DNA lesions and repair response caused by genome editing in Saccharomyces cerevisiae

Abstract:

[Objective] To investigate DNA lesions and repair response caused by the DNA double strand break (DSB) generated by the genome editing tools including CRISPR/Cas9 and CRISPR/Cpf1 in Saccharomyces cerevisiae, we used the damage and repair of S. cerevisiae genomic DNA caused by a chemical substance methyl methane sulfonate (MMS) as a comparison and elucidated the changes of edited cells at the cellular and transcriptional levels. [Methods] Initial cells were divided into two situations, including unsynchronized cell cycle and synchronized cell cycle to G0/G1 phase by α-factor. We measured the growth profiles of CRISPR/Cas9-and CRISPR/Cpf1-mediated edited cells. We employed flow cytometry to detect the arrested cell cycle of edited cells. We used Real-time PCR to quantify the transcriptional expression changes of key genes involved in DNA damage response in edited cells and MMS-treated cells. [Results] Growth of initial cells, which were either unsynchronized or synchronized cell cycle by α-factor, were inhibited by genome editing. Cell viabilities of edited cells decreased, and the cell cycles were arrested at the G2/M phase. Furthermore, along with the prolongation of editing time, mutation efficiency of edited cells increased while cell viabilities decreased. The mutation efficiency and viabilities of CRISPR/Cpf1 edited cells were lower than those of CRISPR/Cas9, and thus the damage induced by CRISPR/Cpf1 was stronger than that of CRISPR/Cas9. Both these two editing tools induced significantly up-regulated transcriptional expressions of RNR3 and HUG1, which are key genes involved in DNA damage response in yeast. Additionally, the extent of CRISPR/Cpf1-mediated up-regulation was higher than that of CRISPR/Cas9, but both were lower than MMS treatment. [Conclusion] This study analyzed DNA lesions and repair response caused by CRISPR/Cas9-and CRISPR/Cpf1-mediated genome editing at the cellular and transcriptional levels, and preliminarily revealed the divergent extents of S. cerevisiae in response to different DSBs, thus providing an important guidance for improving the editing capacity and estimating the safety of genome editing.

Key words:Saccharomyces cerevisiae;CRISPR/Cas9;CRISPR/Cpf1;methyl methane sulfonate;cell cycle;DNA damage response