Increasing the efficiency and range of prime editing for disease modeling in zebrafish

斑马鱼是模拟人类先天缺陷和疾病的主要模式生物之一。单碱基 配对改变是调节或蛋白质改变突变的常见候选因素，这些突变是许多常见的和 罕见的疾病。然而，编辑内源性斑马鱼基因来模拟错义突变或其他离散的 人类的变异仍然具有挑战性。Prime Editing(PE)是一个突破性的基于Cas9的应用程序 建立基于动物和细胞的疾病模型和开发人类遗传病的治疗方法 因为它能够进行精确的基因组编辑，如碱基替换、小插入和删除。素数 编辑酶(PE2)由pegRNA定向到一个互补的基因组位置，该基因座包含适当的 Protspacer邻近基序(PAM)识别位点，5‘-NGG-3’。PE2的镍酶活性引入了一个缺口 靶序列的非靶向链。PE2的逆转录酶(RT)然后读取 含有所需编辑和合成的DNA链的pegRNA。鉴于最近的一项研究表明 传递PE2蛋白可以诱导斑马鱼发生PE，因为PE2酶还没有商业化， 许多斑马鱼实验室仍然无法使用这种方法。此外，目前的方法效率不高。 可编辑的PE范围是一个相对较小的区域，包括PAM上游3个BP到下游29个BP 识别站点。 为了克服PE的这些局限性，并使其在斑马鱼实验室中广泛发挥作用，在我们的 初步研究表明，在斑马鱼中使用PE2的RNA形式的改良PE方法是可行的 和Cas9-RT实现了高达20%的注入F0胚胎的编辑。我们的目标是1)优化生产条件 通过注射不同剂量的三种RNA成分PE2，Cas9-RT， 和pegRNA，导入斑马鱼；2)建立PE转基因系。我们的目标2是扩大主要的编辑 射程。为此，我们将首先测试Cas9 D10A镍酶是否可以作为PE2酶应用于Access PAM位点上游的序列。 而不是使用切割非靶标的Cas9 H840A镍酶 对于链，我们将测试Cas9 D10A突变形式，它切割目标链。剪断目标链将 触发pegRNA的引物结合位点与PAM位点附近的序列的杂交。因此， PegRNA的RT模板将位于PAM位点的更上游，从而带来上游序列 在可编辑范围内。同时，我们将调查是否 从犬链球菌(ScCas9)中分离到的Cas9 需要一个鸟嘌呤(G)核苷酸作为PAM，可以在PE2中取代标准的Cas9，它需要5‘- NGG-3‘。通过这些研究，本项目将对素材编辑的修改方法进行优化 斑马鱼，并扩大其范围，以促进生成准确的斑马鱼模型，以改善诊断， 机械学研究和治疗学筛选。

Zebrafish is among the premier model organisms for modeling human birth defects and diseases. Single base pair changes are common candidates for regulatory or protein altering mutations underlying many common and rare diseases. However, editing the endogenous zebrafish genes to model missense mutations or other discrete human variants remains challenging. Prime editing (PE) is a breakthrough Cas9-based application for the creation of animal and cell-based models of disease and for developing treatments of human genetic diseases because it enables precise genome editing such as base substitutions, small insertions, and deletions. Prime editing enzyme (PE2) is directed by pegRNA to a complementary genomic locus that contains an appropriate protospacer adjacent motif (PAM) recognition site, 5’-NGG-3’. Nickase activity of PE2 introduces a nick to the non-target strand of the target sequence. The reverse transcriptase (RT) of PE2 then reads the RT template of the pegRNA containing the desired edit and synthesizes the DNA strand. Whereas a recent study showed that delivery of PE2 protein can induce PE in zebrafish, because the PE2 enzyme is not yet commercially available, this approach remains inaccessible to many zebrafish laboratories. Moreover, the current method is not efficient and editable PE range is a relatively small region encompassing 3 bp upstream to 29 bp downstream of the PAM recognition site. To overcome these limitations of PE and make it broadly functional in zebrafish laboratories, in our preliminary studies we showed feasibility in zebrafish of a modified PE method that uses the RNA forms of PE2 and Cas9-RT achieving editing in up to 20% of injected F0 embryos. Our Aim 1 is 1) to optimize conditions for the modified prime editing method by injecting different doses of the three RNA components, PE2, Cas9-RT, and pegRNA, into zebrafish and 2) to establish a PE transgenic line. Our Aim 2 is to expand the prime editing range. To this end, we will first test whether Cas9 D10A nickase can be applied as a PE2 enzyme to access sequences upstream of the PAM site. Instead of using the Cas9 H840A nickase that cleaves the non-target strand, we will test the Cas9 D10A mutant form, which cleaves the target strand. Nicking the target strand will trigger hybridization of the primer binding site of pegRNA to the sequences near the PAM site. Consequently, the RT template of the pegRNA will be located further upstream of the PAM site, bringing the upstream sequence within editable range. In parallel, we will investigate whether Cas9 from Streptococcus canis (ScCas9), which requires a single guanine (G) nucleotide as a PAM, can substitute in PE2 the standard Cas9, which requires 5’- NGG-3’. Through these lines of research, this project will optimize the modified method of prime editing in zebrafish and expand its range to facilitate generation of accurate zebrafish models for improved diagnosis, mechanistic studies and therapeutics screens.