Evidence for Leishmania essential genes included in Table 1 using the rRNA-P stable CRISPR protocol.

PCR shows the retention of the wild type gene for all surviving clones tested and at least one allele of the essential gene in the surviving clones (+/-) was successfully targeted and disrupted by CRISPR. Observation of the dying and dead promastigotes clumps of the essential gene null mutants’ clones (-/-) in 96 well plates. In the rRNA-P stable CRISPR protocol, the gRNA and Cas9 are constantly expressed, the gRNA/Cas9 complex will continue to scan the genome until the last copy of the target gene is deleted or disrupted. If the gene is required for viability, once the remaining copy of the essential gene has been disrupted by CRISPR after cloning into a 96 well plate, null mutants in those wells would stop growing or multiply slowly to form clumps until the gene products are diluted and degraded to the minimum level required for survival. Depending on the relative importance, and initial abundance of the essential gene product in the cell and how soon the individual promastigote was cloned into a 96 well plate after the complete gene disruption, the dying (dead) cell clump size could vary from only a few cells to more than hundreds of cells, the earlier the complete gene disruption took place before cloning, the smaller the size of the dying cell clumps was expected. In this approach, by combining observation of the dying gene null mutant clones and detection of the WT gene band in all surviving clones, using rRNA-P CRISPR protocol, we provide evidence that 14 out of 22 genes listed in Table 1 are essential for promastigote viability. Those essential genes include ten L. mexicana kinase genes: LmxM.02.0290 (Mitogen-activated kinase kinase); LmxM.03.0780 (serine/threonine-protein kinase); LmxM.08.0530; LmxM.08_29.1330 (serine/threonine-protein kinase; Aurora kinase 2, AUK2); LmxM.17.0790 (polo-like protein kinase, PLK); LmxM.20.0960; LmxM.24.2010 (phosphatidylinositol 3-kinase, PI3K); LmxM.25.2340 (AGC essential kinase 1, AEK1); LmxM.30.2860 (Tousled-like kinase, TLK); LmxM.30.2960 (Repressor of differentiation kinase 2, RDK2) and the Calmodulin gene LmxM.09.0910; and three L. donovani genes LdBPK_111030 (hypothetical protein); LdBPK_260650 (Protein of unknown function (DUF2012)) and LdBPK_354780 (Hsp70 protein) (also see Figs 2 and 3).

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