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<div><p>The ability to determine the essentiality of a gene in the protozoan parasite <i>Leishmania</i> is important to identify potential targets for intervention and understanding the parasite biology. CRISPR gene editing technology has significantly improved gene targeting efficiency in <i>Leishmania</i>. There are two commonly used CRISPR gene targeting methods in <i>Leishmania</i>; the stable expression of the gRNA and Cas9 using a plasmid containing a <i>Leishmania</i> ribosomal RNA gene promoter (rRNA-P stable protocol) and the T7 RNA polymerase based transient gRNA expression system in promastigotes stably expressing Cas9 (T7 transient protocol). There are distinct advantages with both systems. The T7 transient protocol is excellent for high throughput gene deletions and has been used to successfully delete hundreds of <i>Leishmania</i> genes to study mutant phenotypes and several research labs are now using this protocol to target all the genes in <i>L</i>. <i>mexicana</i> genome. The rRNA-P stable protocol stably expresses the plasmid derived gRNA and has been used to delete or disrupt single and multicopy <i>Leishmania</i> genes, perform single nucleotide changes and provide evidence for gene essentiality by directly observing null mutant promastigotes dying in culture. In this study, the rRNA-P stable protocol was used to target 22 <i>Leishmania</i> genes in which null mutants were not generated using the T7 transient protocol. Notably, the rRNA-P stable protocol was able to generate alive null mutants for 8 of the 22 genes. These results demonstrate the rRNA-P stable protocol could be used alone or in combination with the T7 transient protocol to investigate gene essentiality in <i>Leishmania</i>.</p></div>