Cas9/RNA-mediated gene targeting. (A) Schematic diagram of Cas9/RNA-mediated gene targeting. Chimeric RNA, a single engineered RNA molecule combining crRNA and tracrRNA, can guide Cas9 to cleave the target site of ∼20 nt. PAM, an NGG motif shown in purple, is essential for the activity of the complex. (B) Schematic representation of EGFP-A chimeric RNA (chiRNA) binding to pEGFP-N1 plasmid through spacer sequence. PAM is highlighted in purple. (C) Chimeric RNA guides Cas9 to cleave pEGFP-N1 at the target site in zebrafish. Four hundred nanograms per litre of Cas9 mRNA, 100 ng/μl of EGFP-A chimeric RNA, and pEGFP-N1 plasmid were co-injected into one-cell zebrafish embroys. Cleavage assays were performed 12 h after injection. Arrowheads indicate that two cleavage bands (about 295 bp and 198 bp) were detected in zygotes treated with Cas9 and non-annealed EGFP-A chimeric RNA. The Cas9 and chimeric RNA were added as indicated. (D) Sequencing results of T-A colonies of targeted fragments amplified from the sample of lane 4 in C. Two colonies with 1-bp insertion were detected in 48 colonies. Target sequence is capitalized and highlighted in red. Red lower case represents insert sequence. (E) PCR amplification of targeted fragment in the EGFP gene in founder mice treated with Cas9/RNA microinjection. Founder mice were generated as described in Supplementary information, Data S1. PCR amplification of the targeted fragment was performed using genomic DNA extracted from the tails of the founders as templates. Primers used were listed in Supplementary information, Figure S2. Arrowhead indicates a truncated band in founder #5. Ng, negative control. (F) The PCR products from founder #5 were subjected to T-A cloning. Twenty colonies were randomly selected for DNA sequencing. A 108-bp deletion was detected in nine colonies. Target sequence is capitalized and highlighted in red.