Genome Editing Of Zebrafish Hoxb4 Gene Using CRISPR/Cas9 System And Its Mutant Screening

Objective: Hematopoietic stem cells(HSCs) have the unique property of being able to self-renew and differentiate into cells of all hematopoietic lineages. Expansion of HSCs has numerous potential clinical applications. The use of the homeodomain-containing protein HOXB4 to expand HSCs is one of the most promising strategies. HOXB4 is normally expressed in immature hematopoietic progenitor cells and is rapidly downregulated during differentiation. How to expand hematopoietic stem cells in vitro, not only retain its self-renewal characteristics and can differentiate into blood cells are important and difficult in the current study. Based on the reported potency of hoxb4 to stimulate the ex vivo expansion of HSCs, we wished to find the direct target gene of hoxb4 and investigate the mechanism between hoxb4 gene with the hematopoietic system, This research established a knocking out zebrafish hoxb4 gene model by the efficient CRISPR/Cas9 gene editing system and help us to discuss its fuction better. Methods: Three 20 bp g RNAs were designed, which were target 192#(sense strand), target 244#(antisense strand), and target 313#( antisense strand). Three of them were chemically synthesized, and inserted into the linearized plasmid p T7-g RNA by Bbs I Enzyme. The g RNAs were transcribed in vitro by the T7 promoter. The Cas9 m RNA was transcribed in vitro using Cas9 expression vector p SP6-2s NLS-sp Cas9. Following completion of transcription, the poly(A) tailing reaction and DNase I treatment were performed. Both the g RNA and the Cas9-encoding m RNA were then purified and microinjected into the one cell stage of zebrafish embyos. Targeted genomic loci were amplified from genomic zebrafish DNA using primers and tested by T7 Endonuclease I assays. Finally, the PCR products were ligated into p MD19-T simple vector, the positive clones were taken by colony PCR and Sanger sequencing to detect mutations. Results: 1. hoxb4 target nucleic acid sequence of the genomic DNA by sequencing PCR products is consistent with the expected results; 2.g RNA oligonucleotide duplexes successfully connected to the target site of plasmid p T7-g RNA and the sequence is correct; 3. hoxb4 targeted Exon I 313# site can successfully edit hoxb4 genes in zebrafish. T7 EI detect the knockout efficiency up to 26.5%; 4.313# target sequence nucleic acid amplification products successfully ligated into p MD19-T simple vector and get four kinds of positive mutants by sequencing. 5. 30% of the zebrafish can be seen increased pericardium、thined heart chamber wall and blood flowed slowly. Conclusion: 1.As homeobox gene--- hoxb4 whose expression may exhibit functional redundancy to some extent, that is an abnormal gene function may be used to compensate for other gene without causing disease. 2.Knocking out the hoxb4 gene is successfully estabished by the CRISPR/Cas9 system and its mutations were identified and sequenced. The study provide experimental materials to investigate the mechanism between the HOXb4 gene function with the hematopoietic stem cells.