The clustered regularly interspaced short palindromic repeats (CRISPR) - Cas associated protein 9 (Cas9) system is very precise, efficient and relatively simple in creating genetic modifications at a predetermined locus in the genome. Genome editing with Cas9 ribonucleoproteins (RNPs) has reduced cytotoxic effects, off-target cleavage and increased on-target activity and the editing efficiencies. The unicellular alga Chlamydomonas reinhardtii is an emerging model for studying the production of high-value products for industrial applications. Development of C. reinhardtii as an industrial biotechnology host can be achieved more efficiently through genetic modifications using genome editing tools. We made an attempt to target MAA7 gene that encodes the tryptophan synthase β-Subunit using CRISPR-Cas9 RNPs to demonstrate knock-out and knock-in through homology-dependent repair template at the target site. In this study, we have demonstrated targeted gene knock-out in C. reinhardtii using programmed RNPs. Targeted editing of MAA7 gene was confirmed by sequencing the clones that were resistant to 5-Fluoroindole (5-FI). Non-homologous end joining (NHEJ) repair mechanism led to insertion, deletion, and/or base substitution in the Cas9 cleavage vicinity, encoding non-functional MAA7 protein product (knock-out), conferring resistance to 5-FI. Here, we report an efficient protocol for developing knock-out mutants in Chlamydomonas using CRISPR-Cas9 RNPs. The high potential efficiency of editing may also eliminate the need to select mutants by phenotype. These research findings would be more likely applied to other green algae for developing green cell factories to produce high-value molecules.
Keywords: CRISPR/Cas9; Genome editing; Guide RNA (gRNA); Ribonucleoprotein (RNP); Transformation.