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

细胞内的基因组信息（基因组）绘制了细胞的结构和功能能力。随着更复杂的基因编辑工具的出现（例如，CRISPR），有可能通过在特定位置添加、去除或修饰DNA来改变基因组，希望获得具有所需功能的细胞。通过多轮筛选选择具有独特基因组的单细胞，可以选择能够产生有价值产物的菌株（例如，大麻素或生物燃料）。 对于更先进的细胞表征，有必要分离和单独生长细胞，如单克隆。 目前，很少有技术可用于执行选择和隔离。目前用于产生和分离克隆的方法的主要问题是（i）高成本，（ii）冗长的步骤，（iii）缺乏对细胞分选参数的控制，以及（iv）缺乏用于将克隆转移到孔板的整合方法，从而存在丢失所选克隆的风险。 在Shih实验室，我们创造了一种名为“D2-isolate”的发明，这是一种集成了两种微流体范例的手持设备：通道中的液滴和数字微流体（DMF）。 通道内液滴微流体可以在高通量范围内操作以产生含有0或1个细胞的液滴，但仅导致待分析的液滴的大混合物，而其中仅约10%含有细胞。 DMF系统可以单独处理每个液滴。两者的整合使得能够以高效率产生包含1个细胞的液滴（没有空液滴），同时独立地控制/移动包含单个细胞的每个液滴。基于预编程的标准，细胞可以自动丢弃或转移到96孔板中进行进一步表征。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，在本提案中，我们的目标是评估我们技术的市场，以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好准备。

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.