SpEDIT: A fast and efficient CRISPR/Cas9 method for fission yeast
Torres-Garcia, S., Di Pompeo, L., Eivers, L., Gaborieau, B., White, S.A., Pidoux, A.L., Kanigowska, P., Yaseen, I., Cai, Y., and Allshire, R.C.
SpEDIT, an improved CRISPR/Cas9 method for fission yeast, markedly enhances the capabilities of genome editing in this popular model fungal system.
Summary of Paper by Lori Koch
The CRISPR/Cas9 method has revolutionized research biology. In 2020, the Nobel prize in chemistry was awarded to Drs. Doudna and Charpentier for their research which identified and developed it for widespread use. The method enables precise editing of a cell’s DNA by targeting a bacteria enzyme called Cas9 to the sequence of interest via a small guide RNA (sgRNA). Cas9 creates a double-strand break in the DNA which is then repaired either through non-homologous end-joining (NHEJ) or homology-directed repair (HDR). In their recent publication in Wellcome Open Research, scientists in the Allshire group led by Sito Torres-Garcia present an optimized strategy for its use to modify the genome of the model single-cell fungus Schizosaccharomyces pombe. Previous CRISPR strategies in S. pombe had several significant drawbacks. The first is that they required time-consuming cloning methods to produce the sgRNA vector. To address this, Torres-Garcia and colleagues created a plasmid vector where the sgRNA sequence can be inserted via a simple Golden Gate reaction. Insertion of the desired sequence is verified by loss of fluorescence in transformed colonies, which speeds up the process significantly. Another drawback of previous methods was that they introduced a relatively high amount of Cas9 enzyme in cells, which leads to excessive cutting and cell toxicity. The scientists therefore placed Cas9 under the control of a medium-strength inducible promoter, adh15, and they showed that this increases the size of Cas9-expressing yeast colonies compared to the previous method, indicating that toxicity is reduced. The new strategy was able to create mutations with 100% efficiency and outperformed the previous in terms of specificity, creating mutations only when appropriate DNA templates were introduced and not otherwise. The SpEDIT method will undoubtedly improve future fission yeast research.