Optimization of homology-directed repair in zebrafish

项目总结 用位点特异性核酸酶进行基因组编辑促进了反向遗传方法的应用 斑马鱼模型。最近出现的高度特异和活性的RNA引导的核酸酶 降低了基因组编辑的成本和技术障碍，使反向遗传学能够应用于 几乎所有的斑马鱼实验室。在大多数已发布的应用程序中，都使用了这些工具 通过双链断裂的DNA修复引入定向插入或缺失(INDELs) 通过切割后的非同源末端连接(NHEJ)。数量更有限的研究已经 利用DNA供体的同源定向修复(HDR)和序列特异性 核酸酶将感兴趣的序列引入斑马鱼基因组。虽然研究人员已经有了 在这方面，HDR调解的修复事件的成功率始终比INDEL低得多 来自NHEJ的频率。此外，许多网站仍然顽固地拒绝以可接受的速度敲门。 这个问题给高效生产所需的敲入等位基因带来了很大的障碍。 斑马鱼以及这项技术在野外的广泛应用。因此，更多的研究是 需要改进HDR，以精确编辑斑马鱼的基因组。在建议的研究中，我们会 优化定向敲入的条件，以便在斑马鱼中进行高效精确的基因组编辑。在……里面 目标1，我们将扩大通过初步研究确定的新参数的应用 显示他们极大地提高了敲击率，到一组10个基因座来确认他们是 一般适用。在所有情况下，我们都将应用基于确定和准确的深度测序 分析体细胞编辑率，同时评估生殖系传递以确定最佳 可在所有目标上泛化的参数。在目标2中，我们将使用优化的敲击 参数，以产生一系列带有生物标记的内源基因座的斑马鱼，以使细胞类型- 特异性增强子和转录组分析。这些线路与优化的参数一起用于 精确的基因组编辑，将成为斑马鱼界的宝贵资源。

PROJECT SUMMARY Genome editing with site-specific nucleases has facilitated application of reverse genetic approaches to the zebrafish model. The recent advent of highly specific and active RNA-guided nucleases have reduced cost and technical hurdles for genome editing, allowing reverse genetics to be applied in nearly all zebrafish laboratories. In the majority of published applications, these tools have been used to introduce targeted insertions or deletions (indels) by virtue of DNA repair of double strand breaks via non-homologous end-joining (NHEJ) following cleavage. A more limited number of studies have utilized homology-directed repair (HDR) with a DNA donor together with a sequence-specific nuclease to introduce sequences of interest into the zebrafish genome. While researchers have had success in this regard, rates of HDR-mediated repair events are consistently much lower than indel frequencies from NHEJ. Furthermore, many sites remain recalcitrant to knock-in at acceptable rates. This issue raises a significant barrier to the efficient production of desired knock-in alleles in the zebrafish and the widespread use of this technique in the field. Therefore, additional studies are needed to improve HDR for precise genome editing in zebrafish. In the proposed studies, we will optimize conditions for targeted knock-in to allow efficient precise genome editing in the zebrafish. In Aim 1, we will expand our application of novel parameters, identified through preliminary studies showing they that drastically improve knock-in rates, to a set of 10 loci to confirm that they are generally applicable. In all cases, we will apply definitive and accurate deep sequencing-based analysis of somatic editing rates, along with assessment of germline transmission to identify optimal parameters that are generalizable across all targets. In Aim 2, we will use optimized knock-in parameters to generate a series of zebrafish bearing "Biotagged" endogenous loci to enable cell type- specific enhancer and transcriptome analysis. These lines, together with the optimized parameters for precise genome editing, will serve as valuable resources for the zebrafish community.