Gene editing construct
The gene encoding the allograft inflammatory factor of B. glabrata, BgAIF (2,226 bp; accession number BGLB005061, https://www.vectorbase.org/) includes five exons interrupted by four introns (Fig. 1a). A guide RNA (gRNA) for Cas9-catalyzed gene editing specific for the target B. glabrata gene locus, BgAIF, was identified in the BGLB005061 sequence using the ‘CHOPCHOP’ v3 tool, https://chopchop.cbu.uib.no/, with default parameters compatible for the protospacer adjacent motif, NGG, of Cas9 from Streptococcus pyogenes (49-51) and screened for off-target sites against the Biomphalaria glabrata genome (29). Based on the guidance from the CHOPCHOP analysis, we chose the top ranked guide RNA (gRNA), AGACTTTGTTAGGATGATGC, specific for exon 4 of the AIF gene, with predicted high CRISPR/Cas9 efficiency for double-stranded cleavage in tandem with an absence of off-target activity in the genome of B. glabrata (Fig. 1a). A CRISPR/Cas9 vector encoding the gRNA targeting exon 4 of BgAIF under the control of the mammalian U6 promoter and encoding Cas 9, with nuclear localization signals 1 and 2, driven by the human cytomegalovirus (CMV) immediate early enhancer and promoter was assembled using the GeneArt CRISPR Nuclease Vector system (Thermo Fisher Scientific, Waltham, MA), according to the manufacturer’s protocol. Briefly, the 20 nt of either target (including ‘gtttt’ on the 3’ end) or complementary to target (including ‘cggtg’ on the 3’end) sequences were synthesized commercially, and annealed according to manufacturer’s protocol. The annealed double strand DNA (dsDNA) was ligated into the linearized GeneArtÒ CRISPR Nuclease vector via BamHI and BsmBI restriction sites, respectively, and the construct was termed pCas-BgAIFx4 (Fig. 1b). (The sequence of GeneArt CRISPR nuclease vector backbone is available at https://www.thermofisher.com/order/catalog/product/A21174#/A21174). Chemically competent TOP10, E. coli cells (Invitrogen, Thermo Fisher Scientific) were transformed with pCas-BgAIFx4 by the heat shock method and cultured on LB-agar supplemented with ampicillin at 100 µg/ml. Subsequently, the integrity of the recombinant plasmids from several single colonies of ampicillin-resistant E. coli transformants was confirmed by amplicon PCR-based Sanger direct nucleotide sequence analysis using a U6 gene-specific primer for gRNA ligation and orientation (Fig. 1b).
Biomphalaria glabrata embryonic (Bge) cell line culture
The Bge cell line was provided by the Schistosomiasis Resource Center (SRC), Biomedical Research Institute (BRI), Rockville, MD. Historically, the Bge cell line was sourced by the SRC from the American Type Culture Collection (Manassas, VA), catalog no. ATCC CRL 1494, and thereafter maintained at BRI for >10 years. Bge cells were maintained at 26ºC in air in ‘Bge medium’, which is comprised of 22% (v/v) Schneider’s Drosophila medium, 0.13% galactose, 0.45% lactalbumin hydrolysate, 0.5% (v/v) phenol red solution, 20 µg/ml gentamycin, and supplemented with 10% heat-inactivated fetal bovine serum (24, 52). Bge cells were grown to 80% confluence before transfection by electroporation with pCas-BgAIFx4. The Bge cells were free of contamination with Mycoplasma, as established with a PCR-based test (LookOut® Mycoplasma PCR Detection kit, Sigma-Aldrich, St. Louis, MO).
Transfection of Bge cells by square wave electroporation
Bge cells were harvested using a cell scraper, washed twice in Bge medium, counted, and resuspended at 20,000 cell/µl in Opti-MEM medium (Sigma-Aldrich, St. Louis, MO). Two million cells were transferred into 0.2 mm path length electroporation cuvettes (BTX Harvard Apparatus, Hollister, MA) containing 6 µg pCas-BgAIFx4 in ~100 µl Opti-MEM. The cells were subjected to electroporation using one pulse at 125 volts for 20 milliseconds, using a square wave pulse generator (ECM 830, BTX Harvard Apparatus). Immediately thereafter, the Bge cells were maintained in 12-well plates (Greiner Bio-One) at 26ºC. The mock control included Opti-MEM only for electroporation. The presence of transcripts encoding the B. glabrata actin and the Cas9 was monitored daily for nine days following transfection by electroporation (Fig. 1c).
Sequential isolation of total RNA and genomic DNA
To monitor the transfection of Bge cell by pCas9-BgAIFx4, we investigated the expression of Cas9 in Bge cells by reverse transcription PCR (RT-PCR). Both total RNA and genomic DNA were extracted sequentially from cell pellets, as described (53, 54). In brief, each sample of total RNA sample was extracted using the RNAzol® RT reagent (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer’s protocol. Subsequently, the DNA/protein pellet retained after recovery of RNA was resuspend in DNAzol® solution (Molecular Research Center, Inc), from which total DNA was recovered. The RNAs and DNAs were dissolved in nuclease-free water and their concentration and purity established by spectrophotometry (Nanodrop 1000, Thermo Fisher Scientific).
Expression of Cas9 in Bge cells
To investigate transcription from the pCas-BgAIFx4 vector following transfection of Bge cells, levels of transcribed Cas9 were investigated by semi-quantitative RT-PCR. The Cas9-specific primers were Cas9-F, 5’- agcatcggccttgatatcgg-3’ and Cas9-R, 5’- agaagctgtcgtccaccttg-3’ (Fig. 1b). Total RNA from the non-transfected, mock (Opti-MEM electroporated-), and pCas-BgAIFx4 DNA electroporated-Bge cells were treated with DNase I (Ambion, Thermo Fisher Scientific) to digest any residual vector pCas-BgAIFx4 DNA and contaminating genomic DNAs. The RNAs were reverse transcribed to cDNA using ProtoScript II reverse transcriptase with oligo dT and random primers (First Strand cDNA Synthesis kit New England Biolabs, Ipswich, MA). RT-PCRs specific for the Cas9 or actin gene of B. glabrata, BgActin (GenBank accession number U53348.1) were undertaken, with BgActin serving as the positive control for RNA integrity. The primer pairs used for the BgActin coding sequences were termed actin-F, 5’-aagcgacgttttcttggtgc-3’ and actin-R, 5’- acccataccaaccatcacacc-3’. Amplicons and molecular size standards were separated by electrophoresis through Tris-acetate-EDTA-buffered agarose 1% stained with ethidium bromide (Fig. 1c).
Analysis of programmed mutation of the allograft inflammatory factor gene of B. glabrata
Genomic DNA samples from the mock-transfected and pCas-BgAIFx4-transfected cells were amplified by PCR using AIF-F (5’-gcagatttgcaattcaacactta-3’) and AIF-R (5’-tgccagctagcttactgcat-3’) primers that flank the CRISPR/Cas9 programmed double-stranded break (DSB) site (Fig. 1a). Amplicons of 568 nt in length (from residues 489 to 1,056 of the BgAIF_BGLB0055061 gene) were obtained using the AIF-F and -R primer pair. Amplicons were isolated from the agarose gel using the PCR cleanup and gel extraction kit (ClonTech, Takara USA, Mountain View, CA) and the nucleotide sequence of amplicons determined by Sanger direct sequencing (GENEWIZ, South Plainfield, NJ). Chromatograms of the sequence reads from the control and experimental groups in each replicate experiment were subjected to online analysis using the TIDE algorithm, https://tide.deskgen.com/ (55, 56) and also using the Inference of CRISPR v2 Edits analysis (ICE) software, https://ice.synthego.com/#/ (Synthego Corporation, Redwood City, CA) (57). Estimates of CRISPR efficiency, insertion-deletion (INDEL)-substitution percentages, and the nucleotide sequence of mutant alleles were obtained using both the TIDE and the ICE platforms (55, 56) (Fig. 2a, 2b).
Quantitative real time PCR analysis of transcription of BgAIF
To evaluate the differential levels of the BgAIF transcript among the control and experimental groups, total RNAs were extracted and treated with DNase I, as above. DNase I treated-RNA (200 ng) was reverse transcribed to cDNA, followed by quantitative RT-PCR, using the ViiA7 Real Time PCR System (Applied Biosystems, Scientific), and the SSoAdvanced Universal SYBR Green Supermix reagents (Bio-Rad), according to the manufacturer’s recommendations. The following nucleotide primers used BgAIF gene-specific primers amplify 119-257 nt of BgAIF GenBank accession number EX001601.1; BgAIF-rt-F, 5’-cctgcttttaacccgacaga-3’ and BgAIF-rt-R, 5’-tgaatgaaagctcctcgtca-3’. Differential BgAIF gene expression were calculated after normalizing with BgActin (primers as above) and comparison with the non-treated (control) cells. The ΔΔCt method was used to calculate the differential gene expression (58), with assistance of the GraphPad Prism 8 software (San Diego, CA) (Fig. 2c).
Miracidia of the NMRI strain of S. mansoni were hatched from eggs recovered from livers of schistosome infected mice (Schistosomiasis Resource Center, Biomedical Research Institute, Rockville, MD) under axenic conditions (28), primary sporocysts were transformed from the miracidia in vitro, as described (26). Briefly, miracidia were immobilized by chilling on ice for 25 min, following by pelleting using centrifugation, 500´g at 4°C, 60 sec. The miracidia were washed with ice cold Chernin’s balanced salt solution (28 mM NaCl, 0.5mM Na2HPO4, 2mM KCl, 1.8mM MgSO4.7H2O, 0.6mM NaHCO3, 3.6 mM CaCl2.2H2O) with 1 mg/ml glucose, trehalose, and antibiotic, 10 µl/ml of 100×´ penicillin/streptomycin (Thermo Fisher Scientific), termed CBSS+. Approximately 5,000 miracidia per well of 24-well plates were cultured in CBSS+ at 26°C for 24 hrs, after which the sporocysts were washed to remove shed ciliated epidermal plates and other debris, followed by transfer to a 1.5 ml microcentrifuge tube (26).
Sporocyst-Bge cell binding assay and cell adhesion index (CAI)
To investigate the if BgAIF would affect the ability of cell adhesion to S. mansoni sporocyst, we co-cultured the non-transfected Bge cell or non-selected-, transfected-pCas-BgAIFx4 cells (BgAIF depleted- cells named ‘ΔBgAIF-Bge’) with in vitro transformed sporocysts, then the cell adhesion index (CAI) were calculated as described (26, 59). With the limitations in this study, we were not be able to select or enrich for BgAIF edited-cells, and hence the ΔBgAIF-Bge cell populations can be considered to be a population of gene mutated mixed with non-modified (wild type) cells. CAI is a semi-quantitative method of cell adhesion to primary sporocysts using the four categories of scores ranging from one to four - lower to higher numbers of cells adherent to the parasite’s surface. In brief, we mixed single cell suspensions of 500,000 Bge cells with 200 freshly prepared-sporocysts (total volume 200 µl of CBSS+) in sterile, siliconized tubes (Bio Plas, Thomas Scientific, Swedesboro, NJ). The Bge cell-sporocyst co-culture was maintained at 26°C for 24 hrs. Cellular morphology and adhesion of the cells to the surface of the sporocysts was monitored and recorded using an inverted microscope, at 20× magnification (Zeiss Axio Observer A1, Carl Zeiss LLC, White Plains, NY) after gently transferring the parasite-cell suspension to the tissue culture plate (Greiner Bio-One). Scoring of the adherence index was carried out in a blinded fashion to the investigator reading the score; ³50 sporocysts from each experimental group were counted each time, and triplicates of each treatment group were scored. Seven independent biological replicates of this CAI-based sporocyst-Bge cell binding assay were carried out. In total, ³400 sporocysts were examined from each treatment and control group. Averages for the CAI values were calculated from the cell adhesion scores ranging from 1 to 4 (examples presented in Fig. 3a) according to the formula, CAI = total binding value per number of sporocysts (26).