Figure 1
CRISPR-generated knockouts in the predicted P. tricornutum uracil biosynthesis pathway. (A) A portion of the predicted P. tricornutum biosynthesis pathway for conversion of carbonic acid to uracil and uridine triphosphate, with the PtUMPS enzyme highlighted in blue. The competitive inhibitor, 5-fluoroorotic acid (5-FOA), is shown in a dashed box at the position where it enters the pathway. Abbreviated names for molecules and enzymes are indicated in parentheses, and the predicted corresponding P. tricornutum gene names are indicated in square brackets. (B) Example image of T7EI editing assay to screen exconjugants for potential editing events in the PtUMPS gene. Substrate indicates PtUMPS gene fragments amplified by the PCR, while T7 product indicates exconjugants with evidence of Cas9 editing. WT, wild-type P. tricornutum genomic DNA used in the T7EI editing assay. M, 100 bp ladder with sizes indicated in basepairs (bp). This image was cropped from a larger image (Supplementary Fig. S10). (C) Example of phenotypic screening of one PtUMPS knockout strain (\(\Delta\)UMPS2) plated on L1 alone or L1 supplemented with uracil at the indicated dilution of initial concentration. (D) Example of screening for loss of the zeocin-resistant Cas9 editing plasmid in a \(\Delta\)UMPS2 knockout strain by plating on L1 supplemented with uracil or L1 supplemented with uracil and zeocin. (E) Sanger sequencing traces of characterized PtUMPS knockouts with the position (below trace) and type of insertion or deletion (above trace) indicated for each allele of the three strains. (F) Graphical map of the position and extent of indels for each of the three PtUMPS knockouts relative to the wild-type UMPS gene (shown at top). Red rectangles indicate nucleotide deletions, green triangles indicate nucleotide insertions, the yellow and blue rectangles on the WT gene indicate the position of the PtUMPS active sites (orotate phosphoribosyl transferase and orotidine-5’-phosphate decarboxylase), and the white rectangles with dashed lines represent introns.
