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.

Photosystem II antenna complexes CP26 and CP29 are essential for nonphotochemical quenching in Chlamydomonas reinhardtii

“…3 | RESULTS 3.1 | Generation of C. reinhardtii mutants on monomeric CP26 and CP29 subunits Monomeric PSII antenna mutants in C. reinhardtii were generated using CRISPR-Cas9 technology (Baek et al, 2016). First, the cp29 gene (Cre17.g720250) was mutated by non-homologous end joining: Two different sgRNA targets gave successful results, leading to introduction of insertions or deletions (Figures 1 and S1).…”

Cas-analyzer: an online tool for assessing genome editing results using NGS data

“…However, these methods do not allow analysis of mutant sequences and are limited by relatively poor sensitivity. Recently we and other groups have used targeted deep sequencing to detect programmable nuclease-induced mutations with high sensitivity and precision and to analyze mutation patterns (Baek et al , 2016). …”

Using YFP as a Reporter of Gene Expression in the Green Alga Chlamydomonas reinhardtii

“…Recently, the use of CRISPR/Cas9 to engineer the C. reinhardtii nuclear genome has been met with some success (41,42)( 1, 2 ), however, this technique is still in its infancy and has not be applied to the creation of a genomic “landing site” for specific integration of the promoter fusion construct, which may aid in reducing the observed transformant-to-transformant expression variability.…”