Graphical abstract
Einhaus, A., Krieger, A., Köhne, L., Rautengarten, B., Jacobebbinghaus, N., Saudhof, M., Baier, T., & Kruse, O. (2025). Genome editing of epigenetic transgene silencing in Chlamydomonas reinhardtii. Trends in Biotechnology.
Created in BioRender. Kruse, O. (2025) https://BioRender.com/ucys7zl
Green microalgal biotechnology is limited by strong transgene silencing.
CRISPR-mediated combinatorial disruption of epigenetic factors in Chlamydomonas reinhardtii identified transgene silencing factors and improved transgene expression strength and stability.
In addition, an intein-based split selectable marker system was established for dual-targeted CRISPR, reducing the need for selectable markers.
Figure 4. Engineered terpenoid production from improved mutant strains.
(A) Top expressing transformants from each mutant strain were used for the production of patchoulol. The top-ten expressing transformants of 288 isolated transformants per biological triplicate for each knockout (KO) mutant strain (based on the initial evaluation, see Figure 2A in the main text) were combined into the respective boxplots. Volumetric production in mg l–1 is depicted in box and whiskers plots and was compared with the CC4350 wild-type (WT) strain by two-sided Student’s t-test for mean production assuming non-homogenous variances; ***P <0.001, *P <0.05, n.s. P >0.05.
(B) Initial transgene expression capacity of the ΔA51 and control strains depicted as fluorescence/OD750 normalized to the mean expression of the WT (left). The top-20 expressing transformants of 288 isolated transformants per biological triplicate of ΔA51 were combined into the respective boxplots. UVM4 and ΔA51 were compared with the WT by two-sided Student’s t-test for mean expression assuming non-homogenous variances; ***P <0.001, *P <0.05, n.s. P >0.05. Long-term stability of transgene expression (right) was assessed by repeated quantification of mVenus fluorescence after 3 months and compared with the initial quantification by two-sided Student’s t-test for mean expression assuming non-6homogenous variances; ***P <0.001, *P <0.05, n.s. P >0.05. The percentage change in mean expression of respective strains is detailed.
(C) Engineered terpenoid production from high cell-density cultivation of the nitrate-complemented UVM4 and ΔA51 strains. The top-ten expressing transformants of 288 isolated transformants each strain (initial evaluation) were analyzed. Volumetric terpenoid production from the ΔA51 strain was compared to UVM4 by two-sided Student’s t-test for mean production assuming non-homogenous variances; ***P <0.001, *P <0.05, n.s. P >0.05.
Abbreviations: DMC5= 5 HLM4= 4 MUT9= 9 SRTA= A VIG1= 1
Interesting work by Einhaus et al. (2025) on how CRISPR/Cas9-mediated combinatorial disruption of #epigenetic factors in the green alga #Chlamydomonas (e.g. ΔA51 strain = ΔSRTA, ΔDMC5 + ΔVIG1) improved transgene expression strength and stability after 3 months.
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