Materials and Methods

CRISPR-Cas9-Mediated Genome Editing inLeishmania donovani

MATERIALS AND METHODSLeishmania donovani strain and culture medium. The L. donovani 1S/Cl2D strain used in this study was routinely cultured at 27°C in M199 medium (pH 7.4) supplemented with 10% heat-inactivated fetal bovine serum, 40 mM HEPES (pH 7.4), 0.1 mM adenine, 5 mg liter−1 hemin, 1 mg liter−1 biotin, 1 mg liter−1 biopterine, 50 U ml−1 penicillin, and 50 µg ml−1 streptomycin. Cultures were passaged to fresh medium in a 20-fold dilution once a week. Unless indicated, all Leishmania cultures were carried out at 27°C in this study.Plasmid construction. All primer sequences used in this study are listed in Fig. S10 in the supplemental material.The pLPhygCas9 plasmid was generated by cloning the humanized Streptococcus pyogenes Cas9 nuclease with nuclear localization signals (4.4 kb) from plasmid pX330 (Addgene) (5) into the HindIII and BamHI sites of Leishmania expression vector pLPhyg (26) with primers pX330Cas9F (F stands for forward) and pX330Cas9R (R stands for reverse).The pSPneogRNA plasmid was generated as follows. (i) The 180-bp L. donovani rRNA promoter was derived from L. donovani genomic DNA by PCR with primers LdrRNApF and LdrRNApR. (ii) A 370-bp DNA fragment that contains the guide sequence insertion site and the 82-bp Cas9 binding RNA coding sequence was obtained from the pX330 plasmid with primer pX330gRNAF and primer pX330gRNAR. (iii) The 180-bp LdrRNA promoter sequence from step 1 and the 370-bp guide RNA coding sequence from step 2 (there are 26-bp overlap sequence between these two sequences) were joined together by PCR with primer pair LdrRNApF and pX330gRNAR. (iv) The joined sequence (550 bp) from step 3 was digested with HindIII and BamHI and cloned into the corresponding sites of pSPneo (32) to generate gRNA expression plasmid pSPneogRNA.The pSPneogRNAH plasmid was generated as follows. (i) The 313-bp DNA sequence which contains the L. donovani rRNA promoter and the complete gRNA coding sequence from the pSPneogRNA plasmid was amplified by PCR with primer LdrRNApF and primer HDVRiboR1 which also contains at its 5′ end 28 nucleotides that overlap the HDV ribozyme coding sequence. (ii) The 313-bp PCR fragment of the LdrRNA promoter and gRNA sequence from step 1 was mixed with the following two HDV ribozyme coding sequence oligonucleotides, HDVRiboF1 and HDVRiboR2. Note that HDVRiboF1 and HDVRiboR2 have 19-nucleotide complementary sequences at their 3′ ends. (iii) The PCR fragment and the HDV ribozyme coding sequence oligonucleotide mixture from step 2 were joined together by PCR with primer pair LdrRNApF and HDVRiboR3. (iv) The joined sequence (359 bp) from step 3 was digested with HindIII and BamHI and cloned into the corresponding sites of pSPneo (32) to generate gRNA expression plasmid pSPneogRNAH.The pSPneoHHgRNAH plasmid was generated as follows. (i) The following five oligonucleotides, {"type":"entrez-nucleotide","attrs":{"text":"HH241510","term_id":"304784317","term_text":"HH241510"}}HH241510+1, HHBbsI+2, HHBbsI-1, HH241510-2, and HH241510-3, which contain a HindIII site and a BglII site, Hammerhead ribozyme sequence, and two BbsI sites were annealed in T4 DNA ligase buffer. (ii) The annealed DNA fragment from step 1 was ligated into BbsI-digested pSPneogRNAH plasmid after the unique BglII site was eliminated with DNA polymerase I Klenow fragment.The pSPneogRNA5′H-HHgRNA3′H plasmid (gRNA5′&3′ construct) was generated by inserting the 360-bp HindIII and BamHI fragment, which contains LdrRNAP and Ld1315905′gRNA coding sequence from pSPneogRNA5′H, into the HindIII and BglII sites of pSPneoHHgRNA3′H after removing the 180-bp LdrRNAP sequence.gRNA and primer design and synthesis. The gRNA-targeting sequences were selected manually or with the gRNA designer tool (http://www.broadinstitute.org/rnai/public/analysis-tools/sgrna-design). The primers for PCR were picked manually or by Primer3 (http://gmdd.shgmo.org/primer3). All primers and oligonucleotides used in this study were synthesized and purchased from Alpha DNA (Montreal, Canada).Cloning guide sequence into various gRNA expression vectors. The complementary oligonucleotides of guide sequence were first phosphorylated in T4 DNA ligase buffer with T4 polynucleotide kinase and then annealed by heating at 95°C for 5 min in a 1-liter water glass beaker, which was allowed to cool to room temperature for about an hour. The annealed guide DNA linkers (4 µM) were then cloned into the various BbsI-digested gRNA expression vectors (see Fig. S1, S2, and S9 in the supplemental material).Leishmania transfection and limiting dilution assay. Leishmania transfections were performed as described previously (21, 56). Four to 10 µg of plasmid DNA, 10 µl of 100-µm single-strand oligonucleotide donor, and 2 to 4 µg of purified PCR products were used for each transfection. To generate a cell line that constantly expresses Cas9, L. donovani promastigotes transfected with pLPhygCas9 were selected and maintained at culture medium containing 100 µg/ml hygromycin. The Cas9-expressing Leishmania cells transfected with various gRNA expression plasmids were selected with G418 (100 µg/ml) and maintained in medium containing both hygromycin and G418 (100 µg/ml each) as a pooled population. Aliquots (<100 µl) of these cultures were taken out for MLF resistance assay by limiting dilution culture at various time points as indicated in the figures (for example, 4 and 6 weeks posttransfection). To determine whether transfection of an oligonucleotide donor would increase LdMT inactivation frequency, the gRNA transfectant culture was split into two flasks. One flask remained in a 27°C incubator. Cells from the other flask were harvested, transfected with the oligonucleotide donor, and then resuspended in culture medium containing both hygromycin and G418 in a 27°C incubator. After continuous culture for 3 days, the cells were taken out from the non-oligonucleotide donor-transfected or oligonucleotide donor-transfected culture flasks, counted, and then directly used for MLF resistance assay by limiting dilution culture. To determine whether culture at 37°C would improve the LdMT inactivation frequency, the gRNA transfectant cultures with or without the oligonucleotide donor were split into two flasks, one remained in a 27°C incubator, and the other flask was transferred to a 37°C incubator. After 4 days of culture, cells were taken out from the 27°C and 37°C culture flasks, counted, and then used for MLF resistance assay by limiting dilution culture.Limiting dilution cultures used to clone the transfectants and to quantify MLF resistance and Ble donor insertion rates were performed at 27°C in 96-well plates that contained Leishmania culture medium (100 µl/well) with 40 µM miltefosine or 100 µg/ml phleomycin in addition to hygromycin and G418 (100 µg/ml each). The gRNA transfectants were inoculated into the wells in the first column at two thousand to one mi llion cells in 200 µl medium per well and then serially diluted by 2-fold from left to right until column 12. Each sample was serially diluted in triplicate or quadruplicate and cultured at 27°C for 2 to 4 weeks. The resistance rates were determined by counting back from the last well containing a single parasite to determine the number of resistant parasites, and this was compared to the number of parasites added in the first well before drug selection. Unlike phleomycin and hygromycin, which take days to kill wild-type L. donovani promastigote cells, miltefosine requires less than 20 h to completely kill wild-type L. donovani cells.The primer pair 241510BleF and 241510BleR with pSPble plasmid as the PCR template were used to generate the bleomycin resistance gene expression cassette donor (see Fig. S6 in the supplemental material). Two to 4 µg of purified Ble donor PCR products was used to transfect the Cas9- and gRNALd241510-expressing Leishmania cells. Three days posttransfection, the cells were subjected to drug selection with 100 µg/ml phleomycin.The primer pair 131590GFPF and 131590GFPR with pEGFP-N1 (EGFP stands for enhanced green fluorescent protein) plasmid as the PCR template were used to generate the GFP tag donor (see Fig. S8 in the supplemental material). Two to 4 µg of purified GFP donor PCR products was used to transfect the Cas9- and gRNAa-expressing Leishmania cells. Three days posttransfection, the cells were subjected to drug selection with 40 µM miltefosine.Sequencing analysis. All plasmid constructs generated in this study were verified by DNA sequencing.Genomic DNAs from wild-type and gRNA construct-transfected Leishmania promastigotes were extracted with a minipreparation method as described previously (57). The sequences surrounding the gRNA-targeting sites were amplified by KAPA Taq DNA polymerase with various primer pairs listed in the supplemental material. The PCR products were purified from agarose gels and sent directly for sequencing or subcloned into the pSP72 vector before sequencing. All gRNA expression plasmids were sequenced by a modified protocol for DNA with secondary structure. Sequencing was performed at the McGill University and Genome Quebec Innovation Center.Flow cytometry and fluorescence microscopy. Flow cytometric analysis was performed using a FACSariaII cytometer from Becton Dickinson (BD Biosciences, USA), and data were collected by using FACSDiva version 6.1.3. Imaging of live Leishmania promastigotes by confocal microscopy (Olympus) was performed as described previously (58).The pLPhygCas9, pSPneogRNAH, pSPneoHHgRNAH, and pSPble plasmids have been deposited in Addgene with identification (ID) no. 63555, 63556, 63557, and 63561, respectively. Partial sequences for some of these plasmids are provided in Fig. S1, S6, and S9 in the supplemental material.

Article TitleCRISPR-Cas9-Mediated Genome Editing inLeishmania donovani

Abstract

The prokaryotic CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9, an RNA-guided endonuclease, has been shown to mediate efficient genome editing in a wide variety of organisms. In the present study, the CRISPR-Cas9 system has been adapted toLeishmania donovani, a protozoan parasite that causes fatal human visceral leishmaniasis. We introduced the Cas9 nuclease intoL. donovaniand generated guide RNA (gRNA) expression vectors by using theL. donovanirRNA promoter and the hepatitis delta virus (HDV) ribozyme. It is demonstrated within thatL. donovanimainly used homology-directed repair (HDR) and microhomology-mediated end joining (MMEJ) to repair the Cas9 nuclease-created double-strand DNA break (DSB). The nonhomologous end-joining (NHEJ) pathway appears to be absent inL. donovani. With this CRISPR-Cas9 system, it was possible to generate knockouts without selection by insertion of an oligonucleotide donor with stop codons and 25-nucleotide homology arms into the Cas9 cleavage site. Likewise, we disrupted and precisely tagged endogenous genes by inserting a bleomycin drug selection marker andGFPgene into the Cas9 cleavage site. With the use of Hammerhead and HDV ribozymes, a double-gRNA expression vector that further improved gene-targeting efficiency was developed, and it was used to make precise deletion of the 3-kb miltefosine transporter gene (LdMT). In addition, this study identified a novel single point mutation caused by CRISPR-Cas9 inLdMT(M381T) that led to miltefosine resistance, a concern for the only available oral antileishmanial drug. Together, these results demonstrate that the CRISPR-Cas9 system represents an effective genome engineering tool forL. donovani.


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