Generation of mutant mice by pronuclear injection of circular plasmid expressing hCas9/gRNA complex — ASN Events
  1. Schedule
  2. Poster Session 1-Spermatology
  3. Generation of mutant mice by pronuclear injection of circular plasmid expressing hCas9/gRNA complex

Generation of mutant mice by pronuclear injection of circular plasmid expressing hCas9/gRNA complex (#123)

Haruhiko Miyata 1 , Daisuke Mashiko 1 , Yoshitaka Fujihara 1 , Yuhkoh Satouh 1 , Samantha AM Young 1 2 , Masahito Ikawa 1
  1. Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
  2. School of Environmental and Life Science, The University of Newcastle, Callaghan, NSW, Australia
The recombinant CRISPR/Cas system has opened a new era for the mammalian genome editing. Here we generated mutant mice for Cetn1 and Prm1 expressed specifically in the testis using CRISPR/Cas9 system. First, we constructed pX330 plasmids expressing humanized Cas9 (hCas9) and guide RNAs (gRNAs) against Cetn1 and Prm1 and validated their activities in vitro by observing green fluorescence reconstituted by homology dependent repair (HDR) of an EGFP expression cassette in HEK293T cells. Then, we injected the validated pX330 plasmids into mouse zygotes in its circular form. 58.8% (10/17) of the pups carried the mutations at the Cetn1 locus and six of them were homozygously mutated. Co-injection of the plasmids targeting different Cetn1 loci resulted in the successful removal of the flanked region in two out of three mutant pups. The efficient mutagenesis at the Prm1 lucus was also observed. Among the 46 offspring carrying CRISPR/Cas plasmid mediated mutations, only two of them carried the hCas9 transgene. The homozygous Cetn1 mutant males were infertile because of malformed spermatozoa and Prm1 mutant founder male was infertile due to impaired sperm motility and sperm malformation as it has been reported. The pronuclear injection of circular plasmid expressing hCas9/gRNA complex is a rapid, simple, and reproducible method for targeted mutagenesis.