Zinc finger nuclease technology: A stable tool for high efficiency transformation in bloodstream form T. brucei

Schumann, Gabriela; Kangussu-Marcolino, Mônica Mendes; Doiron, Nicholas; Käser, Sandro; Burle-Caldas, Gabriela de Assis; DaRocha, Wanderson D.; Teixeira, Santuza Maria Ribeiro; Roditi, Isabel

doi:10.1016/j.molbiopara.2017.02.002

Cited by 5 publications

(3 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Firstly, compared to the use of I-SceI meganuclease (Glover and Horn, 2009), it allowed us to select any TTTV-PAM containing locus of interest to insert our CBE sgRNA expression construct, simply by just changing the Cas12a crRNA sequence. Secondly, Cas12a editing – just like any CRISPR strategy – is much simpler to design than a zinc-finger nuclease (Schumann et al, 2017) or TALENs approach. Thirdly, compared to a functional SpCas9 nuclease, there was no risk of interference between the Cas9 base editor and Cas9 nuclease sgRNAs.…”

Section: Resultsmentioning

confidence: 99%

“…Strategies to increase transfection rates and stably integrate expression cassettes by homologous recombination have been previously developed in T. brucei . Here, the use of an I-SceI mega- (Glover and Horn, 2009) or zinc finger- (Schumann et al, 2017) nuclease-induced DSB at a safe harbor locus has been shown to increase the transfection efficiency for the integration of RNAi expression constructs significantly. In combination with improved transfection protocols (Burkard et al, 2007; Glover and Horn, 2009), this has enabled RNAi loss-of-function screens on a genome-wide scale (Glover et al, 2015; Horn, 2022; Schumann Burkard et al, 2011).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Improved base editing and functional screening inLeishmaniavia co-expression of the AsCas12a ultra variant, a T7 RNA Polymerase, and a cytosine base editor

May,

Cao,

Schmid

et al. 2024

Preprint

View full text Add to dashboard Cite

The ability to analyse the function of all genes in a genome has obvious appeal. However, this has been challenging inLeishmaniadue to a repetitive genome architecture, limited DNA repair mechanisms and the absence of RNA interference machinery in most species. While our previous introduction of a cytosine base editor (CBE) tool inLeishmaniashowcased the potential for bypassing these limits (Engstler and Beneke (2023)), challenges remained in achieving high transfection efficiencies, overcoming species-specific editing rates, minimizing effects on parasite growth and eliminating competition between deleterious and non-deleterious mutations. Here, we present an optimized approach to address these limitations. Firstly, we identified a T7 RNAP promoter variant that ensures high editing rates acrossLeishmaniaspecies without adversely affecting parasite growth. Secondly, we adjusted the scoring of CBE single-guide RNAs (sgRNAs) to prioritize those ensuring STOP codon generation. Thirdly, we developed a triple-expression construct enabling the integration of CBE sgRNA expression cassettes into aLeishmaniasafe harbor locus via AsCas12a ultra-mediated DNA double-strand breaks. This facilitates the generation of stable CBE sgRNA expression cell lines and increases transfection rates by ∼400-fold, resulting in up to one transfectant per 70 transfected cells. Lastly, we show how the co-expression of AsCas12a ultra, T7 RNAP and CBE can be utilized for hybrid CRISPR gene replacement and base editing approaches in the same cell line. Overall, we believe that these improvements will broaden the range of possible gene editing applications inLeishmaniaspecies and will enable a variety of loss-of-function screens in the future.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Improved base editing and functional screening inLeishmaniavia co-expression of the AsCas12a ultra variant, a T7 RNA Polymerase, and a cytosine base editor

May,

Cao,

Schmid

et al. 2024

Preprint

View full text Add to dashboard Cite

show abstract

“…Further screens will likely be conducted in other trypanosomatids and in insect or mammalian hosts. Indeed, wider application of RNAi screening will be facilitated by improved protocols for RNAi library assembly [100] and improved genetic tools for other African trypanosomes such as T. congolense [101] or for Leishmania braziliensis [9]. The machinery for RNAi may even be imported into trypanosomatids that naturally lack these factors [9,102], or alternative knockdown approaches may be developed, using Cas13 nuclease for example [103]; Cas13 is related to RNA-programmable Cas9, but is a RNA endonuclease.…”

Section: Concluding Remarks and Future Perspectivesmentioning

confidence: 99%