEJ), and developed the PITCh system.
The researchers have described detailed procedures for constructing a desired vector, transfecting it into cells, selecting knocked-in cells, and checking after insertion together with an actual successful example. A simplified method of gene insertion in frog embryos is also described.
The article can be found at: Sakuma et al. (2015) MMEJ-assisted Gene Knock-in using TALENs and CRISPR-Cas9 with the PITCh Systems.
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Source: Hiroshima University.
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