To study the efficiency of CRISPR/CAS9-mediated gene disruption by integration of a selectable marker, sgUPRT or sgROP18 targeting CRISPR/CAS9 plasmids (see Table S1 in the supplemental material) was combined with various amplicons containing DHFR* expression cassettes for transfection. Amplicons were PCR amplified using primers listed in Table S2 in the supplemental material, purified by agarose gel electrophoresis, and extracted using the Qiaquick gel extraction kit (Qiagen Inc., Valencia, CA). Recovered DNAs were quantified using a Nanodrop 2000 instrument (Nanodrop Instruments, Wilmington, DE). Mixtures of CRISPR/CAS9 plasmids with the purified DHFR* amplicons (5:1 mass ratio) were cotransfected into RH parasites by electroporation. Pyrimethamine-resistant parasites were obtained by selection with 3 µM pyrimethamine, and resistant cells were then used for determination of viability and FUDR resistance by plaque assay as described above. In parallel, single-cell clones were obtained by limiting dilution, and lysates were generated as described previously (4 (link)). The specificity of DHFR* integration and loss of genes by deletion were tested by PCR using primers listed in Table S2. PCRs were performed using Taq DNA polymerase (New England Biolabs, Ipswich, MA) in a 25-µl reaction mixture containing 2 µl of lysate as the template according to the manufacturer’s directions. In general, reactions were performed for 35 cycles with denaturation at 95°C for 30 s, annealing at 57°C for 45 s, and extension for 90 s at 68°C. Products were analyzed by electrophoresis in agarose gels with ethidium bromide. In some cases, PCR3 was conducted using Q5 DNA polymerase (New England Biolabs, Ipswich, MA) to efficiently amplify the inserted DHFR* fragment, giving a band of 4.4 kb, which was inefficiently amplified using Taq polymerase under the conditions described above. When PCR3 was performed with Q5 polymerase, 25-µl reaction mixtures containing 2 µl template were amplified for 35 cycles with denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension for 3 min at 68°C.