A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids

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.

Direction of flagellum beat propagation is controlled by proximal/distal outer dynein arm asymmetry

“…fasciculata PGKB 5' UTR fusion was replaced with the L. mexicana histone 2B intergenic sequence (between LmxM.19.0050 and LmxM.19.0030), the C. fasiculata PGKA/B intergenic was replaced with the L. mexicana histone 2A intergenic (between LmxM.08_29.1740 and LmxM.08_29.1730) and the T. brucei aldolase 3' UTR was replaced with the L. mexicana eukaryotic initiation factor 5 (LmxM.25.0720) 3' UTR. Constructs and sgRNA templates for open reading frame deletion were generated by PCR and transfected as previously described, using pT Blast and pT Neo as templates (66). Primers were designed using LeishGEdit (http://www.LeishGEdit.net) (66).…”

“…Constructs and sgRNA templates for open reading frame deletion were generated by PCR and transfected as previously described, using pT Blast and pT Neo as templates (66). Primers were designed using LeishGEdit (http://www.LeishGEdit.net) (66). Transfectants were selected with the necessary combination of 20 µg/ml puromycin dihydrochloride, 5 µg/ml blasticidin S hydrochloride, 40 µg/ml G-418 disulfate, 50 µg/ml nourseothricin sulfate and 25 µg/ml phleomycin and cloned by limiting dilution in 96 well plates using MM199 as previously described (64).…”

Drug candidate and target for leishmaniasis

“…The range of protein kinases in Leishmania is different from that in humans, so this study provides an impetus to search for other 'druggable' protein kinases in the parasite 7 . Methods such as gene editing 8 using the CRISPR-Cas9 technique have improved researchers' ability to perform large-scale genetic validation of drug targets in Leishmania. However, a major bottleneck in the drug-discovery process for neglected tropical diseases is the identification of highly specific chemical probes that allow chemical validation -evidence that confirms the molecular target of a compound of interest.…”

State‐of‐the‐art CRISPR/Cas9 Technology for Genome Editing in Trypanosomatids

“…), L. donovani (Zhang and Matlashewski ), L. mexicana (Beneke et al. ) , T. brucei (Beneke et al. ; Rico et al.…”

“…) and the system has been successfully used to investigate the role of several proteins in trypanosomatids, but mainly in T. cruzi (Beneke et al. ; Bertolini et al. ; Chiurillo et al.…”