Morn3 knockout zebrafish model established by the CRISPR/Cas9 system

Abstract:

Objective：To establish a Morn3 knockout zebrafish model using the efficient CRISPR/Cas9 gene editing system. Methods：The target sites for the Morn3 gene in zebrafish were chosen by bioinformatics analysis；the gRNA expression vector was constructed，and in vitro transcription was performed；then a mixture of gRNA and Cas9 mRNA was microscopically injected into the one-cell fer－tilized eggs of zebrafish. Genomic DNA was extracted at 24 to 48 hours post microinjection，and target fragments were amplified by PCR. Restriction enzyme digestion was used to detect gene targeting. Finally，sequencing analysis was used to determine the editing effect. Results：Three target sites（i.e.，M1，M2，and M3） were tested，and the two sites with higher concentrations of gRNA（M2 and M3） were injected. Results from restriction enzyme digestion and DNA sequencing revealed that gene editing effect had been produced in M3. Conclusion：The Morn3 knockout zebrafish model has been successfully obtained by the CRISPR/Cas9 gene editing system，which provides a basis for further study of the Morn3 gene.