A CRISPR platform for targeted in vivo screens identifies Toxoplasma gondii virulence factors in mice

• Published in: Nature communications Vol. 10; no. 1; pp. 3963 - 11
• Main Authors: Young, Joanna, Dominicus, Caia, Wagener, Jeanette, Butterworth, Simon, Ye, Xingda, Kelly, Gavin, Ordan, Merav, Saunders, Becky, Instrell, Rachael, Howell, Michael, Stewart, Aengus, Treeck, Moritz
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 03.09.2019; Nature Publishing Group; Nature Portfolio
• Subjects: 42/41; 45/41; 49/47; 631/326/417/2546; 631/326/417/2547; 631/326/417/2550; Animals; Cell Line; Cloning; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR; CRISPR-Cas Systems; Gene Knockout Techniques - methods; Gene Library; Genetic screening; Genome, Protozoan; Genomes; gRNA; Humanities and Social Sciences; Humans; In vivo methods and tests; Libraries; Mice, Inbred C57BL; multidisciplinary; Oligonucleotides; Parasites; Pools; Protozoa; Ribonucleic acid; RNA; RNA, Guide, CRISPR-Cas Systems; Science; Science (multidisciplinary); Toxoplasma - genetics; Toxoplasma - pathogenicity; Toxoplasmosis - genetics; Toxoplasmosis - parasitology; Toxoplasmosis - pathology; Virulence; Virulence factors; Virulence Factors - genetics
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-019-11855-w

Genome-wide CRISPR screening is a powerful tool to identify genes required under selective conditions. However, the inherent scale of genome-wide libraries can limit their application in experimental settings where cell numbers are restricted, such as in vivo infections or single cell analysis. The use of small scale CRISPR libraries targeting gene subsets circumvents this problem. Here we develop a method for rapid generation of custom guide RNA (gRNA) libraries using arrayed single-stranded oligonucleotides for reproducible pooled cloning of CRISPR/Cas9 libraries. We use this system to generate mutant pools of different sizes in the protozoan parasite Toxoplasma gondi and describe optimised analysis methods for small scale libraries. An in vivo genetic screen in the murine host identifies novel and known virulence factors and we confirm results using cloned knock-out parasites. Our study also reveals a potential trans-rescue of individual knock-out parasites in pools of mutants compared to homogenous knock-out lines of the key virulence factor MYR1. Targeted CRISPR libraries expand the use of genetic screens across experimental conditions. Here, the authors develop a method for generating and analysing small scale custom CRISPR libraries and use it in the human and livestock pathogen Toxoplasma gondii to identify virulence factors in mice.