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Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum
Published on: December 4, 2015
Guide RNA selection for CRISPR-Cas9 transfections in Plasmodium falciparum
Jose M Ribeiro1, Meera Garriga1, Nicole Potchen1
1The Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA.
Abstract:
CRISPR-Cas9 mediated genome editing is addressing key limitations in the transfection of malaria parasites. While this method has already simplified the needed molecular cloning and reduced the time required to generate mutants in the human pathogen Plasmodium falciparum, optimal selection of required guide RNAs and guidelines for successful transfections have not been well characterised, leading workers to use time-consuming trial and error approaches. We used a genome-wide computational approach to create a comprehensive and publicly accessible database of possible guide RNA sequences in the P. falciparum genome. For each guide, we report on-target efficiency and specificity scores as well as information about the genomic site relevant to optimal design of CRISPR-Cas9 transfections to modify, disrupt, or conditionally knockdown any gene. As many antimalarial drug and vaccine targets are encoded by multigene families, we also developed a new paralog specificity score that should facilitate modification of either a single family member of interest or multiple paralogs that serve overlapping roles. Finally, we tabulated features of successful transfections in our laboratory, providing broadly useful guidelines for parasite transfections. Molecular studies aimed at understanding parasite biology or characterising drug and vaccine targets in P. falciparum should be facilitated by this comprehensive database.
Insights
This study introduces a genome-wide database of guide RNA sequences for CRISPR-Cas9 genome editing in Plasmodium falciparum malaria parasites. It provides efficiency scores and guidelines to streamline gene modification and target validation.
Area of Science:
- Genetics
- Molecular Biology
- Parasitology
Background:
- CRISPR-Cas9 genome editing offers advancements for Plasmodium falciparum transfection, simplifying molecular cloning and mutant generation.
- However, suboptimal guide RNA selection and poorly characterized transfection protocols necessitate time-consuming trial-and-error methods.
Purpose of the Study:
- To develop a comprehensive, publicly accessible database of guide RNA sequences for P. falciparum.
- To provide on-target efficiency, specificity scores, and genomic site information for optimal CRISPR-Cas9 design.
- To facilitate targeted gene modification, disruption, or knockdown, including for multigene families.
Main Methods:
- A genome-wide computational approach was employed to identify and score potential guide RNA sequences in the P. falciparum genome.
- On-target efficiency, specificity, and paralog specificity scores were calculated for each guide RNA.
- Successful transfection parameters were tabulated from laboratory experiments to establish practical guidelines.
Main Results:
- A comprehensive database of P. falciparum guide RNA sequences with associated efficiency and specificity scores is now available.
- A novel paralog specificity score aids in targeting single or multiple members of gene families.
- Guidelines for successful P. falciparum transfections based on laboratory data are provided.
Conclusions:
- This database and associated guidelines will significantly accelerate molecular studies in P. falciparum.
- It will aid in understanding parasite biology and identifying antimalarial drug and vaccine targets.
- The resource is expected to facilitate efficient gene editing and functional genomics in malaria research.
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