Expanding the Scope of Base Editing

扩大碱基编辑的范围

• 批准号: 9982216
• 负责人: DAVID R LIU
• 金额: $ 42.18万
• 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/9982216
• 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.

项目概要：扩大基础编辑的范围 基因组编辑已经彻底改变了生命科学，并提供了治愈遗传疾病的潜力。我们 最近开发的碱基编辑，一种在靶基因组位点进行单碱基改变的方法， 引入双链断裂或依赖于同源重组。基本编辑器（BE）特别是 与遗传疾病的研究和治疗相关，因为大多数疾病相关的突变是 单基变化。自从我们率先使用C·G-to-T·A编辑器进行基础编辑以来， 提高BE效率和产物纯度，减少脱靶和旁观者碱基编辑，进化出一种新的类别 腺嘌呤碱基编辑器（Adenine base editors，ABE）将A·T碱基对转换为G·C碱基对，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记录细胞事件。 世界各地的数百个其他实验室已经使用碱基编辑来研究遗传疾病， 潜在的治疗策略在这里，我们建议将基本编辑器的功能扩展到 这是一个能够在任何体细胞中的任何靶基因座处实现任何期望的碱基改变的转化目标。 碱基编辑需要存在适当定位的前间区序列邻近基序（PAM），以用于 Cas9结构域的结合。大多数DNA位点仍然无法进行基因组编辑，因为缺乏任何 DNA结合CRISPR蛋白，识别大多数PAM。为了进一步扩展我们的基础编辑能力， 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， 进化出识别目前许多不可靶向的PAM序列的Cas9变体的集合（Aim 1a）。的 BE的靶向范围还受到由于序列背景而导致的某些碱基对的低效编辑的限制。到 进一步扩大基地编辑的目标范围，我们将使用我们最近建立的PACE选择基地 编辑以产生可以有效地修饰具有当前不受欢迎的侧翼序列的靶标的BE（Aim 1b）。 碱基编辑器在编辑窗口内修改碱基，相对于碱基序列定位的~5个核苷酸的范围。 Pam.除了靶C·G或A·T碱基对的转换外，其他“旁观者”C·G或A·T碱基对也被转化。 也在这个窗口中编辑。这些旁观者编辑可能导致不期望的基因组变化。以最小化 旁观者碱基编辑，我们建议进化出一个大的BE集合，它们只会编辑特定碱基内的碱基。 序列上下文（目标2），从而能够在编辑窗口内区分多个C或A。 最后，碱基编辑的一个主要限制是不能产生颠换（嘌呤β-嘧啶） 这些突变是安装或纠正约38%的已知人类致病性SNP所需的。我们建议 开发第一个碱基编辑器，可以使用两个不同的碱基对在靶碱基对上产生颠换突变。 战略（目标3a和3b）。任何一种策略的成功都将极大地扩展碱基编辑的能力， 并且原则上还允许通过单独或顺序使用所有12种可能的碱基到碱基的变化， transition和transversion编辑器