Home › Microbiology › QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii
Microbiology JoVE (Open Access) Citable · DOI

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

DOI: 10.3791/55185-v
What you'll learn
  • ✓ Apply QTL mapping with whole genome sequencing to identify drug resistance genes
  • ✓ Design and execute CRISPR/Cas9 editing to verify genomic targets in parasites
  • ✓ Assess drug resistance phenotypes in Toxoplasma gondii progeny crosses
Protocol

Details are presented on how QTL mapping with a whole genome sequence based genetic map can be used to identify a drug resistance gene in Toxoplasma gondii and how this can be verified with the CRISPR/Cas9 system that efficiently edits a genomic target, in this case the drug resistance gene.

Difficulty
advanced
Total time
~4–6 weeks (includes parasite crosses, progeny screening, genome sequencing, CRISPR construct design/cloning, transfection, clone isolation, and phenotypic validation)
Model organism
Toxoplasma gondii
Biosafety
BSL-2

Steps

1
Assess drug resistance in parasite progeny

Screen progeny from ME49-FUDRr × VAND-SNFr cross for sinefungin resistance phenotypes. Quantify resistance levels in the segregating population to establish baseline genetic variation.

▶ 00:52
2
Design and introduce CRISPR/Cas9 mutations

Create CRISPR/Cas9 constructs targeting the candidate drug resistance gene. Transfect into parasites and select for stable integration to induce targeted genomic mutations.

▶ 02:30
3
Verify CRISPR/Cas9 mutations by sequencing

Clone edited parasites and perform DNA sequencing to confirm mutation induction at the target locus. Validate that expected genetic changes are present.

▶ 06:20
4
Interpret QTL mapping and CRISPR results

Integrate QTL mapping data with CRISPR/Cas9 editing outcomes to confirm SNR1 as the sinefungin resistance gene. Correlate genotype changes with phenotypic resistance loss or gain.

▶ 08:33
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