TABLE 2 .

Disruption of UPRT by DHFR through CRISPR/CAS9-induced homologous recombination

Expt	Trial	sgRNA target	Type of disruptiona	Plaque formationb		% efficiencyc	Frequency of clones by PCR
				Without FUDR	With FUDR		Complete homologous replacementd	Partial homologous replacemente
1	1	none	—f	49/200	0/200,000	0.0	—	—
	2	none	—	43/200	0/200,000	0.0	—	—
2	1	UPRT	Insertion	55/200	38/200	69.0	4/9	2/9
	2	UPRT	Insertion	64/200	50/200	78.1	4/6	1/6
	3	UPRT	Insertion	34/200	51/200	≈100.0	NDg	ND
3	1	ROP18	—	75/200	0/200,000	0.0	—	—
	2	ROP18	—	47/200	0/200,000	0.0	—	—
4	1	UPRT	Deletion	45/200	50/200	≈100.0	4/9	5/9
	2	UPRT	Deletion	55/200	47/200	85.5	3/7	3/7

^aDifferent homology templates were used for gene insertion or deletion as described for Fig. 2A and C.

^bNumber of plaques observed/number of parasites seeded. When used, FUDR was added to a final concentration of 10 µM.

^c(Number of plaques observed in the presence of FUDR/number of plaques observed in the absence of FUDR) × 100.

^dNumber of positive clones/number of total clones checked. Clones that showed correct 5′ integration (positive in PCR1 or PCR4), correct 3′ integration (positive in PCR2 or PCR5), and loss of the endogenous UPRT gene (negative in PCR3) were considered to have undergone complete homologous replacement events.

^eNumber of positive clones/number of total clones checked. Clones that showed loss of the endogenous UPRT gene (negative in PCR3) and correct integration in at least one end (5′ or 3′ but not both) were considered to have undergone partial homologous replacement events.

^f—, not applicable.

^gND, not determined.