Cunfang ZHANG, Linyong HU, Sijia LIU, Wen WANG

Generation of Ectodysplasin A (eda)-targeted Knockout Zebrafish Via the CRISPR/Cas9 System

Ectodysplasin A (EDA) plays a vital role in the development of skin appendages, especially in fish scales. Zebrafish model with the mutation of eda was found using CRISPR/Cas9 system. CRISPR/Cas9 nucleases targeting to two loci in exon 4 of eda, were constructed and injected into zebrafish embryos, respectively. CRISPR-Cas9 mediated mutation frequency toward eda exon 4 was approximately 16%, which was relatively low compared with that of other genes in zebrafish. Five eda mutant types were obtained in F0 generation including a deletion of 5 bp, 6 bp, 8 bp and 87 bp, and an insertion of 11 bp around the targeted site respectively, and all of which happened just in one allele. But the scales of all F0 founders were normal compared with their wild counterparts. In the F1 generation, five scale loss mutants with few scales covered were achieved that were all caused by bi-allelic 11-bp insertion in eda. The insertion results in frameshift mutation of eda and leads to loss of expression and function inactivation of EDA as determined by western blotting. This provides a good model for elucidating the function of EDA in the development of fish scales.

CRISPR/Cas9 Sistemi Kullanılarak Ectodysplasin A (eda) İfade Etmeyen Zebra Balığı Üretimi

Ektodisplasin A (EDA) deri uzantılarının gelişiminde, özellikle balık pullarında, hayati bir rol oynar. CRISPR/Cas9 sistemi kullanılarak eda mutasyonlu zebra balığı modeli bulundu. Eda’nın ekson 4’ünde iki lokusu hedefleyen CRISPR/Cas9 nükleazları oluşturuldu ve zebra balığı embriyolarına enjekte edildi. Eda ekson 4’e yönelik CRISPR-Cas9 aracılı mutasyon sıklığı yaklaşık %16 idi; bu, zebra balığındaki diğer genlerinkine kıyasla nispeten düşüktü. F0 jenerasyonunda, hedeflenen bölge etrafında sırasıyla 5 bp, 6 bp, 8 bp ve 87 bp’lik bir silme ve 11 bp’lik bir ekleme dahil olmak üzere beş eda mutant türü elde edildi ve bunların hepsi sadece bir allelde gerçekleşti. Ancak tüm F0 üyelerinin pulları yabani akranlarına kıyasla normaldi. F1 jenerasyonunda, tamamı eda’ya bi-allelik 11-bp ilavesinden kaynaklanan az sayıda pulla çevrili beş adet pulsuz mutant elde edildi. Ekleme, eda’nın çerçeve kayması mutasyonuna neden olur ve Western Blot tekniği ile belirlendiği üzere EDA’nın ekspresyonunda azalma ve fonksiyon inaktivasyonuna yol açar. Bu çalışma, balık pullarının gelişiminde EDA’nın işlevini açıklamak için iyi bir model oluşturmaktadır.

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