Genome editing using Cas9-RNA ribonucleoprotein complexes in the high-productivity marine alga Picochlorum celeri

Abstract Cas9-guide RNA ribonucleoprotein (RNP) complexes were used to efficiently generate insertions in both alleles of two targeted genes (nitrate reductase and carotenoid isomerase) in the biotechnologically relevant alga Picochlorum celeri. Diploid loss-of-function mutants were present in ~6% of transformants targeting nitrate reductase and in ~50% of transformants targeting carotenoid isomerase. The protocol described here successfully generates mutants within weeks and can be used to mechanistically probe the remarkably fast growth rates and extreme physiological tolerances of this alga.