Shaw Sebastian, Knüsel Sebastian, Hoenner Sarah, Roditi Isabel
Institute of Cell Biology, University of Bern, Bern, Switzerland.
Graduate School of Cellular and Biomedical Science, University of Bern, Bern, Switzerland.
BMC Res Notes. 2020 Jun 3;13(1):268. doi: 10.1186/s13104-020-05089-z.
Generation of knockouts and in situ tagging of genes in Trypanosoma brucei has been greatly facilitated by using CRISPR/Cas9 as a genome editing tool. To date, this has entailed using a limited number of cell lines that are stably transformed to express Cas9 and T7 RNA polymerase (T7RNAP). It would be desirable, however, to be able to use CRISPR/Cas9 for any trypanosome cell line.
We describe a sequential transfection expression system that enables transient expression of the two proteins, followed by delivery of PCR products for gRNAs and repair templates. This procedure can be used for genome editing without the need for stable integration of the Cas9 and T7RNAP genes.
通过使用CRISPR/Cas9作为基因组编辑工具，极大地促进了布氏锥虫基因敲除和原位标记的产生。迄今为止，这需要使用有限数量的稳定转化以表达Cas9和T7 RNA聚合酶（T7RNAP）的细胞系。然而，能够将CRISPR/Cas9用于任何锥虫细胞系将是很理想的。
我们描述了一种顺序转染表达系统，该系统能够使这两种蛋白质瞬时表达，随后递送用于gRNA和修复模板的PCR产物。此程序可用于基因组编辑，而无需Cas9和T7RNAP基因的稳定整合。
