Rodríguez-López María, Cotobal Cristina, Fernández-Sánchez Oscar, Borbarán Bravo Natalia, Oktriani Risky, Abendroth Heike, Uka Dardan, Hoti Mimoza, Wang Jin, Zaratiegui Mikel, Bähler Jürg
Research Department of Genetics, Evolution and Environment, University College London, London, UK.
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, USA.
Wellcome Open Res. 2017 May 5;1:19. doi: 10.12688/wellcomeopenres.10038.3. eCollection 2016.
In the fission yeast the prevailing approach for gene manipulations is based on homologous recombination of a PCR product that contains genomic target sequences and a selectable marker. The CRISPR/Cas9 system has recently been implemented in fission yeast, which allows for seamless genome editing without integration of a selection marker or leaving any other genomic 'scars'. The published method involves manual design of the single guide RNA (sgRNA), and digestion of a large plasmid with a problematic restriction enzyme to clone the sgRNA. To increase the efficiency of this approach, we have established and optimized a PCR-based system to clone the sgRNA without restriction enzymes into a plasmid with a dominant (nourseothricin) selection marker. We also provide a web-tool, CRISPR4P, to support the design of the sgRNAs and the primers required for the entire process of seamless DNA deletion. Moreover, we report the preparation of G1-synchronized and cryopreserved cells, which greatly increases the efficiency and speed for transformations, and may also facilitate standard gene manipulations. Applying this optimized CRISPR/Cas9-based approach, we have successfully deleted over 80 different non-coding RNA genes, which are generally lowly expressed, and have inserted 7 point mutations in 4 different genomic regions.
在裂殖酵母中，基因操作的主要方法基于含有基因组靶序列和选择标记的PCR产物的同源重组。CRISPR/Cas9系统最近已在裂殖酵母中得以应用，它允许进行无缝基因组编辑，无需整合选择标记或留下任何其他基因组“疤痕”。已发表的方法涉及单导向RNA（sgRNA）的人工设计，以及用一种有问题的限制性内切酶消化一个大质粒来克隆sgRNA。为提高这种方法的效率，我们建立并优化了一种基于PCR的系统，无需限制性内切酶即可将sgRNA克隆到带有显性（诺尔丝菌素）选择标记的质粒中。我们还提供了一个网络工具CRISPR4P，以支持sgRNA的设计以及无缝DNA缺失整个过程所需的引物设计。此外，我们报告了G1期同步化和冷冻保存细胞的制备方法，这大大提高了转化的效率和速度，也可能有助于标准的基因操作。应用这种优化的基于CRISPR/Cas9的方法，我们已成功删除了80多个通常低表达的不同非编码RNA基因，并在4个不同基因组区域插入了7个点突变。
