Cámara Elena, Lenitz Ibai, Nygård Yvonne
Division of Industrial Biotechnology, Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 412 96, Gothenburg, Sweden.
Sci Rep. 2020 Sep 3;10(1):14605. doi: 10.1038/s41598-020-71648-w.
Recent advances in CRISPR/Cas9 based genome editing have considerably advanced genetic engineering of industrial yeast strains. In this study, we report the construction and characterization of a toolkit for CRISPR activation and interference (CRISPRa/i) for a polyploid industrial yeast strain. In the CRISPRa/i plasmids that are available in high and low copy variants, dCas9 is expressed alone, or as a fusion with an activation or repression domain; VP64, VPR or Mxi1. The sgRNA is introduced to the CRISPRa/i plasmids from a double stranded oligonucleotide by in vivo homology-directed repair, allowing rapid transcriptional modulation of new target genes without cloning. The CRISPRa/i toolkit was characterized by alteration of expression of fluorescent protein-encoding genes under two different promoters allowing expression alterations up to ~ 2.5-fold. Furthermore, we demonstrated the usability of the CRISPRa/i toolkit by improving the tolerance towards wheat straw hydrolysate of our industrial production strain. We anticipate that our CRISPRa/i toolkit can be widely used to assess novel targets for strain improvement and thus accelerate the design-build-test cycle for developing various industrial production strains.
基于CRISPR/Cas9的基因组编辑技术的最新进展极大地推动了工业酵母菌株的基因工程发展。在本研究中，我们报告了一种用于多倍体工业酵母菌株的CRISPR激活和干扰（CRISPRa/i）工具包的构建及特性。在高拷贝和低拷贝变体的CRISPRa/i质粒中，dCas9单独表达，或与激活或抑制结构域（VP64、VPR或Mxi1）融合表达。通过体内同源定向修复将sgRNA从双链寡核苷酸引入到CRISPRa/i质粒中，无需克隆即可快速对新的靶基因进行转录调控。通过改变两种不同启动子下荧光蛋白编码基因的表达对CRISPRa/i工具包进行了特性分析，表达变化可达约2.5倍。此外，我们通过提高工业生产菌株对小麦秸秆水解产物的耐受性证明了CRISPRa/i工具包的可用性。我们预计，我们的CRISPRa/i工具包可广泛用于评估菌株改良的新靶点，从而加速开发各种工业生产菌株的设计-构建-测试循环。
