Transcriptional gene fusions via targeted integration at safe harbors for high transgene expression in Chlamydomonas reinhardtii
Jacobebbinghaus N, Bigge F, Saudhof M, Hübner W, Kruse O, Baier T (2025)
New Phytologist.
Conventional genetic engineering in green microalgae employs error‐prone nonhomologous end joining to integrate recombinant DNA at double‐strand breaks generated at random positions across the nuclear genome. This typically results in variable transcription strength and requires a labor‐intensive screening procedure to identify transformants with sufficient expression. Current advances in genome editing enable scar‐less integration of DNA at any desired locus for engineered bioproduction.
We optimized construct design for predictable transgene expression at a high level, significantly improved scar‐less integration rates into the nuclear genome via homology arm length optimization and quantified endogenous gene expression
in vivo
. Subsequently, endogenous genes were successfully targeted via Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR‐associated protein 9 (CRISPR/Cas9) to evaluate their capacity for high transgene expression and terpenoid production.
Highest scar‐less homology‐directed repair efficiency was achieved with 50 bp homology arms. The Light harvesting Chl a/b binding protein of LHCII (LHCBM1) locus was found to be differentially expressed under several light intensities and allows an 8.6‐fold increase in transgenic protein accumulation compared with random insertion approaches. Co‐expression of a functional sesquiterpene synthase achieved a 60‐fold increase in valencene production compared with previous attempts.
We showed LHCBM1 locus constitutes a genetic safe harbor for transgene expression and demonstrates the potential of
C. reinhardtii
as a green cell factory.
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