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. 2014 May 29;9(5):e98186. doi: 10.1371/journal.pone.0098186

Figure 4. Overcoming allele biases by inserting stop codon oligonucleotide with injected Cas9/sgRNA complexes.

Figure 4

A. Diagram of Cas9/sgRNA complex injection. B. Purified His-tagged Cas9 protein complexed with sgRNA (n = 12 embryos per target) or Cas9 mRNA and sgRNA (n = 12 embryos per target) were injected and indel frequency measured in single embryos at 1 dpf. C. Diagram of a stop codon oligonucleotide and genomic target site for mezzo. D. Clutches of embryos from adult injected fish were screened by PCR for germline transmission using PCR with a gene-specific primer and a primer specific to the inserted sequence (top panel), or two primers specific to the genome (bottom panel) as a positive control. Specific products indicated with arrows, non-specific product indicated with an asterix. E. DNA sequence of a mezzo insertion allele transmitted through the germline, 6/16 embryos in this clutch contained this mutation.