The overall goal of this quantitative trait locus mapping and CRIPR/Cas9 genome editing methodology, is to identify and subsequently verify a gene involved in sinefungin resistance in Toxoplasma. These methods can help answer key questions in parasitology, such as, the genetic bases for variants, pathogenesis and drug resistance. The main advantage of these techniques is the use of whole genome sequencing data for precise QTL analysis and CRISPR/Cas9 genome editing for efficient genetic manipulation in Toxoplasma.

Demonstrating the procedure will be Dr.Robin Powell, a research associate from my laboratory. The Toxoplasma gondii parasites are grown in confluent human foreskin fibroblast or HFF cells, seeded to T25 flasks with D10 medium. To test individual progeny clones for sinefungin resistance, grow each clone to high parasite density until a majority of the parasites begin to lyse the host cells.

After preparing the parasite solution as described in the text protocol, use a pipetter, to pass 2.5 times 10 to the fifth parasites to a new T25 HFF culture flask containing D10 medium, with a 0.3 micromolar working concentration of the sinefungin drug. Grow the parasites at 37 degree celsius and 5%CO2 for seven to 10 days. Observe the HFF cell monolayer under an inverted phase contrast microscope and score the progeny for the growth phenotype in sinefungin drug.

For example, score zero for no growth and score one for growth and lysis of the monolayer. Once all progeny have been scored, the data will be used as a binary phenotype for quantitative trait locus or QTL mapping. To identify the causal mutation, whole genome sequencing reads of the progeny are aligned to the sinefungin sensitive van strand reference genome, SNPs are called and the QTL locus is scanned for a sinefungin resistance associated SNP.

Confirmation that SNR1 is the sinefungin resistance gene will be performed by inactivating SNR1 in a wild type sinefungin sensitive background using CRISPR/Cas9 induced indel mutations. To construct the SNR1 specific CRISPR plasmid, the UPRT targeting CRISPR plasmid is used as a template in PCR based site directed mutagenesis using specific designed primers. The mutagenesis products containing the gene of interest specific CRISPR plasmids are transformed into E.Coli cells.

Individual clones are then picked and grown in LB medium and the extracted plasmids are analyzed by DNA gel electrophoresis to check their size. Plasmids are subsequently sequenced to confirm the target specific guide RNA sequence. When the target specific CRISPR plasmid is introduced into a Toxoplasma cell, the guide RNA directs the Cas9 nucleus to the target to cause a double strand DNA break out of specific locus in the target.

The DNA break is then fixed by error prone, non-homologous and joining activity, leading to indel mutations in the target gene. Two to three days before transvecting parasites with the SNR1 specific CRISPR plasmid, add enough parasites to a T25 flask containing a confluent HFF cell monolayer to achieve 70%to 80%HFF cell lysis within two to three days. Examine the culture under an inverted phase contrast microscope to confirm the HFF monolayer is 70%to 80%lysed by the parasites.

Gently remove the medium with a pipette and wash the cells off the flask's surface with five milliliters of cytomix buffer. Transfer the parasite solution to a 15 milliliter conical tube. Pass the parasite solution through a 10 milliliter syringe with a 22 gauge blunt needle, two to three times.

To remove HFF cells and cellular debris, filter the parasite solution through a membrane with a pore size of three microns into a new conical tube. Palette the filtered parasites by centrifugation at 400 times G for 10 minutes. Pour off the supernatant and re-suspend the paletted parasites with 10 milliliters of cytomix buffer.

Remove a 10 microliter aliquot, to determine the parasite concentration with a hemocytometer. Palette the rest of the suspension again by centrifugation at 400 times G for 10 minutes. Remove the supernatant and re-suspend the palette inside the mix buffer to obtain a density of four times 10 to the seventh parasites per milliliter.

In a four milliliter gap cuvette, mix 250 to 300 microliters of the parasite solution with 7.5 micrograms of CRISPR plasmid, six microliters of ATP and six microliters of glutathione. Electroporate the parasites, following a standard procedure for T gondii transvection. Include a separate electroporation as a negative control, in which a CRISPR plasmid targets elsewhere.

Grow the electroporated parasites in a T25 flask, seeded with HFF cells at 37 degree celsius for two days. To begin the procedure for examining the induced mutations, first, replace the medium in the flask with five milliliters of D10, containing 0.3 micromoles of sinefungin. Keep the parasites and the selection medium for at least three passages until the resistant pool becomes stable.

As indicated by the absence of parasite growth in the negative control group, but robust parasite growth in the experimental group. Subclone the sinefungin resistant pool to obtain colonial strands. Collect freshly egress parasites purify by three micron membrane filtration, and subclone into 96 well plates, seeded with confluent HFF cells in 150 microliters of D10 medium.

Grow the subcloning cultures in a CO2 incubator at 37 degree celsius for seven to 10 days, without disturbing the plates. After seven to 10 days, check the 96 well plates under an inverted phase contrast microscope. Look for wells that contain only one plaque and mark such wells.

Transfer the cells from each well to 24 well plates, seeded with HFF cells. Incubate at 37 degrees celsius. When parasites begin to lyse the monolayer, pass 50 microliters of the parasite solution to a new well to maintain the strain, and harvest the rest for genomic DNA isolation.

Palette the rest of the parasite solution at 1000 times G for 10 minutes. Wash the paletted parasites with PBS and palette them again. Isolate genomic DNA from paletted cells using a commercial kit or by boiling and re-suspend parasites in 50 to 100 microliters of PBS.

Finally, perform PCR as described in the text protocol to obtain a fragment of SNR1 gene for sequencing. Progeny of the cross-derived, using parental FUDR resistant ME49 and sinefungin resistant van strains, were accessed for resistance to the drug, sinefungin as indicated by growth and lysis of the HFF monolayer. A QTL scan resulted in one significant peak on chromosome nine, spanning approximately one MBP.

The causal mutation is located in this region. Whole genome sequencing led to the identification of the mutation. Progeny SNPs within the QTL locus, were imported into a spreadsheet and scanned for a pattern where the sinefungin resistant progeny shown in yellow, have an SNP and the sinefungin sensitive progeny, shown in green, do not.

Only one SNP indicated in red, matched to this pattern that results in an early stop codone in a putative amino acid transporter gene named, SNR1. When the SNR1 targeting CRISPR case nine plasmid was electroporated into a sinefungin sensitive wild type parasite strain, resistant mutants were obtained when cultured in sinefungin. In contrast, no sinefungin resistant parasites were obtained with a CRISPR plasmid that targeted elsewhere.

Several sinefungin resistant CRISPR mutants were cloned in the region around the SNR1 guide RNA target was sequenced. Representative of results from the RH wild type strain and C5 and C6 sinefungin resistant mutants show that, each mutant had an indel that disrupted the coding sequence of the SNR1 gene. Don't forget that working with Toxoplasma gondii can be hazardous, as it is infectious to humans.

Precaution should be taken during this procedure, such as the proper handling of sharps that may come into contact with the parasite. Following this procedure, other methods like CRISPR/Cas9 mediated site specific exogenous gene insertion can be performed, in order to answer additional questions like, genetic complementation or transgenic parasite construction. After its development, this technique paved the way for researchers in the field of molecular parasitology, to explore the complex biology and mechanisms of pathogenesis in Toxoplasma gondii.

After watching this video, you should have a good understanding of how to perform QTL analysis, using genome sequencing data. As well as, genetic metabulation of Toxoplasma gondii using CRISPR/Cas9 gene editing.

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