A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids

Royal Society Open Science - Tập 4 Số 5 - Trang 170095 - 2017
Tom Beneke1, Ross Madden2, Laura Makin3, Jessica Valli4, Jack Daniel Sunter5, Eva Gluenz6
1Tom Beneke Google Scholar Find this author on PubMed
2Ross Madden http://orcid.org/0000-0003-1448-8441 Google Scholar Find this author on PubMed
3Laura Makin Google Scholar Find this author on PubMed
4Jessica Valli Google Scholar Find this author on PubMed
5Jack Sunter Google Scholar Find this author on PubMed
6Eva Gluenz http://orcid.org/0000-0003-4346-8896 [email protected] Google Scholar Find this author on PubMed

Tóm tắt

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 transcribedin vivoby 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 formLeishmania major,Leishmania mexicanaand bloodstream formTrypanosoma brucei; deleted genes were undetectable in non-clonal populations, enabling for the first time rapid and large-scale knockout screens.

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Royal Society Open Science

Tập 4 Số 5

170095

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