Abstract
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.
Highlights
Genome-wide Clustered Randomly Interspersed Short Palindromic Repeats (CRISPR) screening is a powerful tool to identify genes required under selective conditions
We observed that some gene KOs for virulence factors behave differently when present in a mutant pool than they do as homogenous clones in murine infections
The resulting vector library is transfected into parasites to generate a mutant pool that is propagated in cell culture for subsequent selection strategies in vitro or in vivo (Fig. 1a)
Summary
Genome-wide CRISPR screening is a powerful tool to identify genes required under selective conditions. We develop a method for rapid generation of custom guide RNA (gRNA) libraries using arrayed single-stranded oligonucleotides for reproducible pooled cloning of CRISPR/Cas[9] 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. The dose needs to be sufficiently low to allow parasite replication without killing the mouse, while maintaining coverage of the library In such instances, tailored CRISPR libraries that target a subset of genes provide a powerful tool. The methodology established here provides a versatile and inexpensive platform for CRISPR library generation that is widely applicable
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