Optimization of homology-directed repair in zebrafish

项目摘要 使用位点特异性核酸酶的基因组编辑促进了反向遗传方法的应用 到斑马鱼模型。最近出现的高度特异性和活性的RNA引导的核酸酶， 降低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， 通过双链断裂的DNA修复引入靶向插入或缺失（indels） 通过切割后的非同源末端连接（NHEJ）。更有限数量的研究 利用同源性定向修复（HDR）与DNA供体以及序列特异性 将目的序列引入斑马鱼基因组。虽然研究人员已经 在这方面的成功，HDR介导的修复事件的速率始终比插入缺失低得多。 NHEJ的频率此外，许多网站仍然拒绝以可接受的速度敲入。 这个问题对在哺乳动物细胞中有效产生所需的敲入等位基因提出了显著的障碍。 斑马鱼以及这项技术在该领域的广泛应用。因此，额外的研究 需要改进HDR以实现斑马鱼的精确基因组编辑。在建议的研究中，我们会 优化靶向敲入的条件，以允许在斑马鱼中进行高效精确的基因组编辑。在 目标1，我们将扩大我们的应用新的参数，确定通过初步研究 显示它们大大提高了敲入率，一组10个位点，以确认它们是 一般适用。在所有情况下，我们将应用明确和准确的基于深度测序的 分析体细胞编辑率，沿着评估生殖系传播，以确定最佳的 这些参数可在所有目标中推广。在目标2中，我们将使用优化的敲入 参数，以产生一系列携带“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，连同优化的参数， 精确的基因组编辑，将成为斑马鱼社区的宝贵资源。

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.