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CRISPR-Cas13b mediated gene knockdowns in Leishmania infantum
Marine Queffeulou1, Raouia Fakhfakh1, Fereshteh Fani1
1Centre de Recherche en Infectiologie du Centre de Recherche du CHU Québec and Département de Microbiologie, Infectiologie et Immunologie, Faculté de Médecine, Université Laval, Québec, Québec, Canada.
Abstract:
Chemotherapy is an effective means to control infections caused by the protozoan parasite Leishmania. However, available treatments are limited, expensive, and associated with considerable toxicity. Genomic strategies have contributed to a better understanding of Leishmania's response to drugs and in the characterization of drug targets. Nonetheless, there is no knockdown system operational for Leishmania. In this study, we show that the CRISPR-Cas13 system can be an effective strategy to knockdown expression levels of both exogenous and endogenous transcripts. We succeeded in effectively knocking down the expression of the firefly luciferase gene integrated in the genome of L. infantum. This Cas13-mediated decrease in mRNA was paralleled with a significant reduction in both the luciferase protein level and its activity. Furthermore, we tested the effectiveness of the Cas13 system to target the endogenous miltefosine transporter (MT) and the aquaglyceroporin 1 (AQP1) genes. Knockdown was effective and parasites with less MT or AQP1 mRNA levels exhibited reduced susceptibility to miltefosine or antimonials, respectively. While further optimization is warranted, this knockdown system has the potential to facilitate numerous studies related to various aspects of Leishmania biology.
Insights
Researchers developed a CRISPR-Cas13 system for Leishmania parasites, enabling gene knockdown. This tool effectively reduced target gene expression, impacting parasite drug susceptibility and offering new research avenues.
Area of Science:
- Molecular Biology
- Parasitology
- Genetics
Background:
- Current Leishmania treatments are limited by cost and toxicity.
- Genomic approaches have advanced understanding of drug targets but lack knockdown systems.
- A functional knockdown system is crucial for Leishmania research.
Purpose of the Study:
- To establish and validate a CRISPR-Cas13 system for gene knockdown in Leishmania.
- To assess the system's efficacy on both exogenous and endogenous gene expression.
- To investigate the impact of gene knockdown on parasite drug susceptibility.
Main Methods:
- Utilized the CRISPR-Cas13 system to target specific mRNA transcripts in Leishmania infantum.
- Quantified knockdown of firefly luciferase, miltefosine transporter (MT), and aquaglyceroporin 1 (AQP1) genes.
- Assessed the functional consequences of knockdown on protein levels, enzyme activity, and drug sensitivity.
Main Results:
- Successfully achieved significant knockdown of exogenous firefly luciferase mRNA, protein, and activity.
- Demonstrated effective knockdown of endogenous MT and AQP1 genes.
- Reduced MT and AQP1 expression correlated with decreased susceptibility to miltefosine and antimonials, respectively.
Conclusions:
- The CRISPR-Cas13 system is a viable tool for targeted gene knockdown in Leishmania.
- This system facilitates functional genomics studies and drug target validation.
- Further optimization holds promise for advancing Leishmania biology research and therapeutic development.
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