A transient CRISPR/Cas9 expression system for genome editing in Trypanosoma brucei

Abstract

Objective: 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. Results: 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.

Cyclic AMP signalling and glucose metabolism mediate pH taxis by African trypanosomes

“…We next assessed the requirement for CARP3 in vivo. For these experiments we generated a null mutant in wild-type T. b. brucei Lister 427 using a recently described CRISPR/Cas9 transient expression system 39 . In addition, we engineered an addback ectopically expressing an untagged version of CARP3 (Supplementary Fig.…”

“…Single knockouts, double knockouts and tagged addbacks used for all in vitro experiments were generated in the parental 427 Cas9 cell line 29 . The CARP3 double knockout used for fly experiments was generated in T. b. brucei Lister 427 36 using transiently expressed Cas9 and T7 RNA polymerase 39 . An addback ectopically expressing CARP3 was derived from the null mutant.…”

Elimination of GPI2 suppresses glycosylphosphatidylinositol GlcNAc transferase activity and alters GPI glycan modification in Trypanosoma brucei

“…TbGPI2-KO parasites were generated using CRISPR/Cas9 technique as described before ( 64 ). In addition, TbGPI2-KO parasites were also generated in a T. brucei 427–derived CRISPR-competent background ( 65 ). Briefly, two resistance gene cassettes were generated by PCR using primers 1 and 2 ( Table S2 ) and template plasmid pPOTv6 ( 66 ) containing resistance genes for hygromycin and neomycin, respectively.…”

Persistence of Trypanosoma brucei as early procyclic forms and social motility are dependent on glycosylphosphatidylinositol transamidase

“…The two endogenous copies of TbGPI8 were subsequently deleted using a transient CRISPR/Cas9 transfection system (Shaw et al, 2020), giving rise to GPI8-cKO. The insertion of the inducible copy and deletion of the endogenous copies were previously confirmed by PCR genotyping (Shaw et al, 2020).…”

“…Constructs and oligonucleotides used for knocking out GPI8 and for genotyping PCRs were described previously (Shaw et al, 2020).…”