Schematic overview of TIM-based N-terminal tagging strategy using a single RNP.

The 5’ end of the gene is shown as a grey box. The start codon is shown as a green star. The RNP target cut site is located downstream of the start codon. The PAM is shown as a blue bar on the gene. The donor DNA begins with a drug-resistance gene (green oval), then sequence homologous to the 5’ end of the target gene and continuing through the start codon, then sequence encoding the desired tag (yellow oval), and finally sequence homologous to that of the target gene beginning immediately after the start codon, including the RNP PAM modified with a silent mutation (white box), and continuing downstream for at least 20 bp (the “right” homology arm). The RNP can cut the wild-type gene, creating a DSB, but not the donor DNA or tagged gene if the donor DNA end is used as template to repair the DSB (upper diagram of “successfully targeted gene”). The new 5’ end created by the DSB also can be used as a template for repair, resulting in a tagged allele with a wild-type PAM sequence (lower diagram of “successfully targeted gene”). In these tagged alleles, the drug-resistance cassette is upstream of the gene. A portion (indicated by a red line) of the original gene remains immediately upstream of the drug-resistance cassette. To screen for positive clones, a 5’-end primer (P1) is designed to be complementary to the tag sequence (as illustrated here) or to sequence upstream of the tag; the 3’-end primer (P2) is designed to be complementary to sequence downstream of the homology arm.