CRISPR-Cas9: Taming protozoan parasites with bacterial scissor

Pal, Suchetana; Dam, Somasri

doi:10.1007/s12639-022-01534-x

Cited by 4 publications

(3 citation statements)

References 43 publications

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“…By simplifying previous labour-intensive gene replacement strategies ( Cruz and Beverley, 1990 ), CRISPR/Cas9 has greatly improved loss-of-function experiments in Leishmania ( Beneke et al, 2017 ; Sollelis et al, 2015 ; Zhang et al, 2017 ; Zhang and Matlashewski, 2015 ). Over the past 8 years, multiple CRISPR toolkits for kinetoplastids, including Leishmania , have emerged ( Bryant et al, 2019 ; Yagoubat et al, 2020a ; Pal and Dam, 2022 ), enabling specialised gene editing approaches, such as marker free ( Soares Medeiros et al, 2017 ) or inducible gene deletions ( Damasceno et al, 2020 ; Rico et al, 2018 ; Yagoubat et al, 2020b ). In particular, the introduction of the PCR-based LeishGEdit toolbox ( Beneke et al, 2017 ; Beneke and Gluenz, 2019 ; Beneke and Gluenz, 2020 ) has boosted the number of successful loss-of-function studies in Leishmania species ( Bryant et al, 2019 ; Yagoubat et al, 2020a ; Jones et al, 2018 ).…”

Section: Introductionmentioning

confidence: 99%

Gene editing and scalable functional genomic screening in Leishmania species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEedit

Engstler

Beneke

2023

eLife

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CRISPR/Cas9 gene editing has revolutionised loss-of-function experiments in Leishmania, the causative agent of leishmaniasis. As Leishmania lack a functional non-homologous DNA end joining pathway however, obtaining null mutants typically requires additional donor DNA, selection of drug resistance-associated edits or time-consuming isolation of clones. Genome-wide loss-of-function screens across different conditions and across multiple Leishmania species are therefore unfeasible at present. Here, we report a CRISPR/Cas9 cytosine base editor (CBE) toolbox that overcomes these limitations. We employed CBEs in Leishmania to introduce STOP codons by converting cytosine into thymine and created http://www.leishbaseedit.net/ for CBE primer design in kinetoplastids. Through reporter assays and by targeting single- and multi-copy genes in L. mexicana, L. major, L. donovani, and L. infantum, we demonstrate how this tool can efficiently generate functional null mutants by expressing just one single-guide RNA, reaching up to 100% editing rate in non-clonal populations. We then generated a Leishmania-optimised CBE and successfully targeted an essential gene in a plasmid library delivered loss-of-function screen in L. mexicana. Since our method does not require DNA double-strand breaks, homologous recombination, donor DNA, or isolation of clones, we believe that this enables for the first time functional genetic screens in Leishmania via delivery of plasmid libraries.

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Section: Introductionmentioning

confidence: 99%

Gene editing and scalable functional genomic screening in Leishmania species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEedit

Engstler

Beneke

2023

eLife

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“…The tracrRNA binds the Cas9 protein after complementation with crRNA, forming a CRISPR/Cas9–sgRNA complex that produces a double-stranded break at the target site in the genome (Fig. 1 A) [ 44 – 46 ]. The CRISPR/Cas9 system specifically recognizes and shears foreign DNA or RNA in 3 stages: adaptation, expression, and interference [ 47 ].…”

Section: Crispr/cas9 Gene Editing Systemmentioning

confidence: 99%

Biomaterial-Based CRISPR/Cas9 Delivery Systems for Tumor Treatment

Li,

Chen,

Yang

et al. 2024

Biomater Res

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CRISPR/Cas9 gene editing technology is characterized by high specificity and efficiency, and has been applied to the treatment of human diseases, especially tumors involving multiple genetic modifications. However, the clinical application of CRISPR/Cas9 still faces some major challenges, the most urgent of which is the development of optimized delivery vectors. Biomaterials are currently the best choice for use in CRISPR/Cas9 delivery vectors owing to their tunability, biocompatibility, and efficiency. As research on biomaterial vectors continues to progress, hope for the application of the CRISPR/Cas9 system for clinical oncology therapy builds. In this review, we first detail the CRISPR/Cas9 system and its potential applications in tumor therapy. Then, we introduce the different delivery forms and compare the physical, viral, and non-viral vectors. In addition, we analyze the characteristics of different types of biomaterial vectors. We further review recent research progress in the use of biomaterials as vectors for CRISPR/Cas9 delivery to treat specific tumors. Finally, we summarize the shortcomings and prospects of biomaterial-based CRISPR/Cas9 delivery systems.

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“…This is a major challenge for the field despite the fact that CRISPR/Cas9 gene editing has revolutionised loss-of-function experiments in Leishmania (5–8) and has greatly simplified previously labour-intensive gene replacement strategies (9). Over the past 7 years, multiple CRISPR toolkits for kinetoplastids, including Leishmania , have emerged (10–12), enabling specialised gene editing approaches, such as marker free (13) or inducible gene deletions (14–16). In particular, the introduction of the PCR-based LeishGEdit toolbox (5,17,18) has boosted the number of successful loss-of-function studies in Leishmania species (10,11,19).…”

Section: Introductionmentioning

confidence: 99%

Introducing the CRISPR/Cas9 cytosine base editor toolbox ‘LeishBASEedit’ – Gene editing and high-throughput screening inLeishmaniawithout requiring DNA double-strand breaks, homologous recombination or donor DNA

Beneke

Engstler

2022

Preprint

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CRISPR/Cas9 gene editing has revolutionised loss-of-function experiments in Leishmania, the causative agent of leishmaniasis. As Leishmania lack a functional non-homologous DNA end joining pathway however, obtaining null mutants typically requires additional donor DNA, selection of drug resistance-associated edits or time-consuming isolation of clones. Genome-wide loss-of-function screens across different conditions and across multiple Leishmania species are therefore unfeasible at present. Here, we report a CRISPR/Cas9 cytosine base editor (CBE) toolbox that overcomes these limitations. We employed CBEs in Leishmania to introduce STOP codons by converting cytosine into thymine and created www.leishbaseedit.net for CBE primer design in kinetoplastids. Through reporter assays and by targeting single- and multi-copy genes in L. mexicana, L. major, L. donovani and L. infantum, we demonstrate how this tool can efficiently generate functional null mutants by expressing just one single-guide RNA, reaching up to 100% editing rate in non-clonal populations. We then generated a Leishmania-optimised CBE and successfully targeted an essential gene in a plasmid library delivered loss-of-function screen in L. mexicana. Since our method does not require DNA double-strand breaks, homologous recombination, donor DNA or isolation of clones, we believe that this enables for the first time functional genetic screens in Leishmania via delivery of plasmid libraries.

show abstract

CRISPR-Cas9: Taming protozoan parasites with bacterial scissor

Cited by 4 publications

References 43 publications

Gene editing and scalable functional genomic screening in Leishmania species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEedit

Gene editing and scalable functional genomic screening in Leishmania species using the CRISPR/Cas9 cytosine base editor toolbox LeishBASEedit

Biomaterial-Based CRISPR/Cas9 Delivery Systems for Tumor Treatment

Introducing the CRISPR/Cas9 cytosine base editor toolbox ‘LeishBASEedit’ – Gene editing and high-throughput screening inLeishmaniawithout requiring DNA double-strand breaks, homologous recombination or donor DNA

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