The Effect Of Hbae1.1 Gene On Hemoglobin Production In Zebrafish Knocked Out By CRISPR/Cas9

Many animals,including vertebrates and invertebrates,use hemoglobin to transport oxygen from their lungs,gills,or other respirators to surrounding tissues that need it for efficient metabolism.Hemoglobin exists in the red blood cells of higher organisms.It is an indispensable component of blood and plays an important role in organisms.But the presence of icefish,a natural hemoglobin-free mutant,opens up the possibility of building hemoglobin-deficient mutants in the laboratory.Zebrafish and humans,both vertebrates,have highly homologous zebrafish hemoglobin and human hemoglobin,moreover it has the advantages of transparent embryo,abundant spawning and easy feeding,so it is an ideal organism suitable for studying the development of hemoglobin.There are many known zebrafish hemoglobin genes,and the study of these genes can provide some pathological knowledge for human diseases caused by the mutation of hemoglobin gene.So far,however,little research has been done on the hemoglobin gene of zebrafish.The CRISPR/Cas9 system was modified from the immune defense system of streptococcus pyogenes,which stood out as a widely used gene editing technology with obvious advantages such as simple construction,low cost and high efficiency.The main purpose of this study was to use CRISPR/Cas9 technology to knock out the hbae1.1gene in zebrafish hemoglobin gene family,and to study the effect of the deletion of hbae1.1 gene on the production of zebrafish hemoglobin.The method was as follows: firstly,the target and detection primers were designed according to zebrafish hbae1.1 gene,and the corresponding g RNA was prepared.g RNA and Cas9 protein were mixed in a certain proportion and then injected into zebrafish fertilized egg at a cellular stage by microinjection.Small fish were taken after 72 hours of injection,the effectiveness of the target was detected by T7E1 enzyme digestion andgene sequencing technology.By extracting genomic DNA,using the detection primers with PCR,and cutting PCR products by T7E1 enzyme,The PCR product of the band cut by the enzyme is the screened mutant F0 generation.The sexually mature mutant F0 generation was mated with WT,and the embryo genome DNA was extracted and sequenced to test whether the mutation of F0 generation zebrafish could be successfully inherited to the offspring.The heritable zebrafish of F0 generation were mated with WT,and the F1 generation heterozygotes with a number of non-3 multiples were obtained after screening.The F1 generation heterozygous zebrafish of adult fish with the same mutation type were interbred and the F2 generation homozygous zebrafish were obtained by screening.The embryos of F2 generation homozygous mutant and WT zebrafish were cultured in 28℃ incubator,the embryos cultured for 48 h were stained with o-dianisidine to observe the synthesis of embryonic hemoglobin.The results of this study are as follows: g RNA located in exon 2 of hbae1.1 gene was successfully prepared,the knockout target was detected to have a high knockout efficiency,and the F1 generation with code shift mutation at the target was successfully screened out.After the F1 generation intercrossing,the F2 generation homozygous was successfully screened out.By comparing the o-dianisidine staining of embryos produced by F2 generation homozygous and control group WT zebrafish,compared with control group WT,the results showed the hemoglobin content of F2 generation homozygous mutant significantly decreased,indicating that the deletion of hbae1.1 gene in embryo would affect the production of zebrafish hemoglobin.In summary,this study used the CRISPR /Cas9 technology to knock out the hbae1.1 gene and obtain the homozygous mutant,and successfully established the zebrafish model with hbae1.1 gene deletion.By the o-dianisidine results show that the lack of hbae1.1 gene will seriously affect the production of hemoglobin zebrafish,it also suggests that hbae1.1 genes in the zebrafish hemoglobin gene play an important role in the family,this provides a basis for the research on the production and development of fish hemoglobin,as well as human diseases related to hemoglobin to explore provide a certain amount of research.