Single- and duplex TaqMan-quantitative PCR for determining the copy numbers of integrated selection markers during site-specific mutagenesis in Toxoplasma gondii by CRISPR-Cas9.

Hänggeli, Kai Pascal Alexander; Hemphill, Andrew; Müller, Norbert; Schimanski, Bernd; Olias, Philipp; Müller, Joachim; Boubaker, Ghalia (2022). Single- and duplex TaqMan-quantitative PCR for determining the copy numbers of integrated selection markers during site-specific mutagenesis in Toxoplasma gondii by CRISPR-Cas9. PLoS ONE, 17(9), e0271011. Public Library of Science 10.1371/journal.pone.0271011

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Herein, we developed a single and a duplex TaqMan quantitative PCR (qPCR) for absolute quantification of copy numbers of integrated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) drug selectable marker for pyrimethamine resistance in Toxoplasma gondii knockouts (KOs). The single TaqMan qPCR amplifies a 174 bp DNA fragment of the inserted mdhfr-ts and of the wild-type (WT) dhfr-ts (wtdhfr-ts) which is present as single copy gene in Toxoplasma and encodes a sensitive enzyme to pyrimethamine. Thus, the copy number of the dhfr-ts fragment in a given DNA quantity from KO parasites with a single site-specific integration should be twice the number of dhfr-ts copies recorded in the same DNA quantity from WT parasites. The duplex TaqMan qPCR allows simultaneous amplification of the 174 bp dhfr-ts fragment and the T. gondii 529-bp repeat element. Accordingly, for a WT DNA sample, the determined number of tachyzoites given by dhfr-ts amplification is equal to the number of tachyzoites determined by amplification of the Toxoplasma 529-bp, resulting thus in a ratio of 1. However, for a KO clone having a single site-specific integration of mdhfr-ts, the calculated ratio is 2. We then applied both approaches to test T. gondii RH mutants in which the major surface antigen (SAG1) was disrupted through insertion of mdhfr-ts using CRISPR-Cas9. Results from both assays were in correlation showing a high accuracy in detecting KOs with multiple integrated mdhfr-ts. Southern blot analyses using BsaBI and DraIII confirmed qPCRs results. Both TaqMan qPCRs are needed for reliable diagnostic of T. gondii KOs following CRISPR-Cas9-mediated mutagenesis, particularly with respect to off-target effects resulting from multiple insertions of mdhfr-ts. The principle of the duplex TaqMan qPCR is applicable for other selectable markers in Toxoplasma. TaqMan qPCR tools may contribute to more frequent use of WT Toxoplasma strains during functional genomics.

Item Type:

Journal Article (Original Article)

Division/Institute:

05 Veterinary Medicine > Department of Infectious Diseases and Pathobiology (DIP) > Institute of Parasitology
05 Veterinary Medicine > Department of Infectious Diseases and Pathobiology (DIP) > Institute of Animal Pathology
08 Faculty of Science > Department of Chemistry, Biochemistry and Pharmaceutical Sciences (DCBP)
05 Veterinary Medicine > Department of Infectious Diseases and Pathobiology (DIP)

Graduate School:

Graduate School for Cellular and Biomedical Sciences (GCB)

UniBE Contributor:

Hänggeli, Kai Pascal Alexander; Hemphill, Andrew; Müller, Norbert; Schimanski, Bernd; Olias, Philipp Alexander; Müller, Joachim and Boubaker, Ghalia

Subjects:

600 Technology > 630 Agriculture
500 Science > 570 Life sciences; biology
500 Science > 540 Chemistry

ISSN:

1932-6203

Publisher:

Public Library of Science

Language:

English

Submitter:

Pubmed Import

Date Deposited:

20 Sep 2022 09:31

Last Modified:

20 Sep 2022 09:41

Publisher DOI:

10.1371/journal.pone.0271011

PubMed ID:

36112587

BORIS DOI:

10.48350/173040

URI:

https://boris.unibe.ch/id/eprint/173040

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