Laboratory of Cytoskeletal Logistics, Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsucho, Shinjuku-ku, Tokyo 162-8480, Japan.
Laboratory of Cytoskeletal Logistics, Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsucho, Shinjuku-ku, Tokyo 162-8480, Japan; Faculty of Science and Engineering, Global Center for Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan; Institute for Advanced Research of Biosystem Dynamics, Waseda Research Institute for Science and Engineering, Graduate School of Advanced Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan.
Gene. 2021 Jun 5;784:145595. doi: 10.1016/j.gene.2021.145595. Epub 2021 Mar 26.
CRISPR/Cas9 is a powerful tool for genome editing. Several studies have been conducted to take the benefit of the versatile tool in the fission yeast Schizosaccharomyces pombe. However, the protocols for the CRISPR/Cas9 system proposed in previous studies are complicated in culture conditions compared to traditional genome editing methods. In this study, we introduced vectors for expression of sgRNA as well as Cas9, which employ natMX6 and bsdMX6 dominant selection markers. Using these materials, we examined nutritional conditions of cell cultures and found that nitrogen depletion introduced in previous methods does not affect the efficiency of genome editing. We found that bsdMX6-based plasmids enable us to skip any recovery steps before plating onto medium containing blasticidin S, unlike other antibiotic resistance selection markers. We thus propose easier transformation procedures with natMX6 and particularly bsdMX6 markers. We also simulate prescreening of mutants by genotyping with DNA endonucleases or proofreading PCR instead of relying on existing knowledge of mutant phenotypes. These materials and methods assist easy construction of S. pombe strains using CRISPR/Cas9, thereby accelerating seamless introduction of CRISPR/Cas9 to S. pombe researchers.
CRISPR/Cas9 是一种强大的基因组编辑工具。已有多项研究利用这一多功能工具在裂殖酵母 Schizosaccharomyces pombe 中开展研究。然而，与传统的基因组编辑方法相比，之前研究中提出的 CRISPR/Cas9 系统方案在培养条件方面较为复杂。在本研究中，我们引入了表达 sgRNA 和 Cas9 的载体，它们采用 natMX6 和 bsdMX6 显性选择标记。使用这些材料，我们考察了细胞培养的营养条件，发现之前方法中引入的氮饥饿对基因组编辑效率没有影响。我们发现，基于 bsdMX6 的质粒使我们能够跳过在含有博来霉素 S 的培养基上接种前的任何恢复步骤，这与其他抗生素抗性选择标记不同。因此，我们提出了使用 natMX6 和特别是 bsdMX6 标记的更简单的转化程序。我们还通过 DNA 内切酶或校对 PCR 进行基因型分析来模拟突变体的预筛选，而不是依赖于现有突变体表型的知识。这些材料和方法有助于使用 CRISPR/Cas9 轻松构建 S. pombe 菌株，从而加速将 CRISPR/Cas9 无缝引入 S. pombe 研究人员手中。
