Schematic overview of TIM-based N-terminal tagging strategy using two gRNAs.

The 5’ end of the gene is shown as a grey box, the start codon is indicated by the green star, and the PAMs are indicated by blue bars. RNP1 cuts upstream of, but as near as possible to, the start of the gene, while RNP2 cuts downstream of the start codon. The donor DNA is identical to that of Fig 3 except that it starts with a left arm (L) homologous to genomic sequence just upstream of the RNP1-cut site. In the ideal situation, the DSB created by RNP1 and RNP2 will be repaired by homologous integration of the donor DNA facilitated by the left and right homology arms, resulting in a wild-type gene tagged at its N-terminus, and a drug-resistance gene just upstream of the tagged target gene. During integration, the 3’ end of the donor DNA or the genomic DNA’s new 5’ end created by the DSB can be used as a template, creating either a tagged allele with a mutated RNP2 PAM sequence (top diagram of “successfully targeted gene”) or a tagged allele with a wild-type RNP2 PAM sequence (bottom diagram of “successfully targeted gene”), respectively. Following selection of colonies on antibiotic-containing medium, colonies are screened by PCR using primer pairs P1/P2 and P3/P4 to identify those in which the insertion has occurred as planned. The sizes of the expected PCR products for specific experiments of Figs 8 and 9 are shown between the primers.