Core Benefit
- To produce targeted knock-in animal, such as humanized/reporter mice/rats model, cost and time will be saved by half compared with existing ES cell or CRSPR/Cas9 based method.
- Enables to establish genetically modified large animals.
Features
- For targeted long (>3Kb) cassette insertion in genome of mammalian fertilized egg with more than 50% efficiency without off-targets (published).
- Efficiency has improved to almost 100% (unpublished).
- Targeting RNAs can be easily chemically synthesized.
- No need for cell based assay to evaluate targeted digestion activity.
- Proved with EGFP(2.5kb), CreERT2 (2.5kb), flox (1kb), and other genes (>3Kb), reproduced by other researchers.
Background and Technology
Even though the CRISPR/Cas system is recognized as excellent genome editing tool, application for knock-in animal production has been limited due to low efficiency. Existing ES cell based method requires prolong period for selection from chimera origin.
The new method is based on finding that direct injection of wild type Cas9 complex consist of three components: Cas9 protein, CRISPR RNA (crRNA), and trans activating crRNA (tracrRNA) with targeting vector can make large gene insertion with very high efficiency and accuracy.
Immediate digestion of target DNA site and rapid degradation and deactivation of Cas9 at early one cell stage contribute to efficient knock-in and prevention of off-target and mosaicism. crRNA and tracrRNA is short enough to chemically synthesize, and Cas9 enzyme activity can be tested by cell-free test tube.
Publication and Patent
- Genome Biology 2015 16:87 (DOI: 10.1186/s13059-015-0653-x)
- WO 2016/080097 *100% efficiency technic is not published.
Researcher
Prof. Koichi Tanaka and Associate Prof. Tomomi Aida (Tokyo Medical and Dental University, Japan)
Expectations
We are looking for companies to license-in this technology and expand its commercial applications creating genetically modified animal. Detail can be disclosed under CDA.
Product No. TP0534