Expanding the Scope of Base Editing

扩大碱基编辑的范围

• 批准号: 10227955
• 负责人: DAVID R LIU
• 金额: $ 42.15万
• 依托单位: BROAD INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-23 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10227955
• 关键词: Address; Adenine; Bacteriophages; Base Pairing; Binding; Biological Sciences; Cells; Chemicals; Clinic; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Cytidine; DNA; DNA Binding; DNA Repair Pathway; Deamination; Development; Discrimination; Disease; Effectiveness; Event; Evolution; GTP-Binding Protein alpha Subunits, Gs; Genetic; Genetic Diseases; Genetic study; Genome; Genomics; Goals; Guanine; Human; Individual; Laboratories; Lead; Letters; Location; Mediating; Methods; Mitotic; Mutation; Nature; Nucleotides; Organism; Pathogenicity; Patients; Positioning Attribute; Proteins; Publications; Purines; Pyrimidine; Pyrimidines; Reporting; Research; Science; Site; Somatic Cell; Specificity; Testing; Therapeutic; Variant; base; disease-causing mutation; genome editing; homologous recombination; human disease; improved; in vivo; innovation; next generation; nucleobase; oxidation; success; transition mutation; transversion mutation

Project Summary: Expanding the Scope of Base Editing Genome editing has revolutionized the life sciences and offers the potential to cure genetic diseases. We recently developed base editing, a method of making single-base changes at target genomic sites without introducing double-strand breaks or relying on homologous recombination. Base editors (BEs) are especially relevant for the study and treatment of genetic diseases because the majority of disease-relevant mutations are single-base changes. In the two years since we pioneered base editing with a C•G-to-T•A editor, we have improved BE efficiency and product purity, reduced off-target and bystander base editing, evolved a new class of adenine base editors (ABEs) that convert A•T to G•C base pairs, expanded the targeting scope of BEs, established base editing of post-mitotic somatic cells in vivo, and applied BEs to record cellular events. Hundreds of other laboratories around the world have used base editing to study genetic diseases and to test potential therapeutic strategies. Here we propose to expand the capabilities of base editors towards the transformative goal of enabling any desired base change at any target locus in any somatic cell. Base editing requires the presence of an appropriately positioned protospacer adjacent motif (PAM) for binding of the Cas9 domain. Most DNA sites remain inaccessible for genome editing due to the lack of any DNA-binding CRISPR protein that recognizes the majority of PAMs. To further expand our ability to base edit the broadest range of targets, we will use our phage-assisted continuous evolution (PACE) platform to rapidly evolve a collection of Cas9 variants that recognize currently many untargetable PAM sequences (Aim 1a). The targeting scope of BEs is also limited by inefficient editing of certain base pairs because of sequence context. To further expand the targeting scope of base editing, we will use our recently established PACE selection for base editing to generate BEs that can efficiently modify targets with currently disfavored flanking sequences (Aim 1b). Base editors modify bases within the editing window, a range of ~5 nucleotides positioned relative to the PAM. In addition to conversion of the target C•G or A•T base pair, other “bystander” C•G or A•T base pairs are also edited within this window. These bystander edits can lead to undesired genome changes. To minimize bystander base editing, we propose to evolve a large set of BEs that will only edit bases within specific sequence contexts (Aim 2), thereby enabling discrimination between multiple Cs or As within the editing window. Finally, a major limitation of base editing is the inability to generate transversion (purine ßà pyrimidine) mutations, which are needed to install or correct ~38% of known human pathogenic SNPs. We propose to develop the first base editors that can generate transversion mutations at target base pairs using two distinct strategies (Aims 3a and 3b). Success with either strategy would greatly expand the capabilities of base editing, and would also allow, in principle, all 12 possible base-to-base change via individual or sequential use of transition and transversion editors.

项目摘要：扩大碱基编辑范围 基因组编辑彻底改变了生命科学，并提供了治愈遗传疾病的潜力。 最近开发的碱基编辑，一种在目标基因组位点进行单碱基改变的方法，无需 引入双链断裂或依赖同源重组（BE）尤其如此。 与遗传疾病的研究和治疗相关，因为大多数与疾病相关的突变是 自从我们率先使用 C•G-to-T•A 编辑器进行碱基编辑以来，两年来，我们已经实现了单碱基变化。 提高BE效率和产品纯度，减少脱靶和旁观者碱基编辑，进化出一个新类别 将 A•T 碱基对转换为 G•C 碱基对的腺嘌呤碱基编辑器 (ABE)，扩大了 BE 的靶向范围， 建立了体内有丝分裂后体细胞的碱基编辑，并应用BE来记录细胞事件。 世界各地数百个其他实验室已使用碱基编辑来研究遗传疾病并测试 在这里，我们建议扩展碱基编辑器的能力。 变革性目标是在任何体细胞的任何目标位点实现任何所需的碱基变化。 碱基编辑需要存在适当定位的原型间隔子相邻基序 (PAM) 由于缺乏 Cas9 结构域的结合，大多数 DNA 位点仍然无法进行基因组编辑。 可识别大多数 PAM 的 DNA 结合 CRISPR 蛋白 进一步扩展我们的碱基编辑能力。 为了实现最广泛的目标，我们将使用我们的噬菌体辅助持续进化（PACE）平台快速 进化出一系列 Cas9 变体，可识别当前许多不可靶向的 PAM 序列（目标 1a）。 BE 的靶向范围还受到序列上下文导致某些碱基对编辑效率低下的限制。 进一步扩大碱基编辑的靶向范围，我们将使用我们最近建立的PACE选择碱基 编辑以生成 BE，可以有效地修改具有当前不受欢迎的侧翼序列的目标（目标 1b）。 碱基编辑器在编辑窗口内修改碱基，相对于碱基定位的~5个核苷酸的范围 PAM 除了目标 C•G 或 A•T 碱基对的转换外，还可以转换其他“旁观者”C•G 或 A•T 碱基对。 也在此窗口中进行编辑，这些旁观者编辑可能会导致不良的基因组变化。 旁观者碱基编辑，我们建议发展一大组 BE，它们只会编辑特定范围内的碱基 序列上下文（目标 2），从而能够在编辑窗口内区分多个 C 或 As。 最后，碱基编辑的一个主要限制是无法产生颠换（嘌呤 ßà 嘧啶） 我们建议安装或纠正约 38% 的已知人类致病性 SNP 所需的突变。 开发第一个碱基编辑器，可以使用两种不同的方法在目标碱基对上产生颠换突变 策略（目标 3a 和 3b）。任一策略的成功都将极大地扩展碱基编辑的能力， 原则上，还允许通过单独或连续使用 过渡和颠换编辑器。