Streptococcus pyogenesCas9 ribonucleoprotein delivery for efficient, rapid and marker-free gene editing inTrypanosomaandLeishmania DOI Creative Commons

Asencio Corinne,

Hervé Perrine,

Morand Pauline

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Oct. 26, 2023

SUMMARY Kinetoplastids are unicellular eukaryotic flagellated parasites found in a wide range of hosts within the animal and plant kingdoms. They known to be responsible humans for African sleeping sickness ( Trypanosoma brucei ), Chagas disease cruzi various forms leishmaniasis Leishmania spp.), as well several diseases with important economic impact (African trypanosomes, including T. congolense ). Understanding biology these necessarily implies ability manipulate their genomes. In this study, we demonstrate that transfection ribonucleoprotein complex, composed recombinant Streptococcus pyogenes Cas9 Sp Cas9) an vitro -synthesized guide RNA, results rapid efficient genetic modifications trypanosomatids, marker-free conditions. This approach was successfully developed inactivate, delete mutate candidate genes stages life cycle , promastigotes. The functionality now provides, research community working on parasites, method genome editing, without requiring plasmid construction selection by antibiotics. Importantly, is adaptable any wild-type parasite, field isolates.

Language: Английский

CRISPR-Cas9 high-throughput screening to study drug resistance in Leishmania infantum DOI Creative Commons
Marine Queffeulou,

Philippe Leprohon,

Christopher Fernandez‐Prada

et al.

mBio, Journal Year: 2024, Volume and Issue: 15(7)

Published: June 12, 2024

Parasites of the genus

Language: Английский

Citations

7
Streptococcus pyogenes Cas9 ribonucleoprotein delivery for efficient, rapid and marker‐free gene editing in Trypanosoma and Leishmania DOI Creative Commons
Corinne Asencio, Perrine Hervé, Pauline Morand

et al.

Molecular Microbiology, Journal Year: 2024, Volume and Issue: 121(6), P. 1079 - 1094

Published: April 1, 2024

Abstract Kinetoplastids are unicellular eukaryotic flagellated parasites found in a wide range of hosts within the animal and plant kingdoms. They known to be responsible humans for African sleeping sickness ( Trypanosoma brucei ), Chagas disease cruzi various forms leishmaniasis Leishmania spp.), as well several diseases with important economic impact (African trypanosomes, including congolense ). Understanding biology these necessarily implies ability manipulate their genomes. In this study, we demonstrate that transfection ribonucleoprotein complex, composed recombinant Streptococcus pyogenes Cas9 Sp Cas9) an vitro‐synthesized guide RNA, results rapid efficient genetic modifications trypanosomatids, marker‐free conditions. This approach was successfully developed inactivate, delete, mutate candidate genes stages life cycle T. , promastigotes. The functionality now provides, research community working on parasites, method genome editing, without requiring plasmid construction selection by antibiotics but requires only cloning PCR screening clones. Importantly, is adaptable any wild‐type parasite.

Language: Английский

Citations

5
CRISPR-Cas in Parasites: Advances and Challenges in the Study of Neglected Tropical Diseases DOI
Ana María Mejía‐Jaramillo, Omar Triana‐Chávez

Published: Jan. 1, 2025

Language: Английский

Citations

0
Improved base editing and functional screening in Leishmania via co-expression of the AsCas12a ultra variant, a T7 RNA Polymerase, and a cytosine base editor DOI Open Access

Nicole Herrmann May,

Nan Cao,

Annika Schmid

et al.

Published: Feb. 11, 2025

The ability to analyse the function of all genes in a genome is highly desirable, yet challenging Leishmania due repetitive genome, limited DNA repair mechanisms and lack RNA interference most species. While our introduction cytosine base editor (CBE) demonstrated potential overcome these limitations (Engstler Beneke (2023)), challenges remained, including low transfection efficiency, variable editing rates across species, parasite growth effects, competition between deleterious non-deleterious mutations. Here, we present an optimized approach addressing issues.We identified T7 RNAP promoter variant ensuring high species without compromising growth. A revised CBE single-guide RNAs (sgRNAs) scoring system was developed prioritize STOP codon generation. Additionally, triple-expression construct created for stable integration sgRNA expression cassettes into safe harbor locus using AsCas12a ultra-mediated double-strand breaks, increasing efficiency by ∼400-fold one transfectant per 70 transfected cells. Using this improved small-scale proof-of-principle pooled screen, successfully confirmed essential fitness-associated functions CK1.2, CRK2, CRK3, AUK1/AIRK, TOR1, IFT88, IFT139, IFT140 RAB5A L. mexicana , demonstrating significant improvement over previous method. Lastly, show utility co-expressing ultra, hybrid CRISPR gene replacement within same cell line.Overall, improvements will broaden range possible applications enable variety loss-of-function screens near future.

Language: Английский

Citations

0
Improved base editing and functional screening in Leishmania via co-expression of the AsCas12a ultra variant, a T7 RNA polymerase, and a cytosine base editor DOI Creative Commons

Nicole Herrmann May,

Anh T. Cao,

Annika Schmid

et al.

eLife, Journal Year: 2025, Volume and Issue: 13

Published: Feb. 24, 2025

The ability to analyze the function of all genes in a genome is highly desirable, yet challenging Leishmania due repetitive genome, limited DNA repair mechanisms, and lack RNA interference most species. While our introduction cytosine base editor (CBE) demonstrated potential overcome these limitations (Engstler Beneke, 2023), challenges remained, including low transfection efficiency, variable editing rates across species, parasite growth effects, competition between deleterious non-deleterious mutations. Here, we present an optimized approach addressing issues. We identified T7 RNAP promoter variant ensuring high species without compromising growth. A revised CBE single-guide RNAs (sgRNAs) scoring system was developed prioritize STOP codon generation. Additionally, triple-expression construct created for stable integration sgRNA expression cassettes into safe harbor locus using AsCas12a ultra-mediated double-strand breaks, increasing efficiency by ~400-fold 1 transfectant per 70 transfected cells. Using this improved small-scale proof-of-principle pooled screen, successfully confirmed essential fitness-associated functions CK1.2, CRK2, CRK3, AUK1/AIRK, TOR1, IFT88, IFT139, IFT140, RAB5A mexicana , demonstrating significant improvement over previous method. Lastly, show utility co-expressing ultra, RNAP, hybrid CRISPR gene replacement within same cell line. Overall, improvements will broaden range possible applications enable variety loss-of-function screens near future.

Language: Английский

Citations

0
A comprehensive toolkit for protein localization and functional analysis in trypanosomatids DOI Creative Commons

Athina Paterou,

Julia Sáez Conde,

Jiří Týč

et al.

Open Biology, Journal Year: 2025, Volume and Issue: 15(4)

Published: April 1, 2025

African trypanosomes are medically important parasites that cause sleeping sickness in humans and nagana animals. In addition to their pathogenic role, they have emerged as valuable model organisms for studying fundamental biological processes. Protein tagging is a powerful tool investigating protein localization function. previous study, we developed two plasmids rapid reproducible polymerase chain reaction-based trypanosomes, which enabled the subcellular mapping of 89% trypanosome proteome. However, limited selection fluorescent tags selectable markers restricted flexibility this approach. Here, present an extended set >100 incorporate universal primer annealing sequences, enabling with range fluorescent, biochemical epitope tags, using five different markers. We evaluated suitability various proteins live fixed cell imaging, movies, demonstrate use encoding tandem support expansion microscopy approaches. show series functional other trypanosomatid parasites, significantly increasing its value. Finally, new plasmid glycosylphosphatidylinositol-anchored proteins. anticipate will be toolset

Language: Английский

Citations

0
Co-expression of the AsCas12a ultra variant, a T7 RNA Polymerase and a cytosine base editor greatly increases transfection and editing rates inLeishmaniaspecies DOI Creative Commons

Nicole Herrmann May,

Annika Schmid,

Elisabeth Meiser

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Feb. 28, 2024

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

Language: Английский

Citations

1
Transforming the CRISPR/dCas9-based gene regulation technique into a forward screening tool in Plasmodium falciparum DOI Creative Commons
Amuza Byaruhanga Lucky, Chengqi Wang, Xiaolian Li

et al.

iScience, Journal Year: 2024, Volume and Issue: 27(4), P. 109602 - 109602

Published: March 27, 2024

It is a significant challenge to assess the functions of many uncharacterized genes in human malaria parasites. Here, we present genetic screening tool contribution essential from Plasmodium falciparum by conditional CRISPR-/deadCas9-based interference and activation (i/a) systems. We screened both CRISPRi CRISPRa sets, consisting nine parasite lines per set targeting via their respective gRNAs. By conducting amplicon sequencing gRNA loci, identified each targeted gene fitness upon drug (artemisinin, chloroquine) stress (starvation, heat shock) treatment. The was highly reproducible, libraries were easily generated transfection mixed plasmids expressing different demonstrated that this straightforward, robust, can provide fast efficient study have long presented bottleneck assessing using existing tools.

Language: Английский

Citations

1
Advancing Treatment for Leishmaniasis: From Overcoming Challenges to Embracing Therapeutic Innovations DOI
Vishal Singh, Rahul Tiwari,

Rajneesh

et al.

ACS Infectious Diseases, Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 31, 2024

Protozoan parasite infections, particularly leishmaniasis, present significant public health challenges in tropical and subtropical regions, affecting socio-economic status growth. Despite advancements immunology, effective vaccines remain vague, leaving drug treatments as the primary intervention. However, existing medications face limitations, such toxicity rise of drug-resistant parasites. This presents an urgent need to identify new therapeutic targets for leishmaniasis treatment. Understanding complex life cycle Leishmania its survival host macrophages can provide insights into potential Current treatments, including antimonials, amphotericin B, miltefosine, are constrained by side effects, costs, resistance, reduced efficacy. Exploring novel within parasite's physiology, key metabolic enzymes or essential surface proteins, may lead development more less toxic drugs. Additionally, innovative strategies like repurposing, combination therapies, nanotechnology-based delivery systems could enhance efficacy combat thus improving anti-leishmanial therapies.

Language: Английский

Citations

1
Where is the EXIT? Phenotypic screens for new egress factors in apicomplexan parasites DOI Creative Commons
Elena Jimenéz‐Ruiz, Wei Li, Markus Meissner

et al.

Molecular Microbiology, Journal Year: 2023, Volume and Issue: unknown

Published: Sept. 22, 2023

Apicomplexans, such as Plasmodium and Toxoplasma are obligate intracellular parasites that invade, replicate finally EXIT their host cell. During replication within a parasitophorous vacuole (PV), the establish an extensive F-actin-containing network connects individual is required for material exchange, recycling final steps of daughter cell assembly. After multiple rounds replication, exit involving signalling cascades, disassembly network, secretion microneme proteins activation acto-myosin motor. Blocking process leads to formation large PVs, making screening genes involved in exiting relatively straightforward. Given apicomplexans highly diverse from other eukaryotes, approximately 30% all annotated hypothetical, some apicomplexan-specific factors likely be critical during EXIT. This motivated several labs design perform forward genetic phenotypic screens using various approaches, random insertion mutagenesis, temperature-sensitive mutants and, more recently, CRISPR/Cas9-mediated targeted editing conditional mutagenesis. Here we will provide overview technological developments over recent years most successful stories led identification new gondii.

Language: Английский

Citations

2