National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
Science Center for Future Foods, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
ACS Synth Biol. 2021 Oct 15;10(10):2607-2616. doi: 10.1021/acssynbio.1c00231. Epub 2021 Sep 24.
The resistance markers could ensure the entry of the CRISPR/Cas9 system into cells instead of gene editing. To increase the efficiency of positive colony screening on the primary transformation plates, we designed a visualized multigene editing system (VMS) via a unique tRNA-guide RNA (gRNA) array containing the gRNAs of a pigment gene and target genes. Disruption of produces white colonies, and the sequences of the endogenous tRNA, tRNA, tRNA, tRNA, and tRNA enhance gRNA release. The disruption efficiencies of multigene were analyzed in the strain AG11 using , , , , and as reporters. In white colonies on the primary transformation plates, the disruption rates of one-, two-, three-, four-, and five-target genes reached 89.2, 70.91, 50, 22.41, and 4.17%, respectively. The VMS developed here provides an effective method for screening homokaryotic multigene editing strains of .
抗性标记物可以确保 CRISPR/Cas9 系统进入细胞而不是进行基因编辑。为了提高初级转化平板上阳性菌落筛选的效率，我们设计了一个可视化的多基因编辑系统（VMS），通过一个独特的 tRNA-引导 RNA（gRNA）阵列，该阵列包含一个色素基因和目标基因的 gRNA。破坏 会产生白色菌落，而内源性 tRNA、tRNA、tRNA、tRNA 和 tRNA 的序列会增强 gRNA 的释放。使用 、 、 、 和 作为报告基因，在 AG11 菌株中分析了多基因的破坏效率。在初级转化平板上的白色菌落中，一个、两个、三个、四个和五个靶基因的破坏率分别达到 89.2%、70.91%、50%、22.41%和 4.17%。这里开发的 VMS 为筛选 的同源多基因编辑株系提供了一种有效的方法。
