Application of CRISPR/Cas9-Based Reverse Genetics in Leishmania braziliensis: Conserved Roles for HSP100 and HSP23

Abstract

The protozoan parasite Leishmania (Viannia) braziliensis (L. braziliensis) is the main cause of human tegumentary leishmaniasis in the New World, a disease affecting the skin and/or mucosal tissues. Despite its importance, the study of the unique biology of L. braziliensis through reverse genetics analyses has so far lagged behind in comparison with Old World Leishmania spp. In this study, we successfully applied a cloning-free, PCR-based CRISPR-Cas9 technology in L. braziliensis that was previously developed for Old World Leishmania major and New World L. mexicana species. As proof of principle, we demonstrate the targeted replacement of a transgene (eGFP) and two L. braziliensis single-copy genes (HSP23 and HSP100). We obtained homozygous Cas9-free HSP23- and HSP100-null mutants in L. braziliensis that matched the phenotypes reported previously for the respective L. donovani null mutants. The function of HSP23 is indeed conserved throughout the Trypanosomatida as L. majorHSP23 null mutants could be complemented phenotypically with transgenes from a range of trypanosomatids. In summary, the feasibility of genetic manipulation of L. braziliensis by CRISPR-Cas9-mediated gene editing sets the stage for testing the role of specific genes in that parasite's biology, including functional studies of virulence factors in relevant animal models to reveal novel therapeutic targets to combat American tegumentary leishmaniasis.

Keywords: CRISPR–Cas9; Leishmania braziliensis; gene targeting; heat shock proteins; phenotyping; reverse genetics.

Figure 1

CRISPR–Cas9-mediated disruption of eGFP gene as proof-of-principle test in L. braziliensis. (A) Generation of Cas9–eGFP-expressing parasites. Left panel: plasmid pTB007 [17] bearing hSpCas9 and T7 RNAP transgenes was transfected as circular episome into L. braziliensis PER005cl2 wild-type parasites. Transfectants were selected under Hygromycin B pressure. Right panel: schematic depiction of the double cross-over homologous recombination strategy to integrate the linearised pIR–eGFP construct into the SSU rRNA locus of L. braziliensis Cas9-expressing parasites. Regions shown are the SSU rRNA sequences on either ends resulting from SwaI restriction digest, the eGFP ORF, and the nourseothricine resistance gene ORF (SAT, encoding streptothricin-acetyltransferase). (B) Schematic representation of the eGFP locus and locations of the six 20-nt guide RNA sequences used for gene disruption; the guide sequence pairs with the DNA target (blue bar), directly upstream of a requisite 5’-NGG-3’ adjacent motif (PAM). The green arrowhead indicates the predicted Cas9 cleavage sites. Only the coding strand is shown. Binding sites of primers used for genotyping of genetically engineered parasites are denoted by arrows. The PCR fragment size depended on the pair of single guide RNAs (sgRNAs) tested. Sets of sgRNAs tested: set 1 = eGFP-52-5’sgRNA and eGFP-253-3’sgRNA; set 2 = eGFP-52-5’sgRNA and eGFP-612-3’sgRNA; set 3 = eGFP-52-5’sgRNA and eGFP-639-3’sgRNA; set 4 = eGFP-553-5’sgRNA and eGFP-639-3’sgRNA; set 5 = eGFP-378-5’sgRNA and eGFP-612-3’sgRNA; set 6 = eGFP-378-5’sgRNA and eGFP-639-3’sgRNA. (C) Flow cytometry analysis of eGFP–Cas9-expressing parasites before and after transfection of eGFP-targeting sgRNAs. Efficiency of eGFP disruption using 6 different sets of sgRNAs in L. donovani (left panel) and L. braziliensis (right panel) as quantified by GFP expression. Each set of two sgRNAs was co-transfected with two donor DNAs; transfections were done in triplicate. Sets of sgRNAs tested (labelled as set 1 to 6 in the graphs) consisted of pairs as described in Figure 1B. P, parental cell line Cas9/T7/eGFP. The gating scheme, a representative histogram, and all FACS plots showing the percentage of GFP-positive cells are shown in Supplemental Figures S2 and S3.

Figure 2

CRISPR–Cas9-mediated disruption of the endogenous HSP23 gene in L. braziliensis. (A) Schematic representation of the LbrHSP23 locus depicting the locations of 20-nt guide sequences that worked efficiently to disrupt the LbrHSP23 ORF. Two sets of sgRNAs were tested (set 1 and set 2): set 1 = LbrHSP23-70-5’sgRNA and LbrHSP23-171-3’sgRNA; set 2 = LbrHSP23-183-5’sgRNA and LbrHSP23-323-3’sgRNA. Both pairs are designed to disrupt the conserved functional alpha-crystallin domain of HSP23 (amino acid positions 6–104). The guide sequence pairs with the DNA target (blue bar) directly upstream of a requisite 5′–NGG–3′ adjacent motif (PAM). The green arrowhead indicates the predicted Cas9 cleavage sites. Only the coding strand sequence is shown. (B) NGS analysis of the HSP23 locus after CRISPR–Cas9-mediated gene replacement. Genomic DNA of L. braziliensis PER005cl2 wild-type parasites (WT), the parental cell line WT [Cas9] and HSP23^–/– mutant clones was isolated and subjected to NGS analysis. Resulting NGS reads were aligned to the HSP23 gene locus (LbrM.20.0220) in the L. braziliensis M2904 reference genome using the Bowtie 2 algorithm. The read coverages (Y-axis) for the gene locus are shown in blue. The arrow represents the position and direction of the coding sequence. The X-axis numbering refers to the nucleotide position (bp) on chromosome 20. Grey-shaded areas denote lack of aligned reads. (C) Verification of HSP23 gene replacement by Western blot analysis. 1 × 10⁷ cells of WT, WT [Cas9], and of 3 HSP23^–/– clones were lysed and the cell lysates were analysed by SDS-PAGE and Western blot using anti-HSP23 (1/500, lower panel). Anti-HSP100 (1/1000, upper panel) was used as loading control. MW = Molecular weight in kilodalton.

Figure 3

CRISPR–Cas9-mediated disruption of the endogenous HSP100 gene in L. braziliensis. (A) For targeting LbrHSP100 (LbrM.29.1350), two sets of sgRNAs tested (set 3 and set 4) worked efficiently. sgRNAs set 3 (LbrHSP100-513-5’sgRNA and LbrHSP100-712-3’sgRNA) targeted disruption of the LbrHSP100 ORF in the N terminus. sgRNAs set 4 targeted 5’ and 3’ non-coding flanking sequences for LbrHSP100 whole-gene deletion. Two cloned L. braziliensis HSP100^–/– lines were studied, HSP100^–/– cl.1 and HSP100^–/– cl.2, derived from transfection of set 3 or set 4 of LbrHSP100-targeting sgRNAs, respectively. (B) Whole genome sequencing of HSP100-null mutant lines. Sequence reads from each analysed strain were aligned to the reference DNA sequence consisting of chromosome 29 of L. braziliensis M2904 reference genome using Bowtie 2 software. The Y-axis represents the number of reads and the X-axis shows the nucleotide position (bp) on chromosome 29. Grey shaded areas denote complete lack of aligned reads. (C) Verification of HSP100-null mutants by Western blot analysis using anti-HSP100 (1/1000) antibody. Anti-HSP23 antibody (1/500) served as loading control. MW = Molecular weight in kilodalton.

Figure 4

Phenotypic analyses of L. braziliensis HSP23^–/– and HSP100^–/– clones. For growth curves, promastigotes of WT, WT (Cas9), HSP23^–/– clones, and HSP100^–/– clones were seeded at a density of 1 × 10⁶ parasites/mL into 5 ml of complete M199 medium and grown for 4 days. Cell density was measured on day 4 and is shown as a percentage of WT cell density (set at 100%). Parasites were grown at 25 °C (A) and 30 °C (B). The HSP23^–/– clones incubated for 4 days at 30 °C were also stained with mouse anti-tubulin antibody (1/4000) and DAPI (1/50) (C). Images were taken on an EVOS FL Auto Cell Imaging System and processed using the ImageJ Software (https://fiji.sc). Scale bar: 10µm. Additional cultures were grown at 25 °C and pH 7.4 with the addition of 2% ethanol (D). The horizontal black lines in panels A, B, and D indicate the median of 6 biological samples from 3 separate experiments. Significance was tested using the Kruskal–Wallis test; * p < 0.05, ** p < 0.01, *** p < 0.001. (E) Primary mouse bone-marrow-derived macrophages were differentiated and infected with stationary-phase promastigotes of WT, WT [Cas9], HSP23^–/– clones, and HSP100^–/– clones at a MOI of 1:8 (macrophage-to-parasite ratio). After 4 h, free parasites were washed away and the infected macrophage cultures were further incubated at 34 °C under 5% CO₂ for 44 h. Genomic DNA from Leishmania-infected macrophages was isolated at 4.5 h and at 48 h post-infection, and parasite load was determined by TaqMan qPCR quantifying parasite actin gene DNA relative to host macrophage actin gene DNA. Shown is intracellular parasite survival [%] after 48 h, with the bar indicating the median of n = 5. Ratio-paired, one-sided Student’s t-test: * p < 0.05, ** p < 0.01, *** p < 0.001 between data pairs. ns = not significant.

Figure 5

Phenotypic analysis of L. major HSP23^–/– mutants and complementation strains. 1 × 10⁶ or 5 × 10⁶ parasites/ml were seeded in 10 ml complete M199 medium and parasite density was assessed at day 4. Parasites were grown at 25 °C (A), 34 °C (B), and 25 °C with 2% EtOH (C). Cell density is shown as percentage of WT (set at 100%). (D) Complementation studies in LmjHSP23^–/– mutants. Null mutants were transfected with the pCL1S over expression vector harbouring the HSP23 gene of L. major, L. donovani, L. infantum, L. braziliensis, and Trypanosoma brucei or with the empty vector only. Complementation populations were subjected to growth experiments at 34 °C. Cell density was assessed at day 4 and is shown normalised to Lmj WT growth (set at 100%). * = p < 0.05.