Efficient Single-Gene and Gene Family Editing in the Apicomplexan Parasite Eimeria tenella Using CRISPR-Cas9

Frontiers in Bioengineering and Biotechnology
Dandan HuXianyong Liu

Abstract

Eimeria species are pathogenic protozoa with a wide range of hosts and the cause of poultry coccidiosis, which results in huge economic losses to the poultry industry. These parasites encode a genome of ∼8000 genes that control a highly coordinated life cycle of asexual replication and sexual differentiation, transmission, and virulence. However, the function and physiological importance of the large majority of these genes remain unknown mostly due to the lack of tools for systematic analysis of gene functions. Here, we report the first application of CRISPR-Cas9 gene editing technology in Eimeria tenella for analysis of gene function at a single gene level as well as for systematic functional analysis of an entire gene family. Using a transgenic line constitutively expressing Cas9, we demonstrated successful and efficient loss of function through non-homologous end joining as well as guided homologous recombination. Application of this approach to the study of the localization of EtGRA9 revealed that the gene encodes a secreted protein whose cellular distribution varied during the life cycle. Systematic disruption of the ApiAp2 transcription factor gene family using this approach revealed that 23 of the 33 factors expressed b...Continue Reading

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Citations

Dec 3, 2020·International Journal for Parasitology·Monica Mendes Kangussu-MarcolinoUpinder Singh
Jan 1, 2021·Frontiers in Cellular and Infection Microbiology·Bruno Martorelli Di Genova, Laura J Knoll
Oct 2, 2021·PloS One·Matthew S TuckerBenjamin M Rosenthal
Oct 12, 2021·Frontiers in Genetics·Peipei ChengFeng Gu

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Methods Mentioned

BETA
fluorescence microscopy
transfection
Transgenic
confocal microscopy
PCR
FACS
flow cytometry
Fluorescence
transfections
transfecting

Software Mentioned

samtools
OPTIMIZER
GATK
OFFinder
freebayes
Cas
mem
bwa

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