Generation of double knockout mice using single CRISPR guide RNA targeting two locus

抄録

Emergence of CRISPR-Cas9 engineered nuclease technology evidently improves production of knockout (KO) animals in various species. Recent report showed that multiple gene KO animals can be generated by injection of multiple CRISPR guide RNA. It is particularly interesting because generating multiple KO animal using traditional technique is extremely inefficient. In the present report, we generate multiple knockout mouse using only one CRISPR guide RNA targeting common sequence of two different locus. We selected two genes located at two different locus on mouse chromosome 7. We designed single guide RNA that targeting common sequence of the two genes. For reducing off-target mutation, we select guide RNA that has at least two mismatches compared with whole mouse genome sequences. Microinjection of the guide RNA with recombinant Cas9 protein into superovulated mouse zygotes was performed and the microinjected embryos were surgically transferred to oviduct of surrogate mother. Among the all produced offspring, one mouse (F0) showed that deletion of genomic sequence at the both target locus in sequencing analysis. To confirm absence of off-target mutation, we select 12 potential off-target site and analyzed by T7E1 assay. No mutation was found in all 12 sites. Offspring (F1 and F2) from the F0 mouse is produced and germ line transmission of the mutated gene was confirmed. In conclusion, we generate double knockout mice using single CRISPR guide RNA. Off-target mutation is not found and germ line transmission was confirmed. This work was supported by the Cooperative Research Program for Agriculture Science and Technology Development (no. PJ009802), Rural Development Administration, Korea.