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Mol Biochem Parasitol. 2018 Jun;222:21-28. doi: 10.1016/j.molbiopara.2018.04.003. Epub 2018 Apr 21.

A Cas9 transgenic Plasmodium yoelii parasite for efficient gene editing.

Author information

1
State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signal Network, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China.
2
Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, 20892, United States.
3
State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signal Network, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China. Electronic address: cuihuiting@xmu.edu.cn.
4
State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signal Network, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China. Electronic address: yuanjing@xmu.edu.cn.

Abstract

The RNA-guided endonuclease Cas9 has applied as an efficient gene-editing method in malaria parasite Plasmodium. However, the size (4.2 kb) of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for genome editing in the parasites only introduced with cas9 plasmid. To establish the endogenous and constitutive expression of Cas9 protein in the rodent malaria parasite P. yoelii, we replaced the coding region of an endogenous gene sera1 with the intact SpCas9 coding sequence using the CRISPR/Cas9-mediated genome editing method, generating the cas9-knockin parasite (PyCas9ki) of the rodent malaria parasite P. yoelii. The resulted PyCas9ki parasite displays normal progression during the whole life cycle and possesses the Cas9 protein expression in asexual blood stage. By introducing the plasmid (pYCs) containing only sgRNA and homologous template elements, we successfully achieved both deletion and tagging modifications for different endogenous genes in the genome of PyCas9ki parasite. This cas9-knockin PyCas9ki parasite provides a new platform facilitating gene functions study in the rodent malaria parasite P. yoelii.

KEYWORDS:

Cas9; Genome modifications; Knockin; Plasmodium yoelii; Rodent malaria

[Indexed for MEDLINE]

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