BiPoD, a new knock-in technique that allows for Donor DNA insertion at a target site developed by Waseda University

A research group led by Professor Daisuke Arai and Professor Yoichi Nakao at the Faculty of Science and Engineering, Waseda University, has developed a new highly accurate and efficient biallelic knock-in method that inserts donor DNA at the target location in the genome. This new method is named "BiPoD." Techniques enabling disruption of specific genes in genomic DNA, the blueprint of life, or insertion of (knock-in) foreign donor DNA into target sites, are essential for basic research. The method is also expected to be used for cutting-edge medical treatments such as gene therapy and cell therapy.

A similar genome editing technology, the "CRISPR/Cas9 system," was developed in 2012, which specifically cuts only the target location of genomic DNA by introducing a protein called Cas9 bound to an RNA, called guide RNA, into the DNA sequence. Knock-in, which combines the CRISPR/Cas9 editing technology with homologous recombination, the original DNA repair mechanism in cells, is currently used worldwide as a major genome editing technology.

Human cells harbor two sets of chromosomes, which carry a set of genes meaning that two of the same genes are present in the form of alleles. Therefore, when 'knocking in' to manipulate the function of a gene, it is necessary to insert donor DNA into both chromosomes. However, the probability of simultaneously knocking in two chromosomes using homologous recombination combined with the CRISPR/Cas9 editing technology is very low at less than a few percent, which hinders further studies. Concerns have also been raised about conventional techniques since homologous recombination knock-in may also result in random insertion of donor DNA into a site unrelated to the target, and the original gene at the site of insertion may be disrupted, causing an unexpected effect on the cell. This is a challenge that must be overcome when knock-in is applied clinically.

From this backdrop, a research group at Waseda University has developed and established a new method that can almost completely suppress the off-target insertion of donor DNA by other mechanisms while increasing the efficiency of homologous recombination compared to that with the conventional method. This has enabled precise and safe knock-in, wherein the genome is not disrupted because of partial insertion. The new method was also successful in simultaneously knocking in both sets of chromosomes with a high probability of more than 90%. This new method, which is highly accurate and efficient, will accelerate research based on mouse embryonic stem cells, and is a simple method that does not require special equipment or expensive reagents for achieving homologous recombination knock-in. The research group hopes that it will soon become a new standard method and be used widely. The study was published online on September 13 in Scientific Reports.