Zebrafish is a model organism widely used for the understanding of gene function, including the fundamental basis of human disease, enabled by the presence in its genome of a high number of orthologs to human genes. CRISPR/Cas9 and next-generation gene-editing techniques using cytidine deaminase fused with Cas9 nickase provide fast and efficient tools able to induce sequence-specific single base mutations in various organisms and have also been used to generate genetically modified zebrafish for modeling pathogenic mutations. However, the editing efficiency in zebrafish of currently available base editors is lower than other model organisms, frequently inducing indel formation, which limits the applicability of these tools and calls for the search of more accurate and efficient editors. Here, we generated a new base editor (zAncBE4max) with a length of 5560 bp following a strategy based on the optimization of codon preference in zebrafish. Our new editor effectively created C-to-T base substitution while maintaining a high product purity at multiple target sites. Moreover, zAncBE4max successfully generated the Twist2 p.E78K mutation in zebrafish, recapitulating pathological features of human ablepharon macrostomia syndrome (AMS). Overall, the zAncBE4max system provides a promising tool to perform efficient base editing in zebrafish and enhances its capacity to precisely model human diseases.

中文翻译：

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经过优化的基础编辑器，可在斑马鱼中进行高效的C-to-T基础编辑

斑马鱼是一种模式生物，广泛用于了解基因功能，包括人类疾病的基本基础，这是由于其基因组中存在大量与人类基因的直系同源物而实现的。CRISPR / Cas9和使用胞嘧啶脱氨酶与Cas9切口酶融合的下一代基因编辑技术提供了快速有效的工具，能够在各种生物体中诱导序列特异性单碱基突变，也已被用于生成遗传修饰的斑马鱼以模拟致病性突变。但是，当前可用的基础编辑器在斑马鱼中的编辑效率低于其他模型生物，从而经常导致插入缺失的形成，这限制了这些工具的适用性，并要求寻找更准确和有效的编辑器。这里，我们基于优化斑马鱼密码子偏好性的策略，生成了一个新的碱基编辑器（zAncBE4max），长度为5560 bp。我们的新编辑人员有效地创建了C-to-T碱基替代，同时在多个目标位点上保持了较高的产品纯度。此外，zAncBE4max在斑马鱼中成功产生了Twist2 p.E78K突变，从而概括了人类canpharon macrostomia综合征（AMS）的病理特征。总体而言，zAncBE4max系统提供了一种有前途的工具，可在斑马鱼中进行有效的碱基编辑，并增强了其精确建模人类疾病的能力。zAncBE4max成功地在斑马鱼中产生了Twist2 p.E78K突变，概括了人类ablepharon口臭综合征（AMS）的病理特征。总体而言，zAncBE4max系统提供了一种有前途的工具，可以在斑马鱼中进行有效的碱基编辑，并增强了其精确建模人类疾病的能力。zAncBE4max成功地在斑马鱼中产生了Twist2 p.E78K突变，概括了人类ablepharon口臭综合征（AMS）的病理特征。总体而言，zAncBE4max系统提供了一种有前途的工具，可以在斑马鱼中进行有效的碱基编辑，并增强了其精确建模人类疾病的能力。