Evidence for gene essentiality in Leishmania using CRISPR

Abstract

The ability to determine the essentiality of a gene in the protozoan parasite Leishmania is important to identify potential targets for intervention and understanding the parasite biology. CRISPR gene editing technology has significantly improved gene targeting efficiency in Leishmania. There are two commonly used CRISPR gene targeting methods in Leishmania; the stable expression of the gRNA and Cas9 using a plasmid containing a Leishmania 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 Leishmania genes to study mutant phenotypes and several research labs are now using this protocol to target all the genes in L. mexicana genome. The rRNA-P stable protocol stably expresses the plasmid derived gRNA and has been used to delete or disrupt single and multicopy Leishmania 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 Leishmania 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 Leishmania.

Fig 1. CRISPR gene targeting strategy (rRNA-P stable protocol) used to disrupt the essential and non-essential Leishmania genes in this study.

(A) Schematics of Leishmania CRISPR plasmid pLdSaCN and strategy used for gene disruption. In pLdSaCN CRISPR plasmid, L. donovani rRNA promoter (rRNAP) controls the stable transcription of the gRNA and Staphylococcus aureus Cas9 (SaCas9). The target gene specific gRNA leads Cas9 to generate a double strand break which is then repaired following the introduction of the donor DNA (black bars) containing an antibiotic resistance gene, resulting in disruption of the target gene. A2-IGS, A2 intergenic sequence; NEO, Neomycin resistance gene. Note, the pLdSaCN plasmid is structurally like the other stable expression Leishmania CRISPR plasmid pLdCN also used in this study. Instead, pLdCN expresses the commonly used Streptococcus pyogenes Cas9 (SpCas9). (B) PCR analysis with primers L and R showing the disruption of a non-essential L. donovani gene (LdBPK_100590). The higher disrupted gene band with the inserted donor DNA but not the lower WT gene band was detected in three Ld100590(-/-) clones. (C) Complete disruption of a non-essential L. donovani gene (LdBPK_230540).

Fig 2. Evidence the AGC essential kinase 1 gene (LmxM.25.2340, AEK1) is essential for L. mexicana.

(A) The dying promastigotes clumps observed in some of the 96 well plate wells after the G418 and Puromycin double resistance transfectants were cloned into a 96 well plate. Scale bar, 15 μm. (B) PCR analysis showing the wild type (WT) AEK1 gene band persists in all surviving clones in the 96 well plate and at least one of the gene alleles was correctly disrupted by CRISPR.

Fig 3. Evidence the Calmodulin gene is essential for L. mexicana.

(A) Calmodulin gene locus in chromosome 9 contains three Calmodulin genes (LmxM.09.0910; 0920 and 0930) in tandem array. (B) Strategy used by rRNA-P stable protocol to delete and disrupt L. mexicana Calmodulin gene clusters, and the outcome of one of the Calmodulin gene targeting clones. The PCR primers used to verify gene deletion and disruption are indicated. (C) PCR analysis of the Calmodulin gene targeting clone shown in B. After CRISPR gene targeting, the PCR product size with primers 10R1+30L1 reduced from 4.2 Kb of the WT band size to 2.8 Kb and no PCR product could be detected with primers 30R1+L, indicating LmxM.09.0930 gene had been deleted. The PCR product sizes of primers R+10 L1 increased from 700 bp to 900 bp and 1100 bp respectively, indicating both LmxM.09.0910 alleles had been disrupted. Both the WT band and size increased disruption band of primers R+30L1 were detected, indicating one LmxM.09.0920 allele had been successfully disrupted and one WT LmxM.09.0920 allele (the only WT Calmodulin gene allele) remained in this clone. Note: a smaller than the expected size (R+10R1) band (900 bp) was detected in this Cal (+—/—) clone, indicating a recombination deletion had occurred in one of the two disrupted LmxM 09.0910 alleles, which explains the additional fainter band running at 700 bp detected in the PCR with (R+L) primer pair. Likewise, the fainter band (the middle band) detected in the PCR with (R+30L1) primer pair could be derived from some of the cells in this Cal (+—/—) clone cell population where recombination deletion took place in the disrupted LmxM 09.0920 allele. (D) The dead cell clumps observed after the CRISPR gene targeting transfectants were cloned into a 96 well plate, suggesting all the Calmodulin genes in the dead clones had been deleted or disrupted. (E) Compared with the WT promastigotes, one of the surviving Calmodulin CRISPR gene targeting clones (+—/—) in the 96 well plate is shown with a smaller and round morphology. Scale bar, 15 μm.

Fig 4. Disruption of non-essential Leishmania genes in polyploid chromosomes with rRNAP-P stable protocol.

(A) CRISPR disruption of L. donovani genes LdBPK_310120 and LdBPK_312380 in the tetraploid chromosome 31. A total of four independent Cas9 cleavage and insertion events were required to disrupt all four copies of the genes (see left panel). (B) Disruption of the L. mexicana LmxM.16.1550 (CMF6) gene family in the trisomic chromosome 16. Three Cas9 cleavage and insertion events were required to disrupt all three copies of the genes (see left panel). Note, the LmxM.16.1550 gene was completely disrupted in only two of the 13 CRISPR gene targeting clones examined, highlighting the importance of cloning to isolate gene disruption mutants.

Fig 5. Promastigote growth curves of the L. donovani and L. mexicana null mutants generated in this study.

Equal numbers of Leishmania promastigotes (1 million promastigotes per ml) were inoculated in flasks each containing 4 ml culture medium. The parasite growth was monitored by microscope counting once a day for 8 days. The data shown are the mean plus Standard Error of the Mean (SEM). Note, as examples, the cell density differences between LdWT and Ld100590(-), Ld230540(-), Ld310120(-) and Ld312380(-) cells, and between LmxWT and Lmx161550(-) cells at day 3 post inoculation are statistically significant (Student’s t-test P<0.001). This is the representative data of three independent experiments.

Fig 6. Mobility defects were observed in L. mexicana null mutants generated in this study.

(A) A swimming assay was performed on a 7 cm VINYL Tubing with both ends in an up position as described in methods. The tube was first filled with 900 μl PBS, then 2 million L. mexicana promastigotes in 50 μL culture medium were gently loaded at the left end of the tube. The data shown in the table are the mean number (plus Standard Deviation, SD) of promastigotes detected in 3 μL solution taken from the right end of the tube after incubation for 1, 1.5, 2 and 3 hours. The formalin fixed L. mexicana promastigotes (Dead LmxM WT) was used as the negative control. The differences of promastigote number detected in the 3 μL solution between L. mexicana WT and the various mutant cell lines are statistically significant (Student’s t-test, * P<0.05; ** P< 0.005; *** P< 0.0005; **** P< 0.0001). (B) The Giemsa-stained L. mexicana promastigotes. The promastigote size and its flagellar length appear normal for the null mutants of LmxM.16.1550, LmxM.34.4010 and LmxM.20.1180. However, the promastigotes of Calmodulin CRISPR gene targeting clone (+—/—) are smaller than WT L. mexicana promastigotes though the normal flagellar lengths are still retained. Scale bar, 5 μm.