Improved Gene Targeting through Cell Cycle Synchronization

Abstract

Gene targeting is a challenge in organisms where non-homologous end-joining is the predominant form of recombination. We show that cell division cycle synchronization can be applied to significantly increase the rate of homologous recombination during transformation. Using hydroxyurea-mediated cell cycle arrest, we obtained improved gene targeting rates in Yarrowia lipolytica, Arxula adeninivorans, Saccharomyces cerevisiae, Kluyveromyces lactis and Pichia pastoris demonstrating the broad applicability of the method. Hydroxyurea treatment enriches for S-phase cells that are active in homologous recombination and enables previously unattainable genomic modifications.

CRISPRi repression of nonhomologous end‐joining for enhanced genome engineering via homologous recombination in Yarrowia lipolytica

“…Transient strategies have also been demonstrated, notably the chemical inhibition of DNA ligase IV and shRNA‐mediated suppression of KU70 and KU80, critical proteins in NHEJ, have been showed to increase HR (Chu et al, ; Maruyama et al, ). HR is also known to be more active during DNA replication than other DNA repair pathways (Heyer, Ehmsen, & Liu, ; Mathiasen & Lisby, ), evidence of which has led to chemically‐induced synchronization of cells in G2 or S‐phase to enhance HR (Lin, Staahl, Alla, & Doudna, ; Tsakraklides, Brevnova, Stephanopoulos, & Shaw, ). The mutational and transient strategies outlined above have been primarily demonstrated with model yeasts, mammalian cells, and in model plants, and have not yet been fully explored in many industrial biotechnology and non‐traditional yeasts (Chu et al, ; Endo, Mikami, & Toki, ; Maruyama et al, ; Qi, Zhang, et al, ).…”

Pichia pastoris protease‐deficient and auxotrophic strains generated by a novel, user‐friendly vector toolbox for gene deletion

“…Integration events usually occur with >70% efficiency at the correct locus (Gueldener, Heinisch, Koehler, Voss, & Hegemann, ). Nonconventional yeasts such as P. pastoris often display a high ratio of NHEJ‐to‐HR activity, which is the main reason why targeted gene insertions are difficult to achieve (Näätsaari et al, ; Schwarzhans et al, ; Tsakraklides, Brevnova, Stephanopoulos, & Shaw, ). Several strategies were shown to improve HR activity in P. pastoris such as hydroxyurea‐mediated cell cycle arrest (Tsakraklides et al, ), increasing the genetic redundancy of host cells by providing extra copies of the gene to be deleted on a helper plasmid (Chen et al, ) or deletion of KU70 , a key player in NHEJ improved targeting (Näätsaari et al, ).…”

“…Integration events usually occur with >70% efficiency at the correct locus (Gueldener, Heinisch, Koehler, Voss, & Hegemann, 2002). Nonconventional yeasts such as P. pastoris often display a high ratio of NHEJ-to-HR activity, which is the main reason why targeted gene insertions are difficult to achieve (Näätsaari et al, 2012;Schwarzhans et al, 2016;Tsakraklides, Brevnova, Stephanopoulos, & Shaw, 2015).…”

“…Several strategies were shown to improve HR activity in P. pastoris such as hydroxyurea-mediated cell cycle arrest (Tsakraklides et al, 2015), increasing the genetic redundancy of host cells by providing extra copies of the gene to be deleted on a helper plasmid or deletion of KU70, a key player in NHEJ improved targeting (Näätsaari et al, 2012). Deletion of KU70 was also shown to drastically improved gene modifications using CRISPR/Cas9 tools (Weninger et al, 2018).…”

Finite time synchronization of stochastic Markovian jumping genetic oscillator networks with time-varying delay and Lévy noise

“…For instance, in Ref. [8] it has been found that synchronization can significantly increase the rate of homologous recombination during transformation in gene targeting, and in Ref. [9] it is showed that synchronization can enhance the anticancer activity of 2-deoxyglucose in breast cancer cells due to an increase in cellular glucose metabolism.…”