A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids

Beneke, T., Madden, R., Makin, L., Valli, J., Sunter, J. and Gluenz, E. (2017) A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids. Royal Society Open Science, 4(5), 170095. (doi: 10.1098/rsos.170095) (PMID:28573017) (PMCID:PMC5451818)

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.

• Item Type: Articles
• Additional Information: Funding: This study was supported by the John Fell Oxford University Press (OUP) Research Fund (project 132/029). E.G. is a Royal Society University Research Fellow, T.B. and J.V. are supported by MRC PhD studentships (T.B.: 15/16_MSD_836338; J.V.: 13/14_MSD_OSS_363238); L.M. is supported by a Wellcome Trust PhD studentship (102052/Z/13/Z). This work was supported by resources from Wellcome Trust grant nos. WT066839MA and 104627/Z/14/Z to Prof. Keith Gull and J.S. was also supported by these Wellcome Trust grants
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Gluenz, Dr Eva
• Authors: Beneke, T., Madden, R., Makin, L., Valli, J., Sunter, J., and Gluenz, E.
• College/School: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
• Journal Name: Royal Society Open Science
• Publisher: Royal Society
• ISSN: 2054-5703
• ISSN (Online): 2054-5703
• Published Online: 03 May 2017
• Copyright Holders: Copyright © 2017 The Authors
• First Published: First published in Royal Society Open Science 4(5):170095
• Publisher Policy: Reproduced under a Creative Commons license