FIGURE

Fig. 5

ID

ZDB-FIG-221214-64

Publication

Carrington et al., 2022 - A robust pipeline for efficient knock-in of point mutations and epitope tags in zebrafish using fluorescent PCR based screening

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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

Expression Data

Expression Detail

Antibody Labeling

Phenotype Data

Phenotype Detail

Acknowledgments

This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ BMC Genomics