TABLE 2 

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

ExptTrialsgRNA
target
Type of
disruptiona
Plaque formationb%
efficiencyc
Frequency of clones by PCR
Without
FUDR
With
FUDR
Complete
homologous
replacementd
Partial
homologous
replacemente
11nonef49/2000/200,0000.0
2none43/2000/200,0000.0
21UPRTInsertion55/20038/20069.04/92/9
2UPRTInsertion64/20050/20078.14/61/6
3UPRTInsertion34/20051/200100.0NDgND
31ROP1875/2000/200,0000.0
2ROP1847/2000/200,0000.0
41UPRTDeletion45/20050/200100.04/95/9
2UPRTDeletion55/20047/20085.53/73/7
  • a Different homology templates were used for gene insertion or deletion as described for Fig. 2A and C.

  • b Number 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.

  • d Number 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.

  • e Number 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.

  • g ND, not determined.