Construction of akap12α or β specific knockout zebrafish using CRISPR/Cas9 system

Abstract

AKAP12 (A-Kinase Anchoring Protein 12) is a scaffolding protein which regulates various biological processes. It has been reported that AKAP12 regulates the cytokinesis progression and involves in the formation of brain-barrier by regulating angiogenesis and tight junction formation. Also, AKAP12 has a tumor suppressor function and relates to cell migration. AKAP12 has several binding partners such as PK (protein kinase) C and A, calmodulin, cyclins, phosphoinositides and β-1,4 galactosyltransferase. It has been identified that there are three types of AKAP12 isoforms (designated α, β and γ) with different promoters in human. In zebrafish, two types of AKAP12 isoforms, AKAP12α and AKAP12β, have been reported. We confirmed that AKAP12α and AKAP12β were expressed similarly but distinctly. However, each function of two isoforms is not known so far. To elucidate the role of AKAP12 isoforms, we constructed akap12α or β specific knockout zebrafish. To construct knockout zebrafish, we adopted CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated nuclease 9) system which is powerful and efficient tool for genome modifications at the targeted genomic locus. Here, we reported the construction of akap12α or β specific knockout zebrafish using the latest genome editing tool. To confirm the mutagenesis, we performed T7E1 (T7 endonuclease 1) assay. As a result, expected fragments were detected and indicated the CRISPR/Cas9-mediated modification. Also, we confirmed the presence of indel mutation by DNA sequencing of the akap12α or β specific target region. The results displayed that several bases were deleted or inserted. Then, we identified developmental abnormalities of akap12α or β specific knockout zebrafish. Trunk defect, hemorrhage and disrupted heart laterality were observed in akap12α or β specific knockout zebrafish. However, there was slight difference between akap12α knockout zebrafish and akap12β knockout zebrafish in quantitative analysis. In akap12β knockout zebrafish, abnormal embryos were more observed relatively. Taken together, these data suggested that AKAP12α and AKAP12β performed similar functions, but there was slight difference in expression period and amounts. akap12β was expressed form the earlier stage and more abundantly. Moreover, akap12β knockout embryos showed more defects numerically.