Fig. 3.

Multiple gene knock-outs using a DNA-free RNP genome editing approach. a Outline of the genome-editing workflow used here. The first step consists of generating four independent RNP complexes, two of them targeting PtUMPS and the two others targeting PtAureo1a or any other gene of interest. Each RNP complex is prepared in a separate tube by incubating the Cas9 protein with pre-annealed crRNA::tracrRNA complexes (crRNA: base pairing site, tracrRNA: trans-activating crRNA). Next, the four RNP complexes are combined and loaded onto gold particles. The third step consists of the delivery of these coated gold beads into Phaeodactylum cells using a biolistic apparatus. Finally, after two to four days, the transformed cells are re-plated onto selective medium, here F/2 medium supplemented with uracil and 5-FOA. After three to four weeks, 5-FOA-resistants colonies appear and are analyzed for the presence of mutagenic events. b Each allele of the PtUMPS gene was amplified by allele-specific PCRs from the obtained 5-FOA resistant colonies and sequenced. c Frequency of colonies harboring no mutagenic event at the PtUMPS loci (black sector on the pie chart) or a mutagenic event on one allele (light gray sector) or both alleles (dark gray sector). d Additional information on the nature of the mutagenic events is reported, with four classes of mutagenic events being illustrated: small INDELs in one allele, the second being WT (white bar); large deletion corresponding to the DNA sequence between the two targeted sites in one allele, the second allele being WT (light gray bar); small INDELs in both alleles (dark gray bar); deletion of the DNA fragment between the two targeted sites in both alleles (medium gray bar). e Frequency of targeted mutagenesis of the PtAureo1a target sites (black sector: no TM detected, light gray sector: monoallelic mutants, dark gray sector: biallelic mutants). All clones mutated in PtUMPS also carried a mutation in at least one of the PtAureo1a alleles. f Distribution for the various classes of mutagenic events at the PtAureo1a loci