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A transient CRISPR/Cas9 expression system for genome editing in Trypanosoma brucei
BMC Research Notes volume 13, Article number: 268 (2020)
Generation of knockouts and in situ tagging of genes in Trypanosoma brucei has been greatly facilitated by using CRISPR/Cas9 as a genome editing tool. To date, this has entailed using a limited number of cell lines that are stably transformed to express Cas9 and T7 RNA polymerase (T7RNAP). It would be desirable, however, to be able to use CRISPR/Cas9 for any trypanosome cell line.
We describe a sequential transfection expression system that enables transient expression of the two proteins, followed by delivery of PCR products for gRNAs and repair templates. This procedure can be used for genome editing without the need for stable integration of the Cas9 and T7RNAP genes.
The establishment of genome editing by CRISPR/Cas9 in trypanosomatids has greatly increased the ease with which knockouts can be generated, as two copies of a non-essential gene can often be deleted in a single round of transfection. The system most widely used for Trypanosoma brucei entails creating cell lines which express (either constitutively or inducibly) both Cas9 to generate a DNA double-strand break and T7RNAP for the transcription of guide RNAs from a DNA template [1,2,3,4]. The original pTB011 plasmid generated for T. brucei  encodes Cas9 flanked by tubulin sequences, enabling the construct to be integrated into the corresponding multicopy array. We modified this plasmid by replacing the puromycin acetyltransferase gene with that of T7RNAP, giving rise to pTB011_Cas9_T7RNAP_blast (Fig. 1a). This plasmid allows generation of CRISPR/Cas9-competent cell lines, constitutively expressing T7RNAP and Cas9 from the tubulin locus, in a single round of transfection . Further modification of this plasmid, and optimisation of the transfection protocol for transient expression of the proteins and guide RNAs, are described below.
In order to expand the range of cell lines that can be genetically modified by CRISPR/Cas9, without the need for stable transformation, we developed a protocol for transient expression of all three components (Cas9, T7RNAP and guide RNAs) in T. brucei. The plasmid All-in-one-Cas9 (pAi1C9; Fig. 1b) was constructed by replacing the α-tubulin sequence upstream of T7RNAP with 366 bp of the EP procyclin promoter and 5′ UTR .
To test the functionality of the plasmid, we used the LeishGEdit software  to design a guide RNA that allows tagging of the C-terminus of phosphodiesterase B1 (PDEB1) with mNeonGreen (mNG). The repair template, in the form of a purified PCR product, including homology arms of 30 bp on each side, was amplified from pPOTv7 . As a positive control we transfected a derivative of T. brucei Lister 427 procyclic forms that already expresses Cas9 and T7RNAP constitutively (Lister 427/Cas9) . This gave rise to cells with a fluorescent flagellum as expected (tryptag.org) (Fig. 2a).
For genome editing by transient expression of Cas9 and T7RNAP, we tested two different transfection schemes. Initially, all components were transfected simultaneously: the circular plasmid pAi1C9 encoding Cas9 and T7RNAP, the DNA template for in vivo transcription of a guide RNA, and the DNA repair template harbouring mNG for C-terminal tagging and a hygromycin resistance gene. This procedure gave few hygromycin-resistant clones, only 10 clones in total, from 4 separate transfections. These clones either did not express mNG (4 clones), or expressed it as a cytoplasmic protein (2 clones) or expressed it correctly localised to the flagellum (4 clones). Examples are shown in Fig. 2b. We hypothesise that, due to insufficient expression of Cas9 and T7RNAP, the amounts of site-specific guide RNAs and double-strand breaks at the correct locus were too low to drive efficient integration by homology-directed repair.
We therefore tested sequential transfections in which we first used pAi1C9 to enable expression of Cas9 and T7RNAP. A second transfection 20 h later provided the templates for the gRNA and the mNG repair construct. The same electroporation conditions were used for the plasmid and the templates (see Methods and Fig. 3). This procedure yielded 20 clones. There were 16 clones in which mNG was correctly localised to the flagellum, one in which it was cytoplasmic, 2 clones with mixed populations of mNG localised either to the flagellum or the cytoplasm, and one that was negative (see Fig. 2b for representative examples). One clone with flagellar mNeonGreen had a fluorescence intensity twice that of the others (clone B1) and might have been tagged on both alleles.
We have also used this procedure of sequential transfections to simultaneously knock out both copies of trypanin or GPI-anchor transamidase subunit 8 (GPI8), which are non-essential genes in cultured procyclic form T. brucei [8, 9]. Genotyping data is provided in Additional file 1.
Transfection of pAi1C9
4 × 107T. b. brucei 427 procyclic forms were transfected with 10 µg pAi1C9 dissolved in 100 µl TbBSF transfection buffer , using an Amaxa Nucleofector IIb (Lonza), program X-014 . Cells were transferred to 13 ml SDM79 medium  supplemented with 10% FBS and incubated at 27 °C and 2.5% CO2 for 20 h.
Transfection of pooled PCR products for repair constructs and sgRNA templates
The entire culture from the first transfection was centrifuged at 1700 g for 5 min, the supernatant discarded and the cells resuspended in 100 µl TbBSF transfection buffer containing the pooled PCR products (see protocol for PCRs below). For tagging, one sgRNA template (targeted to the 3′ end of the ORF) and one repair template (hygromycin resistance) were provided; to generate the knockouts, we provided two sgRNA templates (targeted to the 5′ and 3′ ends of the ORF) and two repair templates (hygromycin and neomycin resistance genes).
Transfection was performed as described above and the cells were transferred to 10 ml SDM79 supplemented with 10% FBS. The cells were diluted 1:5, 1:50 and 1:500 in conditioned medium (fresh medium + 20% supernatant of a log-phase culture) and distributed into 24-well plates (1 ml in each well). Transformants were selected using 25 µg ml−1 Hygromycin B and/or 15 µg ml−1 Geneticin; stable clones were obtained 2 weeks post selection.
Polymerase chain reactions (PCR)
Reactions were performed with reagents from New England Biolabs: Phusion High-Fidelity DNA polymerase (M0530S), 5 x Phusion HF buffer (B0519S) and dNTP mix (N0447S). Cycling conditions are identical to those previously published [4, 13].
1st PCR: template for sgRNAs; two 20 µl reactions per target.
1 μl G00 primer (0.5 μM)
4 μl 5 x Phusion HF buffer
0.5 μl dNTP mix (250 μM)
1 μl sgRNA primer (0.5 μM)
0.2 μl Phusion High-Fidelity DNA polymerase (0.4U)
13.3 μl H2O
30′′, 98 °C
10′′, 98 °C
30′′, 60 °C
15′′, 72 °C
go to step 2, 35 cycles in total
10′, 72 °C
hold at 10 °C
2nd PCR: template for resistance gene; two 40 µl reactions per resistance gene
2 μl 60 ng pPOTv7 mNG (hygro or G418) plasmid
8 μl 5 x Phusion HF buffer
1 μl dNTP mix (250 μM)
2 μl Upstream forward primer (0.5 μM)
2 μl Downstream reverse KO/TAG primer (0.5 μM)
0.4 μl Phusion High-Fidelity DNA polymerase (0.4U)
24.6 μl H2O
5′, 94 °C
30′′, 94 °C
30′′, 65 °C
2′30′′, 72 °C
go to step 2, 40 cycles in total
10′, 72 °C
hold at 10 °C
DNA purification after PCR
PCR reactions were pooled and extracted with 1 volume water-saturated phenol (pH 8), followed by extraction with 1 volume chloroform. DNA was precipitated from the aqueous phase by addition of 0.1 volume 3 M sodium acetate, pH 5.2, and 3 volumes ice-cold ethanol. The DNA was pelleted by centrifugation, washed twice with 1 ml 80% ethanol, air-dried at room temperature, dissolved in 40 μl Milli-Q-water and stored at − 20 °C until transfection.
PDEB1 3′ sgRNA primer:
Trypanin 5′ sgRNA primer:
Trypanin 3′ sgRNA primer:
GPI8 5′ sgRNA primer:
GPI8 3′ sgRNA primer:
Trypanin upstream forward primer:
Trypanin downstream reverse KO primer:
PDEB1 downstream forward primer:
PDEB1 downstream reverse TAG:
GPI8 upstream forward primer:
GPI8 downstream reverse KO primer:
Transient transfection with pAi1C9 gives rise to fewer clones than cell lines that are stably transformed with the T7RNAP and Cas9 genes. It has the advantage, however, that it can be applied to any T. brucei cell line. Moreover, in contrast to stable integration of the CRISPR/Cas9 machinery, transient transfection does not require additional selectable markers and it has the added advantage that it circumvents possible Cas9 toxicity. In addition to using Cas9 for deletion, mutation, tagging or integration of ectopic copies, this procedure could also be adapted for nuclease inactive Cas9 variants for targeting RNAs or epigenetic modifications [14, 15].
Availability of data and materials
Plasmids are available on request from Isabel Roditi (Isabel.firstname.lastname@example.org). The nucleotide sequences of pTB011_Cas9_T7RNAP_blast and pAi1C9 are provided on this website: https://www.izb.unibe.ch/research/prof_dr_isabel_roditi/index_eng.html.
T7 RNA polymerase
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We thank Eva Gluenz (University of Glasgow) for providing the progenitor plasmid pTB011 and David Schädeli and Peter Bütikofer for initiating us into CRISPR/Cas9 technology.
Swiss National Science Foundation (Grant nos. 31003A_166427 and 310030_184669) and the Canton of Bern.
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Additional file 1: Validation of knockout (KO) clones. A+B Trypanin-KO (Tb427.10.6350). A Clonal selection of stable transformants in 24-well plates. Dilutions from transfected pool cultures and number of cells seeded are indicated. Circles depict wells containing cells with successful (green filled circle) or unsuccessful (○) genome editing. B Assessment of editing by genotyping PCR and agarose gel electrophoresis. Genes amplified from genomic DNA and target loci are indicated. Lanes corresponding to validated KO clones are marked with green circles. C+D GPI8-KO (Tb427.10.13860) in a cell line allowing inducible expression of an ectopic copy of GPI8. C as in A. D as in B. E List of primers used for genotyping PCRs of Trypanin-KO (left panels) and GPI8-KO (right panels).
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Shaw, S., Knüsel, S., Hoenner, S. et al. A transient CRISPR/Cas9 expression system for genome editing in Trypanosoma brucei. BMC Res Notes 13, 268 (2020). https://doi.org/10.1186/s13104-020-05089-z
- Transient transfection