Expanding the Scope of Base Editing

项目总结：扩大基本编辑的范围 基因组编辑已经给生命科学带来了革命性的变化，并提供了治愈遗传病的潜力。我们 最近开发的碱基编辑，一种在目标基因组位置进行单碱基改变的方法，而不需要 引入双链断裂或依赖同源重组。基础编辑(BE)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 单基数变化。自从我们率先使用C·G-to-T·A编辑进行基础编辑以来的两年里，我们已经 提高了BE效率和产品纯度，减少了偏离目标和旁观者的基础编辑，形成了一个新的类别 将A·T碱基对转换为G·C碱基对的腺嘌呤碱基编辑分子(ABES)，扩大了BES的靶向范围， 建立了体内有丝分裂后体细胞的基础编辑，并应用BES记录细胞事件。 世界上数以百计的其他实验室已经使用碱基编辑来研究遗传病和测试 潜在的治疗策略。在这里，我们建议将基础编辑的能力扩展到 变革性的目标是在任何体细胞中的任何目标位置实现任何所需的碱基变化。 碱基编辑需要存在一个适当定位的邻近基序(PAM)，用于 Cas9结构域的结合。大多数DNA站点仍然无法进行基因组编辑，因为缺少 识别大多数PAM的DNA结合CRISPR蛋白。为了进一步扩展我们的基本编辑能力 ，我们将使用我们的噬菌体辅助持续进化(PACE)平台快速 进化一组Cas9变异体，识别目前许多无法靶向的PAM序列(目标1a)。这个 由于序列环境的原因，BES的靶向范围也受到某些碱基对的低效编辑的限制。至 进一步扩大基础编辑的目标范围，我们将用我们最近建立的节奏选择基础 编辑以生成可有效修改具有当前不受欢迎的侧翼序列的目标的BES(目标1b)。 碱基编辑者在编辑窗口中修改碱基，相对于 帕姆。除了目标C？G或A？T碱基对的转换外，其他“旁观者”的C？G或A？T碱基对还有 也可在此窗口中编辑。这些旁观者的编辑可能会导致不希望看到的基因组变化。要最小化 旁观者碱基编辑，我们建议进化出一大组BES，它将只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不能产生颠换(嘧啶 突变，安装或纠正约38%的已知人类致病SNPs所需的突变。我们建议 开发第一个碱基编辑器，可以使用两个不同的方法在目标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基本编辑的能力， 并原则上允许所有12种可能的碱基到碱基的改变通过单独或连续使用 过渡和转换编辑器。

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.