Fig. 5

Workflow of our 3-phase knock-in pipeline. The design phase consists of five steps involving selection of WT fish for injections by sequence analysis of the target region in your WT fish line, finding an active sgRNA and designing the ssODN based on these data. The somatic screening phase occurs following injections. Embryos are collected and CRISPR-STAT or CRISPR-STAT/RFLP analysis is performed to determine if there is an enrichment of the expected peak in the sgRNA/Cas9 + ssODN group. If no enrichment is seen, another sgRNA/ssODN combination needs to be designed and tested. If expected size peaks are observed, representative samples are TOPO cloned and sequenced to confirm knock-in. The last phase is germline screening and begins by pre-screening of adult founder fish by fin biopsies for somatic knock-in. Positive fish are then prioritized for breeding to screen for germline transmission. Founders that transmit knock-in allele to their progeny are bred to grow F1 adults for genotyping, and sequence confirmation of precise knock-in