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

提高斑马鱼疾病建模的主要编辑效率和范围

• 批准号: 10667988
• 负责人: LILIANNA SOLNICAKREZEL
• 金额: $ 19.54万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-15 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10667988
• 关键词: Animal Model; Animals; Base Pairing; Binding Sites; Biological Models; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Congenital Abnormality; DNA; DNA biosynthesis; Data; Development; Diagnosis; Disease; Disease model; Dose; Embryo; Endonuclease I; Enzymes; Funding; Generations; Genes; Genetic Diseases; Genetic Transcription; Genome; Genome engineering; Goals; Guanine; Guide RNA; Human; Human Genetics; In Vitro; Injections; Knock-in; Laboratories; Laboratory Animals; Mammalian Cell; Methods; Missense Mutation; Modeling; Mutation; Nucleotides; Patients; Proteins; RNA; RNA-Directed DNA Polymerase; Rare Diseases; Relaxation; Research; Research Personnel; Site; Streptococcus; System; Techniques; Technology; Testing; Therapeutic; Time; Transgenic Organisms; United States National Institutes of Health; Variant; Vertebral column; Work; Zebrafish; base; cost; disease mechanisms study; experimental study; genetic analysis; genetic variant; genome editing; genomic locus; human model; improved; in vivo; innovation; interest; model organism; mutant; next generation sequencing; novel; novel strategies; precise genome editing; prime editing; prime editor; success; zygote

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.

斑马鱼是建模人类出生缺陷和疾病的主要模型生物。单基础 成对变化是许多常见和蛋白质改变突变的常见候选者 罕见疾病。但是，编辑内源斑马鱼基因以建模错义突变或其他离散 人类变种仍然受到挑战。 Prime编辑（PE）是基于CAS9的突破性应用程序 创建动物和基于细胞的疾病模型，并开发人类遗传疾病的治疗 因为它可以实现精确的基因组编辑，例如基础取代，小插入和缺失。主要的 Pegrna指示编辑酶（PE2）到一个完整的基因组基因座，其中包含适当的 原始探针相邻基序（PAM）识别位点，5'-NGG-3'。 PE2的Nickase活性将刻度引入 目标序列的非目标链。然后，PE2的逆转录酶（RT）读取的RT模板 包含所需编辑并合成DNA链的Pegrna。而最近的一项研究表明 PE2蛋白的递送可以在斑马鱼中诱导PE，因为PE2酶尚未商业上可用，， 对于许多斑马鱼实验室来说，这种方法仍然无法访问。此外，当前方法不是有效的 而可编辑的PE范围是一个相对较小的区域，包括3 bp至29 bp的PAM下游 识别网站。 为了克服这些对PE的局限性并在斑马鱼实验室中广泛发挥作用 初步研究我们显示了使用PE2的RNA形式的修饰PE方法的斑马鱼的可行性 CAS9-RT在注射的F0胚胎的20％中进行编辑。我们的目标1是1）优化条件的 通过注入不同剂量的三个RNA组件PE2，CAS9-RT， 和Pegrna，进入斑马鱼和2）建立PE转基因线。我们的目标2是扩展主要编辑 范围。为此，我们将首先测试CAS9 D10A Nickase是否可以用作PE2酶访问 PAM站点上游的序列。 而不是使用CAS9 H840A切割非目标的nickase 链，我们将测试CAS9 D10A突变体形式，该突变体形式裂解目标链。划分目标链将 触发Pegrna的引物结合位点与PAM位点附近的序列的杂交。最后， Pegrna的RT模板将位于PAM站点的更高上游，带来上游序列 在可编辑范围内。同时，我们将调查是否 CAS9来自Canis链球菌（SCCAS9）， 需要单个鸟嘌呤（G）核苷酸作为PAM，可以在PE2中代替标准Cas9，该标准Cas9需要5'-- NGG-3'。通过这些研究线，该项目将优化修改的主要编辑方法 斑马鱼并扩大其范围，以促进精确的斑马鱼模型，以改善诊断， 机械研究和治疗筛查。