Generation of Endogenous Fusion Proteins using CRISPR-Cas9

CRISPR-Cas9 tagging technology was used for generation of all lines (Supplementary Data 1) that contain endogenous fusions at the C-terminus of proteins, and at the N-terminus of proteins in the parental lines of the RHku80^KO or TIR1 line^{46 (link)}. In brief, the CRISPR/Cas9 sgRNA 3’ plasmids (Supplementary Data 2) and CRISPR/Cas9 sgRNA 5’ plasmids (Supplementary Data 2) can efficiently produce Cas9 and sgRNA to create DNA double strand breaks (DSB) in parasites, and facilitates the integration of a tagging amplicon. The amplicon for the C-terminal tagging was generated from a generic plasmid (with the name pLinker-) containing a tag (6HA, AID-3xHA, AID-3Ty, 6Ty or TurboID-3Ty, etc.) as described above, using a pair of primers L and T (Supplementary Data 3). The amplicon for the N-terminal tagging was generated from a generic tagging plasmid (with the name pN-) containing a tag (Ty-TurboID, Ty-AID, HA-AID, myc-AID) using a pair of primers M and NL. The amplicon was then combined with the corresponding CRISPR/Cas9 sgRNA 3’ or 5’ plasmid (Supplementary Data 2) and transfected into recipient lines. The drug selection was followed on the second day based on the resistance marker used in the amplicons. The resistance markers with LoxP sites were excised by transfection with pmini-Cre^{96 (link)}. The cpl gene in the TIR1- AID lines was deleted using a similar CRISPR-Cas9 strategy^{94 (link)}. The complementation lines were generated by direct transfection with the plasmids containing the wild type gene (Supplementary Data 2). Transfection and drug selection were performed with 25 μg/ml mycophenolic acid (M5255, Sigma-Aldrich) and 25 μg/ml 6-xanthine (X4002, Sigma-Aldrich), 200 μg/ml 6-thioxanthine (S96242, Shanghai Yuanye Biotech), or 3 μM pyrimethamine (46706, Sigma-Aldrich)^{40 (link),46 (link)}. The lines were confirmed by IFA and diagnostic PCR, as illustrated in Supplementary Fig. 3a, b, and the PCR was designed for testing the integration site and the endogenous region with primers listed in Supplementary Data 3.