Use CRISPR/Cas9 Knock Out The Fhl1a Gene In Zebrfish And Preliminary Study On Its Function

To study the function of the fhl1a gene in heart development,we generated fhl1a gene knockout zebrafish by CRISPR/Cas9 system.First,we constructed the sgRNA expression vector targeting zebrafish fhl1a gene that on the third exon.After in vitro transcription of sgRNA and Cas9 vectors,we co-injected them into one-cell fertilized eggs of zebrafish to generate zebrafish with targeted mutation.After 72hinjection,embryos were collected and acquired the genome DNA,and then PCR and sequencing were performed.To further verify the stability of genetic mutations,we selected mutated founder to outcross with wild type zebrafish.And then F1 were analyzed by PCR and sequencing of the caudal fin.The results showed F1 were introduced insertion and deletion mutations to some extent.Then F1 breeds which have the same mutations to get F2 generation homozygote.Now,we have established the knockout model which is 8bp deletion.Then in situ hybridization experiment was reveald the fhl1a gene knockout zebrafish heart looping was disordered.Our study obtained steady fhl1a gene knockout zebrafish can be transmitted by germline which is important basis for further study of fhl1a regulatory mechanism in the heart development.