L2M NSERC - Integrated technology for automated cell-by-cell genetic screening

细胞内的基因组信息(基因组)映射了细胞的结构和功能。随着更复杂的基因编辑工具(如CRISPR)的出现，有可能通过在特定位置添加、移除或修改DNA来改变基因组，希望获得具有所需功能的细胞。通过几轮筛选选择具有独特基因组的单个细胞，就有可能选择能够产生有价值产品(例如大麻素或生物燃料)的菌株。为了更先进的细胞特性，有必要分离和培养单独的细胞，作为单克隆。目前，可用于执行选择和隔离的技术很少。目前用于产生和分离克隆的方法的主要问题是(I)成本高，(Ii)步骤长，(Iii)缺乏对细胞分选参数的控制，以及(Iv)缺乏将克隆转移到孔板上的综合方法，从而有可能丢失所选择的克隆。在施正荣的实验室里，我们创造了一项名为D2-Isolate的发明，这是一种手持设备，集成了两种微流控模式：通道内液滴和数字微流控(DMF)。液滴通道中的微流体可以在高通量范围内工作，产生含有0或1个细胞的液滴，但只产生大量需要分析的液滴混合物，而其中只有~10%的液滴包含一个细胞。DMF系统可以单独处理每个液滴。两者的结合使含有一个电池的液滴的产生效率很高(没有空液滴)，同时独立地控制/移动包含单个电池的每个液滴。根据预先编程的标准，电池可以被自动丢弃，或被转移到96孔板上进行进一步表征。作为一个完全集成和自动化的平台，D2-Isolate将加快与单细胞生成和分析相关的应用程序的工作流程。因此，在这项提案中，我们的目标是评估我们的技术的市场，目标是扩大与单细胞技术相关的应用空间，并帮助我们为商业化做准备。

The genomic information inside a cell (the genome) maps the structural and functional capabilities of a cell. With the advent of more sophisticated gene editing tools (e.g., CRISPR), it is possible to alter the genome by adding, removing or modifying DNA at a particular location with the hopes of obtaining cells with a desired function. By selecting a single cell with a unique genome through rounds of screening, it is possible to select for strains that are capable of producing valuable products (e.g., cannabinoids or biofuels). For more advanced cell characterization, it is necessary to isolate and to grow cells individually, as monoclones. Currently there are very few technologies available to perform the selection and the isolation. The main problems with current methods for generating and isolating the clones are (i) the high costs, (ii) lengthy steps, (iii) lack of control over cell sorting parameters and (iv) the absence of integrated methods for transferring the clones to well-plates, risking the loss of selected clones. In the Shih lab, we have created an invention called "D2-isolate", which is a hand-held device that integrates two microfluidic paradigms: droplet-in-channel and digital microfluidics (DMF). Droplet-in-channel microfluidics can operate in high-throughput ranges to generate droplets containing 0 or 1 cell, but only results in a large mixture of droplets to be analyzed while only ~10% of them contain a cell. DMF systems can address each droplet individually. The integration of the two enables the generation of droplets containing 1 cell with high efficiency (no empty droplet) while controlling/moving each droplet containing a single cell independently. Based on pre-programmed criteria, a cell can automatically be either discarded, or transferred to a 96-well plate for further characterization. As a fully integrated and automated platform, D2-isolate will expedite the workflow for applications related to single-cell generation and analysis. Hence, in this proposal, we aim to evaluate the market for our technology with the goal of expanding the application space related to single-cell technologies and help prepare our efforts towards commercialization.