Dhinoja Sanchi, Mary Jabila, Qaryoute Ayah Al, De Maria Anthony, Jagadeeswaran Pudur
Department of Biological Sciences, University of North Texas, Denton, Texas, USA.
Blood Coagul Fibrinolysis. 2025 Jan 1;36(1):26-33. doi: 10.1097/MBC.0000000000001337. Epub 2024 Nov 29.
This study aimed to create an f9l mutant zebrafish using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and characterize its coagulation properties to investigate its functional similarity to human FX and explore the potential synergy between f9l and f10 .
Three gRNAs targeting exon 8 encoded by the catalytic domain of the f9l gene were injected into 300 single-cell zebrafish embryos using CRISPR/Cas9 technology. DNA from the resulting adults was extracted from tail tips, and PCR was used to detect indels. The identified founder mutant was bred to homozygosity, and functional assays, kinetic Russel viper venom time, bleeding assay in adults, and venous laser injury on larvae were conducted to assess its hemostatic function. Additionally, f10 was knocked down in f9l homozygous embryos using f10 antisense morpholinos to study their interaction by monitoring its survival.
DNA from 60 adults was screened for indels, resulting in a fish with a heritable complex mutation involving one insertion and two deletions in exon 8. The f9l homozygous mutants exhibited impaired F10 activity, mild bleeding after mechanical injury, and developmental deformities in early larval stages. The caudal vein thrombosis assay showed variable occlusion times, indicating a bleeding phenotype with incomplete penetrance. Knocking down f10 in f9l homozygous embryos resulted in 50% mortality within five dpf, compared to f9l homozygous embryos injected with control morpholinos.
In summary, we generated f9l knockout and showed it is a paralog to f10. We also found a synergy between f9l and f10 genes, highlighting its importance in hemostasis.
本研究旨在利用成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白9（Cas9）技术创建f9l突变斑马鱼，并对其凝血特性进行表征，以研究其与人类凝血因子X（FX）的功能相似性，并探索f9l和f10之间的潜在协同作用。
使用CRISPR/Cas9技术将靶向f9l基因催化结构域编码的第8外显子的三种引导RNA（gRNA）注射到300个单细胞斑马鱼胚胎中。从所得成鱼的尾尖提取DNA，并用聚合酶链反应（PCR）检测插入缺失。将鉴定出的奠基突变体培育至纯合状态，并进行功能测定、动态罗素蝰蛇毒时间、成鱼出血试验以及幼虫静脉激光损伤试验，以评估其止血功能。此外，使用f10反义吗啉代寡核苷酸在f9l纯合胚胎中敲低f10，通过监测其存活情况来研究它们之间的相互作用。
对60条成鱼的DNA进行插入缺失筛选，得到一条具有可遗传复合突变的鱼，该突变涉及第8外显子的一个插入和两个缺失。f9l纯合突变体表现出F10活性受损、机械损伤后轻度出血以及早期幼虫阶段的发育畸形。尾静脉血栓形成试验显示闭塞时间可变，表明存在外显不全的出血表型。与注射对照吗啉代寡核苷酸的f9l纯合胚胎相比，在f9l纯合胚胎中敲低f10导致在受精后5天内死亡率达到50%。
总之，我们生成了f9l基因敲除斑马鱼，并表明它是f10的旁系同源基因。我们还发现f9l和f10基因之间存在协同作用，突出了其在止血中的重要性。
