TABLE 3 

Site-specific integration of DHFR into the UPRT locus mediated by CRISPR/CAS9

ExptTrialsgRNA
target
Type of
disruption
Size (bp) of
homology
armsa
Plaque formationb%
efficiencyc
Frequency of clones by PCR3
Without
FUDR
With
FUDR
IntegrationdDisruptione
11UPRTInsertion750, 90072/20065/20090.33/52/5
2UPRTInsertion750, 90086/20076/20088.4NDfND
21UPRTInsertion20, 2025/20022/20088.03/52/5
2UPRTInsertion20, 2053/20049/20092.54/61/6
31UPRTInsertion0, 032/20024/20075.03/82/8
2UPRTInsertion0, 052/20037/20071.24/51/5
  • a The sizes of the fragments flanking the 5′ and 3′ ends of DHFR that are homologous to UPRT, as indicated in Fig. 3A.

  • 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 [10 µM]/number of plaques observed in the absence of FUDR) × 100.

  • d Clones that showed the presence of a 4.4-kb band and absence of a 1.2-kb band in PCR3 were considered integration events. Number of positive clones/number of total clones checked.

  • e Clones that lacked the endogenous 1.2-kb band for the UPRT locus but which did not give the expected band of 4.4 kb, corresponding to insertion of DHFR*, were considered disruptions. Number of positive clones/number of total clones checked.

  • f ND, not determined.