Knockout Zebrafish Lkb1 Gene By CRISPR/Cas9 Technology And Phenotype Analysis

ObjectiveCRISPR/Cas9 technique was used to knock out the LKB1 Gene of zebrafish,observing and to verify homozygote phenotype,and preliminarily explore the effect of Lkb1 on renal tubular development.MethodsThe gRNA was designed and synthesized in vitro.The gRNA and Cas9 mRNA were injected into the wild-type cell-phase embryo yolk by microinjection technique.The high-efficiency gRNA was screened by T7E1 digestion and sequencing,and the F0 mutation was screened by sequencing.In vivo and heritable mutants,wild fish and heritable mutant fish were crossed to produce F1 hybrid heterozygous mutants,and F1 hybrid heterozygous mutants were identified by sequencing,and F2 generation homozygous juveniles were produced by self-crossing of F1 heterozygotes.Homozygous phenotypes were identified;homozygous phenotypes were verified by qPCR and immunoblotting.ResultsThe zebrafish lkb1 gRNA and Cas9 mRNA were prepared,and three heterozygotes with frameshift mutations were screened.It was found that the zebrafish Lkb1 knockout homozygous juveniles violently swayed at the head and tail on the 5th day after fertilization,and the fish carp was missing.Body axis bending,yolk consumption too fast,liver dim,intestinal edema,most death on the 8th day;homozygous g6 pase,pdk2b,pcpck1 mRNA was significantly increased,lkb1 protein was not expressed at all.ConclusionThe zebrafish lkb1 gene was knocked out by CRISPR/Cas9 technology,resulting in liver failure,intestinal edema,body axis bending,loss of swim bladder,and early lethal phenotype.It also indicated that Lkb1 had an effect on the development of renal tubules in zebrafish.