Co-expression of the AsCas12a ultra variant, a T7 RNA Polymerase and a cytosine base editor greatly increases transfection and editing rates in Leishmania species

Опубликована: Июнь 5, 2024

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.

Язык: Английский

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Co-expression of the AsCas12a ultra variant, a T7 RNA Polymerase and a cytosine base editor greatly increases transfection and editing rates in Leishmania species

eLife, Год журнала: 2024, Номер unknown

Опубликована: Июнь 5, 2024

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.

Язык: Английский

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Siah2- and LRSAM1-mediated K63-linked ubiquitination of snakehead vesiculovirus nucleoprotein facilitates viral replication

Journal of Virology, Год журнала: 2024, Номер 98(7)

Опубликована: Июнь 6, 2024

ABSTRACT Nucleoprotein (N) is well known for its function in the encapsidation of genomic RNAs negative-strand RNA viruses, which leads to formation ribonucleoproteins that serve as templates viral transcription and replication. However, N protein other aspects during infection far from clear. In this study, snakehead vesiculovirus (SHVV), a kind fish rhabdovirus, was proved be ubiquitinated mainly via K63-linked ubiquitination. We identified nine host E3 ubiquitin ligases interacted with SHVV N, among seven facilitated ubiquitination protein. Further investigation revealed only two ligases, Siah ligase 2 (Siah2) leucine-rich repeat sterile alpha motif containing 1 (LRSAM1), mediated upregulated expression Siah2 LRSAM1, maintained stability N. Besides, overexpression or LRSAM1 promoted replication, while knockdown inhibited Deletion domain did not affect their interactions but reduced summary, mediate facilitate provides novel insights into role proteins viruses. IMPORTANCE Ubiquitination plays an important nucleoprotein viruses has rarely been investigated. This study aimed at investigating rhabdovirus (snakehead vesiculovirus), identifying related determining found ubiquitination, by (Siah 2) (leucine-rich 1). The data suggested were hijacked ubiquitinate exhibited anti-SHVV targets drug design.

Язык: Английский

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Determination of key hub genes in Leishmaniasis as potential factors in diagnosis and treatment based on a bioinformatics study

Scientific Reports, Год журнала: 2024, Номер 14(1)

Опубликована: Сен. 28, 2024

Язык: Английский

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Molecular characterization of the E2 conjugating enzyme LinfUbc13 in Leishmania infantum.

Archives of Biochemistry and Biophysics, Год журнала: 2024, Номер unknown, С. 110272 - 110272

Опубликована: Дек. 1, 2024

Язык: Английский

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