All primer sequences can be found in Table S1 . Plasmid pX330 encoding N-terminally FLAG tagged Cas9 with nuclear localization sequences, and the guide RNA under mammalian promoters, as previously published [3] (link)–[5] (link),was generously provided by Tim Wang. The TgTUB1 promoter was cloned into the KpnI and NcoI sites upstream of Cas9 using primers P1 and P2. Constructs with protospacers against SAG1 (pU6-SAG1) and PKG (pU6-PKG), as well as a universal plasmid encoding BsaI sites in place of a protospacer, were synthesized by IDT (Figure S1 ). These constructs were amplified using P3 and P4, digested with NcoI and XbaI, and cloned into the PciI and XbaI sites of the Cas9-encoding plasmid. The construct targeting the CDPK3 locus was generated by annealing oligos P25 and P26, phosphorylating the duplex, and cloning it into the BsaI-digested universal plasmid. The universal plasmid has been deposited with Addgene (ID no. 52694).
Oligos used to facilitate homologous recombination in PKG and CDPK3 were generated by heating complementary 90- or 119- nucleotide oligomers ordered from IDT to 99 degrees for 2 minutes in a heat block, then removing the block from the heating apparatus and allowing the DNA to cool to room temperature over the course of a few hours. The sequences for these oligomers are listed inTable S1 as P5 and P6 for the PKG oligo and P7 and p8 for the CDPK3 oligo.
The plasmid used to generate the allelic replacements of PKG was made by amplifying the 5′ end of the locus with P17 and P18 from RH genomic DNA and the 3′ end of the gene with P19 and P20 from RH cDNA. The two fragments were spliced together with AvrII and cloned into the PacI/AscI sites of pLIC-YFP-HXGPRT (provided by V. Carruthers, University of Michigan, USA), thus removing the YFP and introducing the two fragments. The gatekeeper residue in this construct was changed to code for T761M using site-directed mutagenesis.
Control plasmid 1 used in viability experiments was constructed by PCR-amplifying the pyrimethamine resistance cassette from pDHFR-TS [6] (link) using primers P23 and P24, and cloned into the NsiI and SbfI sites of the universal plasmid in place of Cas9. Control plasmid 2 is the universal plasmid. Sequences for both control plasmids are provided inFile S1 .
Oligos used to facilitate homologous recombination in PKG and CDPK3 were generated by heating complementary 90- or 119- nucleotide oligomers ordered from IDT to 99 degrees for 2 minutes in a heat block, then removing the block from the heating apparatus and allowing the DNA to cool to room temperature over the course of a few hours. The sequences for these oligomers are listed in
The plasmid used to generate the allelic replacements of PKG was made by amplifying the 5′ end of the locus with P17 and P18 from RH genomic DNA and the 3′ end of the gene with P19 and P20 from RH cDNA. The two fragments were spliced together with AvrII and cloned into the PacI/AscI sites of pLIC-YFP-HXGPRT (provided by V. Carruthers, University of Michigan, USA), thus removing the YFP and introducing the two fragments. The gatekeeper residue in this construct was changed to code for T761M using site-directed mutagenesis.
Control plasmid 1 used in viability experiments was constructed by PCR-amplifying the pyrimethamine resistance cassette from pDHFR-TS [6] (link) using primers P23 and P24, and cloned into the NsiI and SbfI sites of the universal plasmid in place of Cas9. Control plasmid 2 is the universal plasmid. Sequences for both control plasmids are provided in
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