Fig. 2: Generation of the L. mexicana kinome gene deletion library. | Nature Communications

Fig. 2: Generation of the L. mexicana kinome gene deletion library.

From: Systematic functional analysis of Leishmania protein kinases identifies regulators of differentiation or survival

Fig. 2

a Schematic representation of gene editing by CRISPR–Cas9 in the L. mexicana progenitor cell line showing the integration of repair cassettes containing 30nt homology sites, a unique barcode and antibiotic-resistance genes for puromycin (PAC) and blasticidin (BSD). Diagnostic PCRs for the presence of the coding sequence (CDS) with Oligo 1 and Oligo 2, (depicted in blue), correct integration of the repair cassettes using Oligo 3 and Oligo 4 (depicted in red), and the gene in the endogenous locus with Oligo 5 and Oligo 6 (depicted in green). b Diagnostic PCR for (i) LmxM.25.0853 and (ii) PKAC1 (LmxM34.4010) using primer pairs as above in L. mexicana Cas9T7 (WT) and Populations A and B. c Heat map indicating the presence or absence of target genes after whole-genome sequencing. The ratio of gene coverage to chromosome coverage was used as a measure of gene copy number in selected mutants. A value of 1 represents two alleles of a given gene. d Diagnostic PCR for facilitated gene deletion mutant LmxM.04.0650 using Oligo 1 and 2 for the CDS, Oligo 5 and 6 for the endogenous locus and Oligo 3 and 4 for correct integration. Western blot using anti-myc confirmed the episomal expression of LmxM.04.0650 at the anticipated size of 140 kDa.

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