Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR-associated gene 9 (Cas9) genome editing is set to revolutionize genetic manipulation of pathogens, including kinetoplastids. CRISPR technology provides the opportunity to develop scalable methods for high-throughput production of mutant phenotypes. Here, we report development of a CRISPR-Cas9 toolkit that allows rapid tagging and gene knockout in diverse kinetoplastid species without requiring the user to perform any DNA cloning. We developed a new protocol for single-guide RNA (sgRNA) delivery using PCR-generated DNA templates which are transcribed in vivo by T7 RNA polymerase and an online resource (LeishGEdit.net) for automated primer design. We produced a set of plasmids that allows easy and scalable generation of DNA constructs for transfections in just a few hours. We show how these tools allow knock-in of fluorescent protein tags, modified biotin ligase BirA*, luciferase, HaloTag and small epitope tags, which can be fused to proteins at the N- or C-terminus, for functional studies of proteins and localization screening. These tools enabled generation of null mutants in a single round of transfection in promastigote form Leishmania major, Leishmania mexicana and bloodstream form Trypanosoma brucei; deleted genes were undetectable in non-clonal populations, enabling for the first time rapid and large-scale knockout screens.
Highlights
Kinetoplastid parasites, including the human pathogenic Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp., pose a huge global burden on health and economic development; the need for better diagnostic tools and medicines for these neglected diseases remains urgent
We found no evidence for CRISPR-associated gene 9 (Cas9) toxicity: L. mex Cas9 promastigote cells ml–1 rsos.royalsocietypublishing.org R
We previously showed that integration of DNA fragments by homologous recombination (HR) in Leishmania required homology flanks (HF; sequences at the ends of the donor DNA cassette that are identical to the target locus) of at least 100 nt [12]
Summary
Kinetoplastid parasites, including the human pathogenic Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp., pose a huge global burden on health and economic development; the need for better diagnostic tools and medicines for these neglected diseases remains urgent. Generate targeted mutations in the laboratory to study mutant phenotypes has been key to the dissection 2 of basic parasite biology, pathogenicity mechanisms and drug resistance. These studies were facilitated by powerful forward and reverse genetics methods [1] and whole-genome sequence information. For T. brucei, important new insights into its basic biology [2,3,4] and drug-resistance mechanisms [5] emerged from genome-scale loss-of-function screens using RNA interference (RNAi) libraries. Plasticity in chromosome copy numbers [7,8] further complicates targeted mutations of many potentially important genes
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