A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids. Issue 5 (3rd May 2017)

• Record Type: Journal Article
• Authors: Beneke, Tom; Madden, Ross; Makin, Laura; Valli, Jessica; Sunter, Jack; Gluenz, Eva
• Is Part Of: Royal Society open science. Volume 4:Issue 5(2017)
• Publication Date: 2017-05-03
• Subjects: Leishmania -- Trypanosoma -- CRISPR -- genome editing -- T7 RNA polymerase; Science -- Periodicals; 500
• DOI: 10.1098/rsos.170095
• Languages: English
• ISSNs: 2054-5703

Abstract

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.