Conditional gene deletion with DiCre demonstrates an essential role for CRK3 in <scp><i>L</i></scp><i>eishmania mexicana</i> cell cycle regulation

Duncan, Sheila L. B.; Myburgh, Elmarie; Philipon, Cintia Iana Monteiro da Silva; Brown, Elaine; Meissner, Markus; Brewer, James M.; Mottram, Jeremy C.

doi:10.1111/mmi.13375

Cited by 58 publications

(79 citation statements)

References 40 publications

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“…Using qPCR, we then examined the CRK3 excision rates after induction and showed that the CRK3 loss was achieved at 90% from D2 post induction onwards; yet the excision was never completely achieved and around 10% of the floxed gene copies persisted in the cell population (Figure 3c). These data correlate with those of Duncan et al, , where a slight amount of CRK3 protein remained detectable after induction. The growth rate of [ CRK3 Flox Puro‐Neo] and [ CRK3‐HA Flox Puro‐Neo] was assessed during 8 days after induction (Figures 3d and S2c, respectively), and a severe growth defect was observed in induced cells compared with non‐induced control cells as early as D2.…”

Section: Resultssupporting

confidence: 91%

“…As a proof of concept of the Floxed gene strategy, we first conditionally deleted the cyclin‐related kinase 3 ( CRK3 ) gene (Figure ), which has previously been characterised as an essential gene in L. mexicana (Duncan et al, ; Hassan, Fergusson, Grant, & Mottram, ). We transfected the L. mexicana Di‐Cre‐Cas9 cell line with PCR products containing the sgRNA and the donor DNA with or without an HA tag (see Section 3) to generate the cell lines [ CRK3 Flox Puro‐Neo] and [ CRK3‐HA Flox Puro‐Neo], respectively (Figures and S2b,c).…”

Section: Resultsmentioning

confidence: 99%

“…The cell line used was an L. mexicana (MNYC/BZ/62/M379) modified cell line with Di‐Cre integrated in the ribosomal locus (Duncan et al, ) and hSpCas9 and T7 polymerase episomally expressed from the plasmid pTB007 (Beneke et al, ). L. mexicana promastigotes were cultivated at 27 °C in haemoflagellate‐modified minimal essential medium (HOMEM) (Gibco®, Life technologies) supplemented with 10% heat inactivated fetal calf serum and 0.002% hemin.…”

Section: Methodsmentioning

confidence: 99%

“…As a proof of concept of the Floxed gene strategy, we first conditionally deleted the cyclin-related kinase 3 (CRK3) gene ( Figure 3), which has previously been characterised as an essential gene in L. mexicana (Duncan et al, 2016;Hassan, Fergusson, Grant, & Mottram, 2001). Figure 3b).…”

Section: Conditional Deletion Of Crk3 Affects L Mexicana Survivalmentioning

confidence: 99%

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Universal highly efficient conditional knockout system in Leishmania , with a focus on untranscribed region preservation

Yagoubat

Crobu

Berry

et al. 2020

Cellular Microbiology

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Trypanosomatids are divergent eukaryotes of high medical and economical relevance. Their biology exhibits original features that remain poorly understood; particularly, Leishmania is known for its high degree of genomic plasticity that makes genomic manipulation challenging. CRISPR‐Cas9 has been applied successfully to these parasites providing a robust tool to study non‐essential gene functions. Here, we have developed a versatile inducible system combining Di‐Cre recombinase and CRISPR‐Cas9 advantages. Cas9 is used to integrate the LoxP sequences, and the Cre‐recombinase catalyses the recombination between LoxP sites, thereby excising the target gene. We used a Leishmania mexicana cell line expressing Di‐Cre, Cas9, and T7 polymerase and then transfected donor DNAs and single guide RNAs as polymerase chain reaction (PCR) products. Because the location of LoxP sequences in the genomic DNA can interfere with the function and localisation of certain proteins of interest, we proposed to target the least transcribed regions upstream and/or downstream the gene of interest. To do so, we developed “universal” template plasmids for donor DNA cassettes with or without a tag, where LoxP sequences may be located either immediately upstream the ATG and downstream the stop codon of the gene of interest, or in the least transcribed areas of intergenic regions. Our methodology is fast, PCR‐based (molecular cloning‐free), highly efficient, versatile, and able to overcome the problems posed by genomic plasticity in Leishmania.

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Section: Resultssupporting

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Conditional Deletion Of Crk3 Affects L Mexicana Survivalmentioning

confidence: 99%

See 2 more Smart Citations

Universal highly efficient conditional knockout system in Leishmania , with a focus on untranscribed region preservation

Yagoubat

Crobu

Berry

et al. 2020

Cellular Microbiology

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“…Deletion of the GOI was induced by rapamycin-mediated DiCre activation, as previously reported (82,83).…”

Section: Dna Constructs and Cell Line Generation A Background Cell Lsupporting

confidence: 77%

Conditional knockout of RAD51-related genes inLeishmania majorreveals a critical role for homologous recombination during genome replication

Damasceno

Reis-Cunha

Crouch

et al. 2019

Preprint

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Homologous recombination (HR) has an intimate relationship with genome replication, both during repair of DNA lesions that might prevent DNA synthesis and in tackling stalls to the replication fork. Recent studies led us to ask if HR might have a more central role in replicating the genome of Leishmania, a eukaryotic parasite. Conflicting evidence has emerged regarding whether or not HR genes are essential, and genomewide mapping has provided evidence for an unorthodox organisation of DNA replication initiation sites, termed origins. To answer this question, we have employed a combined CRISPR/Cas9 and DiCre approach to rapidly generate and assess the effect of conditional ablation of RAD51 and three RAD51-related proteins in Leishmania major. Using this approach, we demonstrate that loss of any of these HR factors is not immediately lethal, but in each case growth slows with time and leads to DNA damage, accumulation of cells with aberrant DNA content, and genome-wide mutation. Despite these similarities, we show that only loss of RAD51 and RAD51-3 impairs DNA synthesis, and that the factors act in distinct ways. Finally, we reveal that loss of RAD51 has a profound effect on DNA replication, causing loss of initiation at the major origins and increased DNA synthesis at subtelomeres. Our work clarifies questions regarding the importance of HR to survival of Leishmania and reveals an unanticipated, central role for RAD51 in the programme of genome replication in a microbial eukaryote.

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The current drug discovery landscape for trypanosomiasis and leishmaniasis: Challenges and strategies to identify drug targets

Altamura

Rajesh

Catta-Preta

et al. 2020

Drug Development Research

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Human trypanosomiasis and leishmaniasis are vector‐borne neglected tropical diseases caused by infection with the protozoan parasites Trypanosoma spp. and Leishmania spp., respectively. Once restricted to endemic areas, these diseases are now distributed worldwide due to human migration, climate change, and anthropogenic disturbance, causing significant health and economic burden globally. The current chemotherapy used to treat these diseases has limited efficacy, and drug resistance is spreading. Hence, new drugs are urgently needed. Phenotypic compound screenings have prevailed as the leading method to discover new drug candidates against these diseases. However, the publication of the complete genome sequences of multiple strains, advances in the application of CRISPR/Cas9 technology, and in vivo bioluminescence‐based imaging have set the stage for advancing target‐based drug discovery. This review analyses the limitations of the narrow pool of available drugs presently used for treating these diseases. It describes the current drug‐based clinical trials highlighting the most promising leads. Furthermore, the review presents a focused discussion on the most important biological and pharmacological challenges that target‐based drug discovery programs must overcome to advance drug candidates. Finally, it examines the advantages and limitations of modern research tools designed to identify and validate essential genes as drug targets, including genomic editing applications and in vivo imaging.

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Conditional gene deletion with DiCre demonstrates an essential role for CRK3 in Leishmania mexicana cell cycle regulation

Cited by 58 publications

References 40 publications

Universal highly efficient conditional knockout system in Leishmania , with a focus on untranscribed region preservation

Universal highly efficient conditional knockout system in Leishmania , with a focus on untranscribed region preservation

Conditional knockout of RAD51-related genes inLeishmania majorreveals a critical role for homologous recombination during genome replication

The current drug discovery landscape for trypanosomiasis and leishmaniasis: Challenges and strategies to identify drug targets

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