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

Abstract

Microalgae are versatile organisms capable of converting CO2, H2O, 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.

Figure 1. RGEN RNP-mediated target gene disruption in C. reinhardtii.

(a) Experimental process schematic showing the steps applied in this work. (b) Single-cell colonies of wild type (WT) and RGEN-induced ΔCpFTSY mutant lines grown on minimal agar medium under low-light (50 μmol photons m⁻² s⁻¹) conditions. (c) Chlorophyll (Chl) a to Chl b ratios and total Chl content of wild type (WT) and the ΔCpFTSY mutant lines. Cells were grown photoautotrophically under low light (50 μmol photons m⁻² s⁻¹) conditions. Data are the average and SE from three biological replicates. (d) Alignment of wild type (WT) and the mutant line DNA sequences at the CpFTSY locus. The 20-bp target sequence is underlined and the PAM sequence is shown in red. The column on the right indicates the number of inserted (+) or deleted (−) bases.

Figure 2. ZEP gene knockout via DNA-free RGEN RNPs delivery.

(a) Schematic diagram of the reversible xanthophyll cycle in green algae. (b) Alignment of wild type (WT) and the mutant line DNA sequences at the ZEP locus. The 20-bp target sequence is underlined and the PAM sequence is shown in red. The column on the right indicates the number of inserted (+). (c) Quantification of pigment content and Chl a to Chl b ratios of wild type (WT) and the ΔZEP mutant lines. Cells were grown TAP media under low light (50 μmol photons m⁻² s⁻¹) conditions. Vio, violaxanthin; An, antheraxanthin; Zea, zeaxanthin. Data are the average and SE from three replicates.

Figure 3. The sequential CpFTSY and ZEP two-gene knockout mutants by transfecting RGEN-RNPs.

(a) Targeted indel mutations induced by RGEN RNPs at the ZEP and CpFTSY gene in the ΔZEP/ΔCpFTSY double mutant. (b) Phenotype of wild type, ΔZEP, ΔCpFTSY and ΔZEP/ΔCpFTSY mutants of C. reinhardtii. Cells were grown photoautotrophically under high-light (500 μmol photons m⁻² s⁻¹) conditions. Cell densities were 10 × 10⁶ cells/mL. Western-blot analysis of the ZEP and FTSY proteins in the wild type (WT) and the RGEN-induced transgenic lines. Immuno-detection of proteins with the β-subunit of ATP synthase (ATPβ) of Chlamydomonas was used as the loading control of the samples. (c) HPLC profiles of total pigments from acetone extracts of wild type(blue), ΔZEP(red), ΔCpFTSY(green) and ΔZEP/ΔCpFTSY double mutant(yellow). Lor, Loroxanthin; Neo, neoxanthin; Vio, violaxanthin; An, antheraxanthin; Lut, lutein; Zea, zeaxanthin; Chl b, Chlorophyll b; Chl a, Chlorophyll a; α-Car, α -carotene; β-Car, β-carotene. (d) Light-saturation curves of photosynthesis in wild type (blue) and ΔZEP (red), ΔCpFTSY (green) and ΔZEP/ΔCpFTSY (yellow). Rates of oxygen evolution on a per Chl basis were measured as a function of incident light intensity. (e) Growth curve of wild type and the mutants lines cultured in HS medium at 25 °C with air containing 5% CO₂ under High light (700 μmol photons m⁻² s⁻¹).