Generation of a zebrafish knock-in line expressing MYC-tagged Sox11a using CRISPR/Cas9 genome editing.
Generation of a zebrafish knock-in line expressing MYC-tagged Sox11a using CRISPR/Cas9 genome editing.
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DOI：
10.1016/j.bbrc.2022.03.103
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发表时间：
2022-06-11
影响因子：
3.1
通讯作者：
Morris, Ann C.
中科院分区：
文献类型：
--
作者：
Krueger, Laura A.;Morris, Ann C.
CRISPR-Cas9基因组编辑技术的进步加强了斑马鱼作为遗传学和发育生物学模式生物的作用。这些工具导致功能丧失的突变斑马鱼品系的产量大幅增加。然而，由于同源定向修复(HDR)效率的降低，精确编辑的敲入线的产生在该领域仍然是一个巨大的挑战。在这项研究中，我们克服了其中的一些挑战，将现有的设计工具和合成的、商业上可用的CRISPR试剂结合在一起，在sox11a基因座产生了一个带有框内MYC表位标签的敲入线。斑马鱼Sox11a是一种转录因子，在器官发生、神经发生、颅面和骨骼发育中起重要作用；然而，目前只有少数几个直接的分子靶点被确定。在这里，我们评估了不同HDR供体配置的敲入效率，并展示了所产生的敲入等位基因的成功表达和定位。我们的结果为斑马鱼的敲入实验提供了一种高效、简化的方法，这将使以前难以执行的下游实验应用程序得以扩展。此外，我们生成的MYC-Sox11a线将允许进一步研究Sox11a的功能和直接靶标。
Advances in CRISPR-Cas9 genome editing technology have strengthened the role of zebrafish as a model organism for genetics and development biology. These tools have led to a significant increase in the production of loss-of-function mutant zebrafish lines. However, the generation of precisely edited knock-in lines has remained a significant challenge in the field due to the decreased efficiency of homology directed repair (HDR). In this study, we overcame some of these challenges by combining available design tools and synthetic, commercially available CRISPR reagents to generate a knock-in line carrying an in-frame MYC epitope tag at the sox11a locus. Zebrafish Sox11a is a transcription factor with critical roles in organogenesis, neurogenesis, craniofacial, and skeletal development; however, only a few direct molecular targets of Sox11a have been identified. Here, we evaluate the knock-in efficiency of various HDR donor configurations and demonstrate the successful expression and localization of the resulting knock-in allele. Our results provide an efficient, streamlined approach to knock-in experiments in zebrafish, which will enable expansion of downstream experimental applications that have previously been difficult to perform. Moreover, the MYC-Sox11a line we have generated will allow further investigation into the function and direct targets of Sox11a.
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影响因子：
2.7
作者：
Forbes-Osborne MA;Wilson SG;Morris AC
通讯作者：
Morris AC
影响因子：
7
作者：
Auer TO;Duroure K;De Cian A;Concordet JP;Del Bene F
通讯作者：
Del Bene F
影响因子：
9.2
作者：
Jia S;Wu X;Wu Y;Cui X;Tao B;Zhu Z;Hu W
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Hu W
影响因子：
4.6
作者：
Hisano Y;Sakuma T;Nakade S;Ohga R;Ota S;Okamoto H;Yamamoto T;Kawahara A
通讯作者：
Kawahara A
影响因子：
14.9
作者：
Prykhozhij SV;Fuller C;Steele SL;Veinotte CJ;Razaghi B;Robitaille JM;McMaster CR;Shlien A;Malkin D;Berman JN
通讯作者：
Berman JN