Initial PCR screening of transformants from the LF5-HA C-terminal tagging experiment.

DNA from the wild-type strain (W), 94 transformants, and the plasmid pLF5HA, which contains the LF5-HA gene, were used as templates for PCR screening with primer pairs P1 and P2 as illustrated in Fig 2. The expected size for the wild-type product was 455 bp (marked with red * on the right side of the gels and below the bands). In theory, one small fragment (563 bp) and one large fragment (3576 bp) would be predicted as products resulting from PCR amplification of the tagged gene in this experiment, yet only the small fragment was observed (marked with green arrows on the right side of the gels and below the bands). There are several likely reasons for this. First, we used a PCR elongation time optimal for 563-bp products, so there would not have been enough time for the polymerase to amplify products as large as 3576 bp. Second, to facilitate screening of a large number of clones, we used crude DNA as template, and in our experience it is more difficult to amplify longer PCR products from crude DNA. Third, due to the high GC content of C. reinhardtii DNA, it is harder to amplify long PCR products than short ones. The presence of the wild-type product indicates that the cell line was not edited at the target site, and the antibiotic-resistance gene has inserted into the genome somewhere else. The absence of the wild-type product indicates that the cell line was edited at the RNP-cut site. Among these 94 cell lines, 11 yielded products of the size expected from the tagged gene, suggesting that the donor DNA was inserted as desired. The PCR products from these 11 strains were sequenced for further confirmation: 10 had the sequence expected for HDR; one (here marked with a red S underneath the band) did not (S1 Appendix). Among the 10 clones with the expected sequence, eight expressed LF5-HA (see Fig 6); only two (here marked with a red P) did not. Among the eight clones that expressed LF5-HA, all but one (here marked with a red U) had a complete 3’ UTR (see Fig 7). The complete absence of PCR product or product of a size other than that expected from the wild-type or tagged gene indicates that a more complicated editing event occurred. These are likely to be lf5 mutants. Six products (L1-L6), the sizes of which range from 1235 bp to 1808 bp, are different from the large fragment (3576 bp) expected from an ideal integration of donor DNA. L1 to L6 were the products of alleles in which only a part of the donor DNA, consisting mainly of the drug-resistance cassette, had integrated into the gene (S1 Appendix). Nucleic acid markers (M) (NEB 100 bp DNA ladder, catalog number NO551), from top to bottom, are 1517, 1200, 1000, 900, 800, 700, 600, 500/517, 400, 300, 200, and 100 bp.