A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids

• Published in: Royal Society open science Vol. 4; no. 5; p. 170095
• Main Authors: Beneke, Tom, Madden, Ross, Makin, Laura, Valli, Jessica, Sunter, Jack, Gluenz, Eva
• Format: Journal Article
• Language: English
• Published: England The Royal Society Publishing 01.05.2017; The Royal Society
• Subjects: Cellular And Molecular Biology; Crispr; Genome Editing; leishmania; T7 Rna Polymerase; trypanosoma; CRISPR; T7 RNA polymerase; genome editing; Trypanosoma; Leishmania
• ISSN: 2054-5703; 2054-5703
• DOI: 10.1098/rsos.170095

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.