Random Integration Analysis ”RAISING”
“RAISING”is a high-performance detection method to assess sites of random transgene integration
We identify transgene integration sites by RAISING (Rapid Amplification of Integration Sites without INterference by Genomic DNA contamination) jointly developed with National Institute of Infectious Diseases.
Transgene integrations into a host genome via viral infection, viral vector-transduction, and genome editing, have risks associated with various diseases, including cancers. Currently, next generation sequencing (NGS)-based detection methods for the integration sites have been developed, and clinically used for the safety assessment of transgene transduction manners. These methods are useful but still remain several concerns particularly about the sensitivity, versatility, cost, and time. Herein, we developed RAISING, a high-performance detection method, corresponding to both Sanger sequencing and NGS analysis.
Schematic outline of RAISING
RAISING main steps are shown below. If transgene-specific primer can be designed, RAISING is available. The preparation of integrated DNA fragments is simple because RAISING is based on PCR technology. Since RAISING avoids the troublesome operation and allows for continuous reactions to be performed in a single PCR tube , RAISING enables to minimize human error. And RAISING contributes to obtain extremely reproducible data.
[Case1] HTLV-1 clonality analysis
*The Data provided from Dr, Saito (National Institute of Infectious Diseases) and Dr. Hasegawa (Nagasaki university)
Human T-cell leukemia virus-1 (HTLV-1) mainly infect CD4 positive T-cell and the provirus effectively integrates into the host genome. And this integration often triggers the development of leukemia. This is especially true if integration occurs near a cancer driver gene. Therefore, Southern blotting is currently needed to detect the monoclonal growth of HTLV-1-infected cells. However, it has various problems such as required a large amount of DNA, troublesome operation, long procedure time and insufficient sensitivity. RAISING is overcome these problem and high sensitivity and extremely reproducible data can be obtained for HTLV-1 clonality analysis.
* The clonality value calculation software, CLOVA is available here
[Case2] The detection of off-target on genome editing
We tried to discriminate between on- and off-target effects in genome-edited mice by the combination of RAISING and NGS. In addition to on-targeted chromosome9, we demonstrated that donor DNA was inserted to other sites.
*If the unexpected mutations are existed in position of provided reference sequence for transgene, the results may not be obtained.*RAISING enables to detect some insertion sites at once. However, it can not guarantee the detection of all insertion sites. *the result may include false-negative. Please confirm the validation of the obtained data separately as needed.