Materials and Methods

Optimized CRISPR-Cas9 Genome Editing forLeishmaniaand Its Use To Target a Multigene Family, Induce Chromosomal Translocation, and Study DNA Break Repair Mechanisms

Leishmania strains and culture medium.

L. donovani 1 S/Cl2D, L. major Friedlin v9, and L. mexicana (MNYC/BZ/62/M379) used in this study were 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 at a 20-fold dilution once a week. Leishmania cells after transfection with the CRISPR vector and donor DNA were sometimes cultured at 33°C or 37°C for 2 to 3 days to improve gene-editing efficiency (7).

Plasmid construction.

All primer sequences used in this study are listed in the supplemental material (Data Sets S1 to S6).

The LdU6gRNAaH plasmid was generated as follows. (i) A 184-bp PCR fragment containing the 98-bp L. donovani RNA polymerase III U6 promoter was amplified from L. donovani genomic DNA with primers LdU6pF and LdU6pR, which also contains the LdMT gRNAa guide coding sequence, and digested with HindIII and BbsI. (ii) The fragment from step 1 was cloned into the HindIII and BbsI sites of the pSPneogRNAH plasmid (7) after removing the 180-bp L. donovani rRNA promoter to create the LdU6gRNAaH plasmid.

The humanU6gRNAa plasmid was generated as follows. (i) A 740-bp PCR fragment containing the human U6 promoter and gRNA coding sequence was amplified from plasmid pX330 (26) with primers pX330gRNAF1 and pX330gRNAR. (ii) The fragment from step 1 was digested and cloned into the HindIII and BamHI sites of pSPneo vector to create HumanU6gRNA plasmid. (iii) The LdMT gRNAa guide coding sequence was then inserted into HumanU6gRNA plasmid after BbsI digestion.

The pLdCH plasmid was generated as follows. (i) A 495-bp PCR fragment containing the 180-bp L. donovani rRNA promoter, the gRNA and HDV ribozyme coding sequences, and the 92-bp pyrimidine track was obtained from the pSPneogRNAH plasmid (7) with primers LdrRNApNde1 and pSPneoRHind3. (ii) The PCR fragment from step 1 was cloned into NdeI and HindIII sites of the pSP72 vector. (iii) The 6,595-bp HindIII and BglII fragment containing the Cas9 and hygromycin resistance genes from pLPhygCas9 (7) was subsequently cloned into the corresponding sites of the pSP72 vector in step ii to generate the pLdCH plasmid.

The pLdCN plasmid was generated as follows. (i) A 495-bp PCR fragment containing the 180-bp L. donovani rRNA promoter, the gRNA and HDV ribozyme coding sequences, and the 92-bp pyrimidine track was obtained from the pSPneogRNAH plasmid with primers LdrRNApXho1 and pSPneoRHind3. (ii) The PCR fragment from step i was cloned into the XhoI and HindIII sites of pSP72 vector. (iii) The 4.4-kb CAS9 fragment from the pLPhygCas9 plasmid was cloned into the HindIII and BamHI sites of the pLPneo vector (49) to generate pLPneoCas9. (iv) The 6.4-kb HindIII and BglII fragment containing the Cas9 and neomycin resistance genes from the pLPneoCas9 plasmid was subsequently cloned into the corresponding sites of the pSP72 vector in step ii to generate the pLdCN plasmid.

The gRNA 241510+MT(gRNAa) coexpression vector was generated by inserting the 360-bp HindIII and BamHI fragment, which contains the LdrRNAP and gRNA 241510 coding sequence from the pSPneogRNA241510H plasmid (7), into the HindIII and BglII sites of the pSPneoHHgRNAaH plasmid (7) after removing the 180-bp LdrRNAP sequence.

The gRNA A2a+b coexpression vector was generated as follows. (i) A 276-bp PCR fragment containing gRNAA2a, HDV, and hammerhead ribozymes and the gRNAA2b guide coding sequences was amplified with primers Ld220670a and Ld220670b from the gRNA 241510+MT coexpression vector. (ii) The PCR product from step i was digested with BbsI and inserted into the BbsI-digested pSPneogRNAH vector.

The gRNA A2a+b+MT(gRNAa) triple expression vector was generated by inserting the 579-bp HindIII and BamHI fragment which contained LdrRNAP, gRNA A2a+b, and ribozyme coding sequences from the gRNA A2a+b coexpression vector into the HindIII and BglII sites of pSPneoHHgRNAaH after removing the 180-bp LdrRNAP sequence.

gRNA and primer design and synthesis.

Since current gRNA design tools developed from data for higher-order eukaryotic cells are not necessarily suitable for Leishmania (7), we selected gRNA guide sequences based on the relatively high activity scores in all of the following three design programs and no off-target site. The Eukaryotic Pathogen CRISPR guide RNA design tool (EuPaGDT) (http://grna.ctegd.uga.edu/) provides useful information on off-target sites and microhomology sequences flanking the DSB (9). The microhomology sequences are required for MMEJ but should be avoided if a donor will be used. Sequence scan for CRISPR (SSC) (based on human and mouse data; http://crispr.dfci.harvard.edu/SSC/) and CRISPRscan (based on zebrafish data; http://www.crisprscan.org/?page=sequence) are helpful for predicting gRNA activity based on the guide sequence alone (50, 51).

The primers for PCR were selected manually or via Primer3 (http://bioinfo.ut.ee/primer3/). All primers and oligonucleotides used in this study were ordered from Alpha DNA (Montreal, Canada).

Other experimental procedures.

The following experimental procedures were performed as previously described: single gRNA guide sequence cloning into various gRNA expression vectors (7); Leishmania transfection and a limiting dilution assay to determine miltefosine resistance rates (7); Leishmania genomic DNA extraction, PCR, and sequencing analysis (7); A2 Western blot analysis (14).

RAD51 inhibition assay.

The stock solutions (10 mM) of Rad51 inhibitors B02 and RI-1(catalog numbers SML0364 and 1274; Sigma) were prepared in dimethyl sulfoxide and stored at 4°C (42,–44). Immediately before use, the stock solutions were diluted with Leishmania culture medium to a 1 mM working concentration for RI-1 and 100 µM for B02. These inhibitors were then directly added into Cas9 and LdMT gRNAc-expressing Leishmania culture (1 × 106 promastigotes per ml) to a final concentration 0 to 20 µM for B02 and 0 to 100 µM for RI-1. The proper concentrations of these inhibitors in culture were maintained by adding fresh inhibitors once every 3 days for a total 4 times in a 2-week period before measuring the miltefosine resistance rate.

Generation of LdMT+/− single-knockout cells via the traditional gene replacement method.

The specific LdMT bleomycin-targeting fragment was generated by overlapping PCR. (i) The 666-bp LdMT 5′ flanking sequence with primers Ld1315905′F and Ld1315905′R, the 534-bp bleomycin expression cassette with primers 131590BleF and 131590 BleR, and the 656-bp LdMT 3′ flanking sequence with primers Ld1315903′F1 and Ld1315903′R were PCR amplified separately. (ii) The three PCR fragments from step i were mixed and used as PCR template for primers Ld1315905′F and Ld1315903′R to generate the specific 1,857-bp LdMT bleomycin-targeting fragment. L. donovani promastigotes transfected with this LdMT bleomycin-targeting fragment were selected with 100 µg/ml phleomycin. The LdMT+/− single-knockout clones were verified by PCR with primer pair Ld1315905′F1 and 131590BleR.

Accession number(s).

The pLdCN, pLdCH, and pSPneogRNA241510+MT plasmids have been deposited in Addgene under numbers 84290, 84291, and 84292, respectively. The partial sequences for these plasmids are provided in the supplemental material (Data Sets S3 and S5).

Article TitleOptimized CRISPR-Cas9 Genome Editing forLeishmaniaand Its Use To Target a Multigene Family, Induce Chromosomal Translocation, and Study DNA Break Repair Mechanisms

Abstract

L. donovani1 S/Cl2D,L. majorFriedlin v9, andL. mexicana(MNYC/BZ/62/M379) used in this study were 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−1hemin, 1 mg liter−1biotin, 1 mg liter−1biopterine, 50 U ml−1penicillin, and 50 µg ml−1streptomycin. Cultures were passaged to fresh medium at a 20-fold dilution once a week.Leishmaniacells after transfection with the CRISPR vector and donor DNA were sometimes cultured at 33°C or 37°C for 2 to 3 days to improve gene-editing efficiency (7).


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