CRISPRing protozoan parasites to better understand the biology of diseases

Kirti, Apurva; Sharma, Manish; Rani, Komal; Bansal, Abhisheka

doi:10.1016/bs.pmbts.2021.01.004

Cited by 5 publications

(4 citation statements)

References 133 publications

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“…While genome sequencing helps to dissect the genome and identify its function, by introducing CRISPR, scientists are now able to manipulate genes, create new sequences, and introduce them to the genome, such as the work done on the schistosome genome [88,89]. Recently, gene-editing platforms have emerged as a treatment for parasitic diseases, which can modify the host genes required by the parasite or target the parasitic genes needed for replication [90][91][92]. This system uses Cas9 endonuclease to create a double-strand break (DSB) at a selected locus in the genome.…”

Section: Crispr and Its Application In Parasitologymentioning

confidence: 99%

“…Nevertheless, the absence of some pathways, such as the non-homologous end joining pathway in many parasites, limits the use of CRISPR for genome-wide screening. On other hand, better understanding of repair pathways such as DNA double-strand break repair in parasites may help exploit alternative pathways for extensive genome functional studies [2,90,91,106]. Several other microbes have been targeted successfully by CRISPR methods (Table 2, [107]).…”

Section: Crispr and Its Application In Parasitologymentioning

confidence: 99%

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CRISPR-Cas Technology: Emerging Applications in Clinical Microbiology and Infectious Diseases

et al. 2021

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Through the years, many promising tools for gene editing have been developed including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR-associated protein 9 (Cas9), and homing endonucleases (HEs). These novel technologies are now leading new scientific advancements and practical applications at an inimitable speed. While most work has been performed in eukaryotes, CRISPR systems also enable tools to understand and engineer bacteria. The increase in the number of multi-drug resistant strains highlights a necessity for more innovative approaches to the diagnosis and treatment of infections. CRISPR has given scientists a glimmer of hope in this area that can provide a novel tool to fight against antimicrobial resistance. This system can provide useful information about the functions of genes and aid us to find potential targets for antimicrobials. This paper discusses the emerging use of CRISPR-Cas systems in the fields of clinical microbiology and infectious diseases with a particular emphasis on future prospects.

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Section: Crispr and Its Application In Parasitologymentioning

confidence: 99%

Section: Crispr and Its Application In Parasitologymentioning

confidence: 99%

CRISPR-Cas Technology: Emerging Applications in Clinical Microbiology and Infectious Diseases

et al. 2021

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“…Using Cas9 to precisely engineer a double-strand break (DSB) enhances the efficiency of gene editing in Plasmodium when using a standard length (≤1000 bp) homology region (HR) 14 . Since Plasmodium lacks the pathway for canonical non-homologous end-joining (c-NHEJ), any CRISPR-Cas9 mediated edit requires a homology directed repair (HDR) template to facilitate DSB repair 15 . This prohibits the adoption of standard CRISPR-Cas9 disruption screens that rely on c-NHEJ, which introduces insertion and deletion mutations during repair.…”

Section: Introductionmentioning

confidence: 99%

A scalable CRISPR-Cas9 gene editing system facilitates CRISPR screens in the malaria parasitePlasmodium berghei

Jonsdottir,

Paoletta,

Ishizaki

et al. 2024

Preprint

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ManyPlasmodiumgenes remain uncharacterised due to low genetic tractability. Previous large scale knockout screens have only been able to target about half of the genome in the more genetically tractable rodent malaria parasitePlasmodium berghei. To overcome this limitation, we have developed a scalable CRISPR system called PbHiT, which uses a single cloning step to generate targeting vectors with 100 bp homology arms physically linked to a guide RNA (gRNA) that effectively integrate into the target locus. We show that PbHiT coupled with gRNA sequencing robustly recapitulates known knockout mutant phenotypes in pooled transfections. Furthermore, we provide vector designs and sequences to target the entireP. bergheigenome and scale-up vector production using a pooled ligation approach. This work presents for the first time a tool for high-throughput CRISPR screens inPlasmodiumfor studying the parasite′s biology at scale.

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“…The inclusion of a DNA donor to induce HDR increased the genome editing efficiency to 100%, generating a homogeneous double knockout population by replacement of both alleles with a single resistance marker in a few weeks, a result never achieved in T. cruzi before [6]. This strategy has been successfully used for endogenous tagging of several genes [9] and for the functional characterization of about 20 proteins (reviewed by [17][18][19]), including important players in calcium signaling and homeostasis, [20][21][22][23][24][25][26], which evidences the usefulness of the system to efficiently modify the genome of T. cruzi. It is important to mention that knockout cell lines obtained through this strategy have been complemented with either wild type or mutant versions of the added-back gene [21].…”

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confidence: 99%

The long and winding road of reverse genetics in Trypanosoma cruzi

Chiurillo

Lander

2021

Microb Cell

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Trypanosomes are early divergent protists with distinctive features among eukaryotic cells. Together with Trypanosoma brucei and Leishmania spp., Trypanosoma cruzi has been one of the most studied members of the group. This protozoan parasite is the causative agent of Chagas disease, a leading cause of heart disease in the Americas, for which there is no vaccine or satisfactory treatment available. Understanding T. cruzi biology is crucial to identify alternative targets for antiparasitic interventions. Genetic manipulation of T. cruzi has been historically challenging. However, the emergence of CRISPR/Cas9 technology has significantly improved the ability to generate genetically modified T. cruzi cell lines. Still, the system alone is not sufficient to answer all biologically relevant questions. In general, current genetic methods have limitations that should be overcome to advance in the study of this peculiar parasite. In this brief historic overview, we highlight the strengths and weaknesses of the molecular strategies that have been developed to genetically modify T. cruzi, emphasizing the future directions of the field.

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CRISPRing protozoan parasites to better understand the biology of diseases

Cited by 5 publications

References 133 publications

CRISPR-Cas Technology: Emerging Applications in Clinical Microbiology and Infectious Diseases

CRISPR-Cas Technology: Emerging Applications in Clinical Microbiology and Infectious Diseases

A scalable CRISPR-Cas9 gene editing system facilitates CRISPR screens in the malaria parasitePlasmodium berghei

The long and winding road of reverse genetics in Trypanosoma cruzi

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