FIG 1 .

Sequential transfections of oligonucleotide donors significantly improve CRISPR-Cas9 gene-editing efficiency. (A) Gene-editing strategy by providing the gRNAc-directed Cas9 cleavage site in the LdMT locus with the 61-nt single-strand oligonucleotide donor containing stop codons. (B) Oligonucleotide donor transfection schedule and time points for sampling miltefosine (MLF) resistance rates. (C) The MLF resistance rates of gRNAc-targeted L. donovani cells after sequential transfections of the oligonucleotide donor. The MLF resistance rates were determined in 96-well plates by limiting dilution culture at 27°C for 2 to 4 weeks, as previously described (7). These are representative data of three independent experiments.