Supplementary material from "A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids"
Published on 2017-04-28T07:19:55Z (GMT) by
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 <i>in vivo</i> 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 <i>Leishmania major</i>, <i>Leishmania mexicana</i> and bloodstream form <i>Trypanosoma brucei</i>; deleted genes were undetectable in non-clonal populations, enabling for the first time rapid and large-scale knockout screens.
Cite this collection
Beneke, Tom; Madden, Ross; Makin, Laura; Valli, Jessica; Sunter, Jack; Gluenz, Eva (2017): Supplementary material from "A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids". The Royal Society. Collection.