Department of Chemical and Environmental Engineering, University of California, Riverside, CA, 92521, USA.
iBio Inc., San Diego, CA, USA.
Commun Biol. 2023 Jun 8;6(1):617. doi: 10.1038/s42003-023-04996-8.
High throughput CRISPR screens are revolutionizing the way scientists unravel the genetic underpinnings of engineered and evolved phenotypes. One of the critical challenges in accurately assessing screening outcomes is accounting for the variability in sgRNA cutting efficiency. Poorly active guides targeting genes essential to screening conditions obscure the growth defects that are expected from disrupting them. Here, we develop acCRISPR, an end-to-end pipeline that identifies essential genes in pooled CRISPR screens using sgRNA read counts obtained from next-generation sequencing. acCRISPR uses experimentally determined cutting efficiencies for each guide in the library to provide an activity correction to the screening outcomes via calculation of an optimization metric, thus determining the fitness effect of disrupted genes. CRISPR-Cas9 and -Cas12a screens were carried out in the non-conventional oleaginous yeast Yarrowia lipolytica and acCRISPR was used to determine a high-confidence set of essential genes for growth under glucose, a common carbon source used for the industrial production of oleochemicals. acCRISPR was also used in screens quantifying relative cellular fitness under high salt conditions to identify genes that were related to salt tolerance. Collectively, this work presents an experimental-computational framework for CRISPR-based functional genomics studies that may be expanded to other non-conventional organisms of interest.
高通量 CRISPR 筛选正在彻底改变科学家揭示工程和进化表型遗传基础的方式。在准确评估筛选结果时，一个关键挑战是考虑 sgRNA 切割效率的可变性。针对筛选条件下必需基因的活性不佳的向导会掩盖预期从它们中断裂所产生的生长缺陷。在这里，我们开发了 acCRISPR，这是一个端到端的管道，它使用从下一代测序中获得的 sgRNA 读数来识别 pooled CRISPR 筛选中的必需基因。acCRISPR 使用库中每个向导的实验确定的切割效率，通过计算优化指标，为筛选结果提供活性校正，从而确定基因中断的适应性效应。在非常规产油酵母 Yarrowia lipolytica 中进行了 CRISPR-Cas9 和 -Cas12a 筛选，并使用 acCRISPR 确定了在葡萄糖（用于工业生产油脂化学品的常见碳源）下生长的高度可信的必需基因集。acCRISPR 还用于在高盐条件下量化相对细胞适应性的筛选，以鉴定与耐盐性相关的基因。总之，这项工作提出了一个基于 CRISPR 的功能基因组学研究的实验计算框架，可以扩展到其他感兴趣的非常规生物体。
