title: A single blastocyst assay optimized for detecting CRISPR/Cas9 system-induced indel mutations in mice.
abstract: BACKGROUND
  Microinjection of clustered regulatory interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-related RNA and DNA into fertilized eggs is a novel approach for creating gene-modified mice. Blastocysts obtained just before implantation may be appropriate for testing the fidelity of CRIPSR/Cas9-mediated genome editing because they can be individually handled in vitro and obtained 3days after microinjection, thus allowing researchers to check mutations rapidly. However, it is not known whether indel mutations caused by the CRISPR/Cas9 system can be reproducibly detected in embryos. In this study, we assessed the detection of CRISPR/Cas9-induced mutations in embryos.
  
  
  RESULTS
  T7 endonuclease I was more effective than Surveyor nuclease for detecting mutations in annealed fragments derived from 2 plasmids, which contained nearly identical sequences. Mouse fertilized eggs were microinjected with CRISPR/Cas9-related RNA/DNA to examine whether non-homologous end joining-mediated knockout and homologous recombination-mediated knockin occurred in the endogenous receptor (G protein-coupled) activity modifying protein 2 (Ramp2) gene. Individual blastocysts were lysed to obtain crude DNA solutions, which were used for polymerase chain reaction (PCR) assays. T7 endonuclease I-based PCR and sequencing analysis demonstrated that 25-100% of the embryos were knockout embryos and 7-57% of the embryos were knockin embryos. Our results also established that crude DNA from a single blastocyst was an appropriate template for Whole genome amplification and subsequent assessment by PCR and the T7 endonuclease I-based assay.
  
  
  CONCLUSIONS
  The single blastocyst-based assay was useful for determining whether CRISPR/Cas9-mediated genome editing worked in murine embryos.
authors.affiliation: Department of Cardiovascular Research, Graduate school of Medicine, Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan. tsakurai@shinshu-u.ac.jp.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Sakurai
authors.forename: Satoshi
authors.initials: S
authors.lastname: Watanabe
authors.forename: Akiko
authors.initials: A
authors.lastname: Kamiyoshi
authors.forename: Masahiro
authors.initials: M
authors.lastname: Sato
authors.forename: Takayuki
authors.initials: T
authors.lastname: Shindo
mesh_terms: D000818:Animals; D001483:Base Sequence; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism*; D064130:CRISPR-Associated Proteins / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004247:DNA / Q000032:analysis*; D003850:Deoxyribonuclease I / Q000378:metabolism; D016678:Genome; D054643:INDEL Mutation; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D058263:Receptor Activity-Modifying Protein 2 / Q000235:genetics
chemical_list: D064130:CRISPR-Associated Proteins; D012333:RNA, Messenger; C545506:Ramp2 protein, mouse; D058263:Receptor Activity-Modifying Protein 2; D004247:DNA; D003850:Deoxyribonuclease I