Nanopore and Illumina sequencing of CBE mutated Leishmania

A cytosine base editing (CBE) single-guide RNA (sgRNA) expression cassette targeting the motility-associated gene PF16 was integrated into the 18S rRNA SSU locus of Leishmania mexicana using a Cas12a-mediated double-strand break (DSB). The transfected cell line expressed a tdTomato reporter and harbored an episomal construct (pTB107) enabling the expression of CBE, T7 RNA Polymerase, and Cas12a. Sixteen days post-transfection, genomic DNA was isolated from non-clonal PF16 mutant populations containing the pTB107 construct, the tdTomato reporter, and the PF16-targeting CBE sgRNA expression construct. The isolated DNA underwent sequencing using both Illumina and Oxford Nanopore Technology (ONT) platforms for comprehensive analysis.