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.

斑马鱼是模拟人类出生缺陷和疾病的首要模式生物之一。单碱基 配对变化是调节或蛋白质改变突变的常见候选者， 罕见疾病。然而，编辑内源性斑马鱼基因以模拟错义突变或其他离散突变， 人类变异仍然具有挑战性。Prime editing（PE）是一项突破性的基于Cas9的应用， 建立基于动物和细胞的疾病模型，并开发人类遗传疾病的治疗方法 因为它能够实现精确的基因组编辑，例如碱基替换、小插入和缺失。Prime 编辑酶（PE 2）由pegRNA引导至互补基因组基因座，该基因座含有适当的 前间区序列邻近基序（PAM）识别位点，5 '-NGG-3'。PE 2的切口酶活性在细胞表面引入切口。 靶序列的非靶链。然后，PE 2的逆转录酶（RT）读取PE 2的RT模板。 含有所需编辑的pegRNA并合成DNA链。而最近的一项研究表明， 递送PE 2蛋白可以诱导斑马鱼发生PE，因为PE 2酶尚未市售， 这种方法对于许多斑马鱼实验室来说仍然是无法实现的。而且，目前的方法效率不高 可编辑的PE范围是相对小的区域，包括PAM上游3bp至下游29 bp 识别位点。 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PE 2的RNA形式的改良PE方法是可行的 Cas9-RT在高达20%的注射F0胚胎中实现编辑。我们的目标1是1）优化条件， 通过注射不同剂量的三种RNA组分PE 2，Cas9-RT， 和pegRNA导入斑马鱼和2）建立PE转基因系。我们的目标2是扩大主要编辑 范围为此，我们将首先测试Cas9 D10 A切口酶是否可以作为PE 2酶应用以获得 PAM位点上游的序列。 代替使用切割非靶标的Cas9 H840 A切口酶， 为了检测Cas9 D10 A突变体形式，我们将测试Cas9 D10 A突变体形式，其切割靶链。切割靶链将 触发pegRNA的引物结合位点与PAM位点附近的序列的杂交。因此，委员会认为， pegRNA的RT模板将位于PAM位点的更上游， 在可编辑范围内。与此同时，我们将调查 来自犬链球菌的Cas9（ScCas 9），其 需要单个鸟嘌呤（G）核苷酸作为PAM，可以取代PE 2中的标准Cas9，后者需要5 '- NGG-3“。通过这些研究，本项目将优化改进的主编辑方法， 斑马鱼和扩大其范围，以促进准确的斑马鱼模型的产生，以改善诊断， 机制研究和治疗筛选。