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：扩大基础编辑的范围 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。我们 最近开发的基础编辑，一种在没有目标基因组位点进行单基础变化的方法 引入双链断裂或依靠同源重组。基础编辑（BES）特别是 与遗传疾病的研究和治疗有关，因为大多数与疾病相关的突变是 单基础变化。自从我们使用C•g-t-t启用基础编辑以来的两年中，我们有 提高效率和产品纯度，降低目标和旁观者基础编辑，发展了新类 将A•t转换为g•c碱基对的腺嘌呤基础编辑器（ABE），扩展了BES的目标范围 在体内建立了溶后体细胞细胞的基础编辑，并将其应用于记录的细胞事件。 世界各地数百个其他实验室都使用基础编辑来研究遗传疾病并测试 潜在的治疗策略。在这里，我们建议将基础编辑者的功能扩展到 在任何体细胞中任何目标基因座的任何所需基础上的基础变化的变革目标。 基础编辑需要适当定位的原始探针相邻基序（PAM） Cas9域的结合。由于缺乏任何 识别大多数PAM的DNA结合CRISPR蛋白。进一步扩大我们基础编辑的能力 最广泛的目标，我们将使用噬菌体辅助连续演化（PACE）平台快速 进化的Cas9变体集合，这些变体目前识别许多不可销的PAM序列（AIM 1A）。 由于序列上下文，对BES的靶向范围也受到某些基本对的效率低下的限制。到 进一步扩大了基础编辑的目标范围，我们将使用我们最近建立的步伐选择基础 编辑以生成BES可以有效地通过当前不利的侧翼序列修改目标（AIM 1B）。 基础编辑器修改了编辑窗口中的基础，相对于〜5个核苷酸的范围相对于 帕姆。除了转换目标c•g或a•t碱基对，其他“旁观者” C•g或a•t碱基对 还在此窗口中进行了编辑。这些旁观者的编辑可能导致不希望的基因组变化。最小化 旁观者基础编辑，我们建议进化一大堆BES 序列上下文（AIM 2），从而在多个CS或编辑窗口内歧视。 最后，基础编辑的主要局限性是无法产生横向（嘌呤β嘧啶） 需要安装或纠正约38％已知人类病原SNP的突变。我们建议 开发可以使用两个不同的不同 策略（目标3A和3B）。两种策略的成功将大大扩展基础编辑的能力， 并原则上允许通过个人或连续使用的所有12个可能的基础对基地更改 过渡和转移编辑器。