A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids

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.

Keywords: CRISPR; Leishmania; T7 RNA polymerase; Trypanosoma; genome editing.

Figure 1.

Constitutive expression of Cas9 in L. mexicana. Growth curves of L. mexicana wild-type cells (WT) and L. mex Cas9 clone G2.

Figure 2.

Short HF allow efficient integration of donor DNA. (a) Leishmania mexicana wild-type cells (WT; grey) and L. mex Cas9 clone G2 (red) were transfected with PF16::YFP tagging cassettes containing different length HF (24, 33, 47, 65 and 350 nt). The plot shows the number of transfectants recovered per transfected cell. Large open circles denote blasticidin-resistant cells; small-filled circles denote blasticidin-resistant cells with green fluorescent flagella. For wild-type cells, homology lengths of 24–65 nt yielded no drug-resistant cells. Each data point represents the mean number of transfectants from three independent transfections. (b) Micrographs showing PF16::YFP and PF16::mCherry (mCh) expression in L. mex Cas9 cells transfected with each tagging construct separately or combined. Merged: phase contrast image overlaid with fluorescence channels showing YFP (green), mCh (magenta) and Hoechst-stained DNA (cyan). Scale bar 5 µm.

Figure 3.

Co-transfection of two PCR amplicons allowed precise insertion of marker genes. (a) PCR-amplified donor DNA containing 30 nt HF specific to the target locus, a fluorescent protein tag and a drug-selectable marker gene. (b) Strategy for sgRNA delivery: the sgRNA template is produced by PCR using an oligo encoding the T7 promoter, 20 nt defining the target-site and a sequence complementary to the 3′-end of the second oligo, comprising the sgRNA scaffold [28]. The resulting PCR product is transfected into cells for T7 RNAP-driven transcription of the sgRNA. (c) Summary of outcome of transfections with different combinations of sgRNA templates and donor DNAs and electroporation protocols. Green filled circles denote drug-resistant cells showing the expected fluorescent signal; red open circles indicate failure to produce any drug-resistant transfectants. (d) Micrographs showing correct flagellar localization of PF16::YFP, nuclear localization of H2B::YFP (the white colour indicates co-localization of YFP and Hoechst) and flagellar membrane localization of SMP-1::YFP in cells that were co-transfected with the donor DNA and corresponding sgRNA template. Phase contrast image merged with mCh or YFP fluorescence channels and Hoechst-stained DNA (magenta). Scale bar 5 µm. (e) PCR-detection of the sgRNA template. Top, agarose gel showing the results of a diagnostic PCR to test for the presence of the sgRNA template. Template DNAs were as follows. Input: 1 µl of PF16 sgRNA PCR used for transfection; 5 min-48 h post transfection (p.t.): genomic DNA from cells at different time points post transfection with PF16 sgRNA; ΔPF16, ΔLPG1, PF16::mCh / PF16::YFP: genomic DNA from drug-resistant cell lines reported in this study; parental: genomic DNA from the parental cell line L. mex Cas9 T7. Bottom, diagram showing the sgRNA template and the primers used for PCR detection.

Figure 4.

A modular system for PCR-amplification of targeting fragments. (a) Strategy for using pT and pPLOT to generate donor DNA for repair of Cas9-induced double-strand breaks allowing precise modification of a target locus. To delete a target gene, two sgRNAs direct cuts to sites immediately upstream (5′) and downstream (3′) of the target gene. Repair cassettes with drug-selectable marker genes (DrugR) and 30 nt HF specific to the target locus are PCR-amplified from pT plasmids with primers 1 and 5. The same primer pair can be used to amplify cassettes with different drug-resistance genes. To tag a target gene, one sgRNA directs a cut immediately upstream or downstream of the target gene, for fusing tags to the N- or C- terminus of a protein, respectively. A repair cassette with 30 nt HF specific to the target locus, the desired tag and a drug-selectable marker gene are PCR-amplified from a pPLOT plasmid. Primer pair 1 and 2 is used for N-terminal tagging (indicated by grey arrows), 4 and 5 for C-terminal tagging (dashed arrows). The same primer pairs can be used to amplify a range of different tagging cassettes. Primers 3 and 6 (not shown) are used to amplify the 5′- and 3′-sgRNA templates. (b) Diagrams showing the target gene locus before and after insertion of repair cassettes.

Figure 5.

Knockout of PF16. (a) PCR analysis of the ΔPF16 cell line. (i) PCR products visualized on agarose gel. P, parental cell line L. mex Cas9 T7; KO, ΔPF16 population; AB, cells expressing an ectopic copy of PF16. (ii) Diagram showing the PF16 locus and PCR primers (arrows) used to test for presence of the PF16 CDS or the correct integration of the drug-resistance genes (blue boxes). (b) Transmission electron microscopy cross section showing the 9 + 2 microtubule arrangement in flagellar axonemes of the parental cell line, scale bar 100 nm. (c–e) Axonemes of ΔPF16 cells with a 9 + 2, 9 + 1 or 9 + 0 microtubule arrangement. (f) Measurements of the angle between the plane though the CP and the PFR; parental N = 23, ΔPF16 N = 21. (g) Cartoon illustrating how angles shown in (f) were measured.

Figure 6.

Knockout of LPG1. (a,b) PCR analysis of the ΔLPG1 cell line: test for the presence of the LPG1 CDS; (c) test for correct integration of the blasticidin-resistance gene; (d) test for correct integration of the neomycin-resistance gene, lanes as in (c). Diagrams above the gel pictures show the primers (arrows) used for PCR and size of expected product. (e) Western blot of whole-cell lysates probed with LT22. P, parental cell line L. mex Cas9 T7; KO POP, ΔLPG1 population; F3, ΔLPG1 clonal cell line; +AB, cell lines expressing an ectopic copy of LPG1.

Figure 7.

Targeting GPI-PLC in T. brucei bloodstream forms. (a) Localization of tagged GPI-PLC in T. brucei SmOx B4 Cas9 cells co-transfected with sgRNA templates and donor DNA(s); one or both alleles of GPI-PLC were tagged with mNeonGreen (mNG) or TagRFP S158 T ([51], TagRFPt), as indicated. SmOx B4 is the parental untagged cell line. Scale bar 5 µm. (b) PCR analysis of the following cell lines: three independent ΔGPI-PLC clones, doubly tagged cell line mNG::GPI-PLC/TagRFPt::GPI-PLC, SmOx B4 pTB011 and SmOx B4. (i) PCR amplicons visualized on an agarose gel. (ii) diagrams showing the primers (arrows) used for PCR and size of expected product. (c,d) Cells were subjected to hypotonic lysis for 20 min, separated into pellet (p) and supernatant (s) fractions and run together with whole-cell lysates (w) on an SDS PAGE gel. (c) Western blot probed with anti-GPI-PLC (arrows indicate expected bands: TagRFPt::GPI-PLC, 67.5 kDa; mNG::GPI-PLC 66.5 kDa; GPI-PLC, 40 kDa); (d) Coomassie stained gel (arrow indicates VSG; m, 50 kDa marker).