Genome Editing by CRISPR-Cas: A Game Change in the Genetic Manipulation of Chlamydomonas

Abstract

Microalgae are promising photosynthetic unicellular eukaryotes among the most abundant on the planet and are considered as alternative sustainable resources for various industrial applications. Chlamydomonas is an emerging model for microalgae to be manipulated by multiple biotechnological tools in order to produce high-value bioproducts such as biofuels, bioactive peptides, pigments, nutraceuticals, and medicines. Specifically, Chlamydomonas reinhardtii has become a subject of different genetic-editing techniques adapted to modulate the production of microalgal metabolites. The main nuclear genome-editing tools available today include zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and more recently discovered the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas) nuclease system. The latter, shown to have an interesting editing capacity, has become an essential tool for genome editing. In this review, we highlight the available literature on the methods and the applications of CRISPR-Cas for C. reinhardtii genetic engineering, including recent transformation methods, most used bioinformatic tools, best strategies for the expression of Cas protein and sgRNA, the CRISPR-Cas mediated gene knock-in/knock-out strategies, and finally the literature related to CRISPR expression and modification approaches.

Chlamydomonas POLQ is necessary for CRISPR/Cas9-mediated gene targeting

“…Chlamydomonas reinhardtii is a model algae used for study of photoreceptors, photomovement, photosynthesis, various aspects of metabolism and biosynthetic pathways, chloroplast gene expression, and others, as well as for many applications in biotechnology ( Salome and Merchant 2019 ). Improvements in Chlamydomonas gene editing have been achieved through the use of the CRISPR/Cas endonuclease system ( Ghribi et al 2020 ). Null mutants for genes with selectable phenotypes were obtained with DNA template-free gene targeting ( Jiang et al 2014 ; Baek et al 2016 ; Shin et al 2016 ; Jiang and Weeks 2017 ; Jeong et al 2018 ; Guzmán-Zapata et al 2019 ; Dhokane et al 2020 ).…”

Grand Challenges in Microalgae Domestication

“…Its life cycle relies mainly on haploid asexual divisions; sexual reproduction can be controlled in vitro (Wilson, 2008 ). Nuclear and chloroplastic transformations are possible and multiple methods have been developed for gene editing (Ghribi et al, 2020 ). It is thus considered as a model for synthetic biology (Scaife et al, 2015 ).…”

The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing

“…The green model microalga Chlamydomonas reinhardtii [ 1 ] has become a powerful biotechnological production host for a wide range of recombinant proteins [ 2 , 3 , 4 ] and metabolites [ 5 , 6 , 7 ]. Innovative tools for metabolic engineering, gene editing and synthetic biology are developing rapidly [ 8 , 9 , 10 , 11 , 12 , 13 , 14 ]. However, heterologous gene expression requires selection systems to establish sufficient expression levels and to prevent transgene silencing.…”