Genome Editing of Zebrafish hoxb4 Gene Using CRISPR/Cas9 System and Its Mutant Screening

doi:10.13560/j.cnki.biotech.bull.1985.2015.10.004

Abstract

Abstract: It was to construct the knocking out zebrafish hoxb4 gene model by the efficient gene editing system CRISPR/Cas9 system. Three 20 bp sgRNAs which were designed to insert into linearized plasmid pT7-gRNA, such as：192#（sense strand ）, target 244#（antisense strand）, target 313#（antisense strand）.Then gRNAs were transcribed in vitro. The Cas9 mRNA was transcribed using Cas9 expression vector as templates. Following completion of transcription, the poly（A）tailing reaction and DNase I treatments were performed. Both the gRNA and the Cas9-encoding mRNA were then purified and microinjected into the one cell stage of zebrafish embyos. Targeted genomic loci were amplified from genomic zebrafish DNA using primers then were tested by T7 Endonuclease I assays. Finally, the PCR products were constructed into pMD19-T simple vector, positive clones by colony PCR and Sanger sequencing to detect mutations. Results showed that SgRNA oligonucleotide duplexes successfully connected to the target site of plasmid pT7-gRNA and the sequence is correct, which targeted Exon 313 ^#site can successfully edit hoxb4 genes in zebrafish. T7EⅠ detect the knockout efficiency up to 26.5%. And four kinds of positive mutants were obtained by sequencing. The CRISPR/Cas9 system of knocking out the hoxb4 gene is successfully constructed and its mutations were identified and sequenced. The experiments provide a reliable knockout method for the study of the HOXb4 gene function.

Key words: zebrafish hoxb4 gene, CRISPR/Cas9, genome editing, mutation screening

Yuan Meng, He Zhixu, Shu Liping, Yuan Jiakan, Liu Feng, Li Yan. Genome Editing of Zebrafish hoxb4 Gene Using CRISPR/Cas9 System and Its Mutant Screening[J]. Biotechnology Bulletin, 2015, 31(10): 249-254.

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