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Int J Parasitol. 2018 Sep;48(11):825-832. doi: 10.1016/j.ijpara.2018.03.009. Epub 2018 Jun 15.

Guide RNA selection for CRISPR-Cas9 transfections in Plasmodium falciparum.

Author information

1
The Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA.
2
The Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA. Electronic address: sdesai@niaid.nih.gov.

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.

KEYWORDS:

CRISPR-Cas9 design; DNA transfection; Guide RNA; Malaria; Multigene families; Plasmodium falciparum

PMID:
29906414
DOI:
10.1016/j.ijpara.2018.03.009
[Indexed for MEDLINE]

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