A Report for Applying CRISPR/Cas9 Genome Editing Technologies on Mouse Zygotes from Taiwan Transgenic Mouse Model Core Facility (TMMC)

Abstract

Recent advances have provided the possibility to produce genetically modified animals by directly applying on zygotes using genome editing technologies such as Zinc Finger Nuclease (ZFN), Transcription Activator-Like Effector Nucleases (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems. Here, we showed the results of Taiwan Transgenic Mouse Models Core Facility (TMMC) for applying CRISPR/Cas9 for genetic engineering on mouse zygotes. In CRISPR/Cas9 system, we injected Cas9 plasmid or Cas9 RNA into either nucleus or cytoplasm of mouse zygoutes. Injection of Cas9 RNA with sgRNA into nucleus of zygotes was more efficient to induce indel than injection of Cas9 plasmid DNA (20% versus 12.5%). Moreover, injection of Cas9 RNA with sgRNA into cytoplasm increased indel rates to the average 38.1% in pups (the maximum is 89.4%), respectively. Further, the knock-in efficiency of injection RNA into cytoplasm was 15.3% in average (the maximum is 43.5%). In conclusion, injection of RNAs into mouse zygotes were more efficient to induce indel through non-homology end joining (NHEJ) or Knock-in through homology-directed recombination (HDR) on target genes, but might have cell toxicity when injected into nucleus than cytoplasm. The optimizations of procedure were needed to enhance efficiency and reduce toxicity.