Knockout Of Zebrafish Aftpha, Sertad2a, Si:Ch73-131E21.5, Panel And Si:Ch211-89O9.6 Gene By CRISPR/Cas9 And Rsph3 And Rsph4a Gene By TALEN

Genome editing techniques have been used as a powerful weapon in reverse genetics for the study of gene function, of which TALEN and CRISPR/cas9 being the latest and the most booming ones in recent years. TALEN has a pair of amino acid repeat sequence which could identify specific tandem DNA fragment, guiding endonuclease FokI to the target site. With dimerization, FokI shows endonuclease activity that whereafter induces DNA double strand breaks (DSB). On the other side, CRISPR/cas9 could generate DSB by cas9 endonuclease guided by gRNA which could bond with the specific recognition of target sites. Then DSB could trigger the DNA repairing mechanisms, such as non-homologous end joining (NHEJ) or homologous recombination (HR), producing a series of gene mutations. The two genome editing techniques above have already been successfully applied in many species, mice and zebrafish for instance, profiting from their broad application scope, relatively simple and time saving operation, high efficiency, low price, and have also shown huge potential in the fields of clinical treatment and animal husbandry.As a practical and easy-operating model animal in functional genomics, zebrafish is widely used in exploring the pathogenesis of human diseases, drug development and improvement. Zebrafish is vertebrate, compared with model invertebrate animals, it has genes sharing more similar functions with those orthologous genes of human beings, and organ structure homologous to human beings. In addition, being different from mammalian embryos developing in vivo, zebrafish fertilize and develop in vitro. With high optical transparency, they could conveniently be studied in all periods of embryonic development. Apart from these visible advantages, zebrafish also has several features superior to other species, for the applicable size, rapid growth, strong fecundity, low maintenance costs and equipment maintenance costs, and large number of individuals satisfying the experiment demands.The steps of gene knockout using CRISPR/cas9 in zebrafish are as follow: acquisition of the sequence of target gene, design of the appropriate target sites and primers, construction of the transcription vector of gRNA, transcription and purification of gRNA in vitro, preparation of cas9 transcription vector, transcription and purification of cas9 mRNA in vitro, microinjection, target site efficiency detection, F1 embryos and adults mutation detection. In this study,5 genes on chromosome 1 were knocked out with the help of CRISPR/cas9, respectively are aftpha, sertad2a, si:ch73-131e21.5, panel, si:ch211-89o9.6. And we obtained F1 individuals with mutation types of 7bp deletion mutation of aftpha,16bp deletion mutation of aftpha,4bp deletion mutation of sertad2a,10bp deletion mutation of sertad2a,2bp deletion mutation of si:ch73-131e21.5,25bp insertion mutation of si:ch73-131e21.5, 5bp deletion mutation of pane1,40bp deletion mutation of pane1.5bp deletion mutation of si:ch211-89o9.6,20bp insertion mutation of si:ch211-89o9.6 respectively.The steps of gene knockout using TALEN in zebrafish are as follow:acquisition of the sequence of target gene, design of the appropriate target sites, design of the primers, amplification of the monomer plasmid, assembling of the array plasmids, transcription and purification of mRNA, microinjection, target site efficiency detection, F1 embryos and adults mutation detection. In this study, TALEN was used to knock out the gene rsph3 and gene rsph4a. And a large number of F0 generation with mutations were obtained.