Materials and Methods

Single-Strand Annealing Plays a Major Role in Double-Strand DNA Break Repair following CRISPR-Cas9 Cleavage inLeishmania

MATERIALS AND METHODSLeishmania strains and culture medium. L. donovani 1S/Cl2D, L. major Friedlin V9, and L. mexicana (MNYC/BZ/62/M379) were used in this study. The culture medium was 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 biopterin, 50 U ml−1 penicillin, and 50 μg ml−1 streptomycin. Leishmania promastigotes were routinely cultured at 27°C and passaged to fresh medium at a 40-fold dilution once a week.The primers and oligonucleotide donors used in this study are listed in Table S1 in the supplemental material.Plasmid construction. The pSPneoSagRNAH vector was generated as follows. (i) Primers LdrRNApxho1 and LdrRNApR were used to obtain the 201-bp PCR product of the L. donovani rRNA promoter; primers SagRNAF and SagRNAR were used with the pX601 (Addgene) plasmid as the template to obtain the 144-bp PCR product of the sequence encoding S. aureus gRNA (SagRNA). Primers HDVRiboF1 and pSPneoRHind3 were used to obtain the 195-bp PCR product containing the hepatitis delta virus (HDV) ribozyme-coding sequence. (ii) Primers LdrRNApXho1 and pSPneoRHind3, along with the 3 PCR products from the first step, were used as the templates (they contained 22- to 26-bp overlapping sequences) to get the 492-bp PCR product, which consisted of the L. donovani rRNA promoter, SagRNA, and the HDV ribozyme-coding sequences. (iii) The 492-bp PCR product from the second step was digested with XhoI and HindIII and cloned into the corresponding sites of the pSPneo plasmid (75) to generate pSPneoSagRNAH.The pNeoSagRNAH vector was generated as follows. (i) The pLPneo2 plasmid (76) was digested with HindIII and BglII to get the P-neo fragment. (ii) The P-neo fragment from the first step was inserted into the pSPneoSagRNAH vector after removing the neomycin resistance gene (neo) fragment with HindIII and BglII to generate a new SagRNA expression vector, pNeoSagRNAH.The pLdSaCN vector was generated as follows. (i) With the pX601 plasmid as the template, primers pxSaCas9F1, pxSaCas9R1, pxSaCas9F2, and pxSaCas9R2 were used to obtain the 3,265-bp PCR product of the SaCas9-coding sequence with the internal HindIII site destroyed by a silent point mutation. This SaCas9 sequence derived from plasmid pX601 (catalog number 61591; Addgene) was humanized by use of a 53% GC content and contains the nuclear localization signal of the simian virus 40 (SV40) large T antigen at its N terminus and the bipartite nuclear localization signal from nucleoplasmin at its C terminus (25). (ii) The SaCas9 fragment from the first step was inserted into the HindIII and BamHI sites of the pNeoSagRNAH vector described above to generate the SagRNA and SaCas9 coexpression vector pLdSaCN.The pLPhygSaCas9 vector was generated by inserting the above-described 3,265-bp SaCas9-coding sequence into the HindIII and BamHI sites of the pLPhyg2 plasmid (76).The L. donovani DNA Polθ helicase expression vector was constructed as follows. (i) The L. donovani DNA Polθ helicase gene was PCR amplified from L. donovani genomic DNA with primers Ld231640F and Ld231640R to get the 6,723-bp gene fragment. (ii) The PCR fragment was digested with HindIII and BglII and ligated into the HindIII and BamHI sites of pLphyg2 to generate the Polθ helicase expression vector.gRNA design. The gRNAs used in this study were designed manually or with the aid of the following gRNA design programs: the Eukaryotic Pathogen CRISPR guide RNA Design Tool (EuPaGDT; http://grna.ctegd.uga.edu/), which was developed on the basis of data from mammalian cells and ranks a gRNA on the basis of its activity score, off-target sites in the genome, and microhomology sequences flanking the DSB (53); Sequence Scan for CRISPR (SSC; http://cistrome.org/SSC/) and CRISPRater (https://crispr.cos.uni-heidelberg.de/), which were developed from human and mouse data and which can predict gRNA activity with only the guide sequence information (55, 56); and CRISPRscan (http://www.crisprscan.org/), which was developed on the basis of zebrafish data (54). All the gRNA sequences used in this study are listed in Table S2.Cloning of the gRNA guide-coding sequence into Leishmania CRISPR vector pLdCN or pLdSaCN. Single gRNA guide-coding sequences were ordered as standard oligonucleotides with 5′-TTGT and 5′-AAAC overhangs. It is important to note the optimal guide length for SpCas9 gRNA is 19 or 20 nt, but for SaCas9 it is 21 nt. After phosphorylation and annealing, the gRNA guide-coding sequences were ligated into the BbsI sites of the pLdCN or pLdSaCN CRISPR vector (19) (Fig. S5). The pLdCN366140MT vector, containing gRNA-coding sequences targeting {"type":"entrez-nucleotide","attrs":{"text":"Ld366140","term_id":"958056775","term_text":"LD366140"}}Ld366140 and the MT gene, was constructed as follows: primers Ld366140a and LdMTb and the pSPneogRNA241510+MT plasmid, which was used as the template, were used to get the 276-bp PCR product, which was then digested with BbsI and cloned into the corresponding BbsI sites of the pLdCN vector.FIG S5Leishmania CRISPR vector pLdSaCN and its partial sequence. (A) Schematic of pLdSaCN and its guide sequence insertion site. rRNAP, L. donovani ribosomal RNA promoter; H, HDV ribozyme. The small black box represents the 92-bp pyrimidine track. The first nucleotide of gRNA, U, is highlighted (black), as L. donovani rRNAP initiates transcription at the T residue site. The SP6 promoter (indicated) can be used as the sequencing primer to confirm the insertion. The drawing is not to scale. (B) The partial sequence of pLdSaCN, which includes the 180-bp rRNAP sequence in black and its T transcription initiation site in bold, the 83-bp SaCas9 binding RNA-coding sequence in green, and the 68-bp HDV ribozyme-coding sequence in blue. The restriction enzymes XhoI and BbsI are highlighted in red. Note that the guide sequence insertion sites for Leishmania CRISPR vectors pLdCN, pSPneoSagRNAH, and pNeoSagRNAH are the same as those for the pLdSaCN vector. All vectors are ampicillin resistant. Download FIG S5, TIF file, 0.6 MB.Copyright © 2019 Zhang and Matlashewski.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.Parasite transfection. Leishmania promastigotes (2 × 107; middle log phase to early stationary phase) in 100 μl Tb-BSF buffer (90 mM Na2HPO4, 5 mM KCl, 0.15 mM CaCl2, 50 mM HEPES, pH 7.3) and 2 to 5 μg CRISPR or other vectors were used for each transfection with the Lonza Nucleofector 2b device (program U33). The transfected Leishmania promastigotes were selected with 50 μg/ml G418 or 100 μg/ml hygromycin on the following day. Once the CRISPR vector-transfected cell culture was established, these cells could subsequently be transfected with the donors; 8 μl 100 μM oligonucleotide donor or 2 to 4 μg of purified PCR product was used for each transfection. For a drug resistance gene donor, drug selection was started 2 to 3 days after donor transfection to allow time for donor integration into the genome. Phleomycin (50 μg/ml) was initially added, and later, phleomycin was added to 100 μg/ml if the bleomycin resistance gene donor was used. These Leishmania cultures could sometimes be incubated at 33°C or 37°C for 2 to 3 days to improve gene editing efficiency (18).Determination of MLF resistance rate and cloning of MLF-resistant cells. For Leishmania promastigotes transfected with various CRISPR vectors expressing MT-targeting gRNA, the MLF resistance rate was determined by limiting dilution culture containing 40 μM MLF in 96-well plates. Depending on the estimate of the MLF resistance rate, 2,000 to 8 million Leishmania promastigotes per well were inoculated into the first column of 96-well plates in quadruplicate or all eight wells for each transfected cell line. The cells in the first column were then serially 2-fold diluted from left to right up to the last column in the plate. The MLF resistance rates (or the diallelic MT gene mutation frequencies) were calculated after the plates were sealed and incubated in a 27°C incubator for 2 to 3 weeks. The surviving Leishmania cell clones in the farthest wells after MLF selection were expanded in 24-well plates for subsequent genomic DNA extraction and PCR analysis. To increase the cloning efficiency, Leishmania cells were sometimes limited diluted in a direction from the top to the bottom rows of the plates, or the cells from the whole MLF-resistant population selected in a 4- to 10-ml culture flask at 3 days after MLF selection were diluted and directly inoculated into 96-well plates at 1 MLF-resistant cell per 100 μl culture medium per well.Bioinformatics analysis. The repeat sequences flanking a target gene were initially identified by alignment of the up- and downstream intergenic sequences progressively away from the target gene. The located repeat sequence was then used to perform a BLAST search against the Leishmania genome to identify the additional repeat sequences in the respective chromosome flanking the target gene. Since there are still some gaps and assembly errors in the L. donovani BPK282A1 genome sequence in TriTrypDB (http://tritrypdb.org/tritrypdb/), except for the gene identifiers, the complete sequences of these repeats described in this study and their chromosome locations were derived from the complete L. donovani genome sequence that we recently reported (77) and were verified by comparison with the L. infantum, L. major, and L. mexicana genome sequences (78). Primers flanking these repeats were designed using the Primer3 program (http://bioinfo.ut.ee/primer3-0.4.0/). The optimal primer length was 20 nucleotides with a 60°C melting temperature.Genomic DNA preparation and PCR analysis. The parasite genomic DNAs were extracted from WT Leishmania promastigotes and various MLF-resistant cells with the minipreparation method (79), which includes phenol-chloroform extraction and ethanol precipitation. The purity and quantity of these genomic DNA were assessed by use of a NanoDrop spectrophotometer.The various Taq DNA polymerases used in this study included 2× KAPA Taq HotStart DNA polymerase mix (Sigma-Aldrich), All in One 2× Green PCR master mix (ZmTech Scientific, Montreal, Canada), 2× DreamTaq Green PCR master mix and 2× Platinum SuperFi PCR master mix (Thermo Fisher Scientific), and Q5 high-fidelity DNA polymerase (New England BioLabs). The PCR program was set up according to the manufacturer’s instructions with variations in annealing temperature, extension time, and the total number of PCR cycles. For semiquantitative PCR, an equal amount of genomic DNA (350 ng) was used for each 12-μl PCR mixture, with the total number of cycles ranging from 22 to 35. The PCR products were separated in a 1 to 1.5% agarose gel. The putative MMEJ-, SSA-, or linear duplication-specific bands were extracted from the gel and sent to the Genome Quebec Sequencing Center for confirmation by sequencing.Southern blot analysis. The same amount of genomic DNA extracted from WT- and pLdSaCNgRNAg-, pLdSaCNgRNAh-, and pLdSaCNgRNAi-transfected MLF-resistant cells was digested to completion with the restriction enzyme PstI and separated on a 0.8% agarose gel (5 μg per lane). After denaturation and neutralization, the DNA was transferred to a nylon membrane with 20× SSC (3 M NaCl and 0.3 M Na3 citrate) and a stack of paper towels overnight. The membrane was prehybridized at 42°C for 2 h in a prehybridization solution, consisting of 1% SDS, 1 M NaCl, 10% dextran sulfate, and 50 μg/ml denatured salmon sperm DNA (0.2 ml/cm2). The primer pair Ld131610F3 and Ld131610R3 was used to generate the 615-bp probe, which was biotin labeled using a Thermo Scientific biotin DecaLabel DNA-labeling kit. The membrane was then incubated overnight at 42°C in hybridization solution, containing 1% SDS, 1 M NaCl, 10% dextran sulfate, and 50 ng/ml the biotin-labeled probe. The membrane was washed twice with 2× SSC, 0.1% SDS for 10 min each time at room temperature and with 0.1× SSC, 0.1% SDS once for 20 min at 65°C. The biotin-labeled DNA probe hybridized to the target DNA on the membrane was detected with alkaline phosphatase-conjugated streptavidin and the substrate BCIP-T (5-bromo-4-chloro-3-indolylphosphate, p-toluidine salt) using a biotin chromogenic detection kit (Thermo Scientific).Data availability. The pLdSaCN, pLPhygSaCas9, pSPneoSagRNAH, and pNeoSagRNAH plasmids have been deposited in Addgene with accession no. 123261, 123262, 123265, and 123266, respectively.

Article TitleSingle-Strand Annealing Plays a Major Role in Double-Strand DNA Break Repair following CRISPR-Cas9 Cleavage inLeishmania

Abstract

The pLdSaCN, pLPhygSaCas9, pSPneoSagRNAH, and pNeoSagRNAH plasmids have been deposited in Addgene with accession no.123261,123262,123265, and123266, respectively.


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