DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins

TLDR

Microalgae can convert CO₂, H₂O, and sunlight into fuels and chemicals, and genetic modifications are essential to boost photosynthetic productivity and biomass production, yet targeted CRISPR‑Cas9 mutagenesis in these organisms remains limited. The study reports a one‑step, DNA‑free CRISPR‑Cas9 transformation of *Chlamydomonas reinhardtii* that bypasses plasmid‑encoded Cas9 and guide RNAs. CRISPR‑Cas9 ribonucleoproteins were employed to sequentially knock out the CpFTSY and ZEP genes in the algae. The resulting strain constitutively produces zeaxanthin and exhibits enhanced photosynthetic productivity.

Abstract

Abstract Microalgae are versatile organisms capable of converting CO 2 , H 2 O and sunlight into fuel and chemicals for domestic and industrial consumption. Thus, genetic modifications of microalgae for enhancing photosynthetic productivity and biomass and bio-products generation are crucial for both academic and industrial applications. However, targeted mutagenesis in microalgae with CRISPR-Cas9 is limited. Here we report, a one-step transformation of Chlamydomonas reinhardtii by the DNA-free CRISPR-Cas9 method rather than plasmids that encode Cas9 and guide RNAs. Outcome was the sequential CpFTSY and ZEP two-gene knockout and the generation of a strain constitutively producing zeaxanthin and showing improved photosynthetic productivity.