CRISPR Chemistry

最近发现了一种用于基因编辑的变革性新技术，称为CRISPR-Cas。它允许蛋白质（Cas9）被CRISPR RNA分子编程，以修饰基因组DNA中的特定序列。这可以用来改变细胞中特定蛋白质的水平，甚至可以用来消除它们或改变它们的功能（通过位点特异性突变）。这项技术在生物学和医学的许多领域都有着深远的影响，在未来，它可能被用来成功地治疗人类遗传疾病和癌症。通过创建人工（修饰的）Cas9蛋白，还可以编辑细胞的RNA分子，并实时创建活细胞中基因组的详细图像。然而，CRISPR-Cas技术也存在缺点：执行某些编辑任务所需的CRISPR RNA分子数量成本高昂，难以对基因组中的多个DNA基因座进行成像，并且完全改变细胞的特征（细胞类型）目前尚不可行。此外，基因组中的非预期编辑事件（脱靶效应）是常见的，并且这些是潜在的灾难性的，特别是在治疗竞技场中，当它们用于治疗应用时可能导致癌症。我们将通过以各种方式化学修饰Cas9结合CRISPR RNA分子来解决这些问题。RNA分子文库将通过将短可变基因靶向RNA部分与与Cas9蛋白缔合所需的较大不变RNA序列混合和匹配（化学连接）来创建。通过向这些RNA分子中添加荧光染料，并开发智能激活和编程输出颜色的系统，活细胞成像能力将得到显着提高。为了控制细胞的行为，我们将在CRISPR RNA分子上添加短片段DNA，这将在细胞内招募关键基因调节蛋白，并使我们能够微调基因表达。最后，将研究使用光激活的化学修饰的Cas9结合RNA分子，其只能短暂和微妙地损伤DNA，以减少不期望的脱靶效应。这些核酸化学驱动的目标旨在促进研究，否则使用当前的Cas9技术是不切实际或不可能的，并且从长远来看可以实现令人兴奋的新应用。

A transformative new technology for gene editing, known as CRISPR-Cas, was recently discovered. It allows a protein (Cas9) to be programmed by a CRISPR RNA molecule to modify specific sequences in genomic DNA. This can be used to alter the levels of specific proteins in cells, and can even be used to eliminate them or change their function (via site-specific mutation). This technology has profound implications in many areas of biology and medicine and, in the future, could be used to successfully treat human genetic diseases and cancer. By creating artificial (modified) Cas9 proteins, it is also possible to edit the cell's RNA molecules and to create detailed images of the genome in live cells in real-time. However, there are drawbacks to CRISPR-Cas technology: the number of CRISPR RNA molecules needed to perform some editing tasks is cost-prohibitive, imaging multiple DNA loci in the genome is difficult, and to completely change a cell's characteristics (cell type) is not currently feasible. Moreover, unintended editing events in the genome (off-target effects) are common and these are potentially catastrophic, particularly in the therapeutic arena where they could cause cancer when used in therapeutic applications. We will address these problems by chemically modifying the Cas9-binding CRISPR RNA molecule in various ways. Libraries of RNA molecules will be created by mixing and matching (chemically ligating) the short variable gene-targeting RNA moiety with a larger invariable RNA sequence needed for association with Cas9 protein. By adding fluorescent dyes to these RNA molecules and developing systems that intelligently activate and programme the output colour, live-cell imaging capabilities will be significantly improved. In order to control a cell's behaviour, we will append short pieces of DNA to the CRISPR RNA molecule that will recruit key gene regulating proteins within the cell and enable us to fine-tune gene expression. Finally, the use of light-activated chemically modified Cas9-binding RNA molecules, which can only transiently and subtly damage DNA, will be investigated to reduce undesirable off-target effects. These nucleic acid chemistry-driven objectives are aimed at facilitating research that would otherwise be impractical or impossible using current Cas9 technology and in the long term could enable exciting new applications.