Knockout and phenotype analysis of lkb1 gene in zebrafish by CRISPR/Cas9 technology

Abstract

Objective To knock out lkb1 gene in zebrafish by CRISPR/Cas9 technique, observe and verify homozygote phenotype, and preliminarily explore the effect of lkb1 on the development of renal tubule. Methods The gRNA targeting lkb1gene was designed and synthesized. 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. The F0 mutants and heritable mutants were screened by sequencing. Wild-type fish and heritable mutant fish were used to hybridize to produce F1 hybrid heterozygous mutants, and F1 hybrid heterozygous mutants were identified by sequencing. Then F2 generation homozygous juveniles were produced by self-crossing of F1 heterozygotes. Homozygous phenotypes were identified, and homozygous phenotypes were verified by qPCR and Western blot analysis. Results The zebrafish lkb1 gRNA and Cas9 mRNA were prepared, and 3 heterozygotes with frameshift mutations were obtained. It was found that the zebrafish Lkb1 knockout homozygous juveniles violently swayed at the head and tail on the 5th day after fertilization, and with missed swim bladder, bended body axis, fast yolk consumption, dim liver, and edematous intestinal tract, and most of them died on the 9th day. The mRNA levels of homozygous g6pase, pdk2b and pcpck1 were significantly increased, while lkb1 was not expressed at all. The mRNA levels of homozygous renal markers, trmp7, slc12a1 and slc12a3 were also obviously elevated. Conclusion Knockout of zebrafish lkb1 gene by CRISPR/Cas9 technology results in liver failure, edematous intestinal tract, bended body axis, missed swim bladder, and early lethal phenotype. The knockout also shows effect on the development of renal tubule.

Keywords

• crispr/cas9
• lkb1
• zebrafish