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. 2021 May 3;2(2):100520.
doi: 10.1016/j.xpro.2021.100520. eCollection 2021 Jun 18.

CRISPR screen to determine the in vivo fitness of Toxoplasma genes

Lamba Omar Sangaré  1 , Yifan Wang  1 , David Arranz-Solís  1 , Jeroen P J Saeij  1
Affiliations

CRISPR screen to determine the in vivo fitness of Toxoplasma genes

Lamba Omar Sangaré et al. STAR Protoc. .

Abstract

The virulence of eukaryotic parasites like Toxoplasma gondii depends on their capacity to escape from the host immune response and disseminate throughout the host organism. However, Toxoplasma gene products essential for its in vivo pathogenesis remain uncharacterized. Here, we present the complete workflow of a CRISPR-Cas9 in vivo loss-of-function screen to identify Toxoplasma fitness-conferring genes. This protocol can be used to uncover gene products that play a role in Toxoplasma immune evasion, nutrient acquisition, dissemination, and tissue colonization. For complete details on the use and execution of this protocol, please refer to Sangaré et al. (2019).

Keywords: CRISPR; High Throughput Screening; Microbiology; Sequencing.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
sgRNA amplification Left, genomic DNA was extracted from mice's lungs and used as a template to amplify sgRNAs (Samples 1 to 5). Right, lungs were put in HFF cell culture to allow the parasites to replicate. Subsequently, parasites were isolated, genomic DNA extracted, and used as a template to amplify sgRNAs (samples A to E). The red arrow indicates sgRNA bands 368–369 bp, and the black arrow indicates remaining primers from the PCR reaction. neg= negative control for PCR reactions.
See this image and copyright information in PMC

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