Abstract: Objective To incorporate a tandem sequence of single-guide RNA(sgRNA) of Plasmodium falciparum K13 and NUP116 genes into a plasmid to simultaneously edit the two genes based on the clustered regularly-interspaced short palindromic repeat(CRISPR)/CRISPR-associated protein 9(Cas9) method. Methods Primers for K13(kelch protein) and NUP116(nucleoporin) of P. falciparum were designed, and PCR was performed to amplify the homologous regions of K13 and NUP116. Mutation sites were introduced. Similarly, the sgRNAs of K13 and NUP116 were linked by PCR. The homologous regions and tandem sequence of sgRNAs were incorporated into plasmid pL6cs to construct the expression vector pL6-K13-NUP116, which was subsequently transfected into P. falciparum. The genes were edited by the CRISPR/Cas9 system. Transgenic P. falciparum were selected by drugs WR and BSD, from which genome DNA was then extracted. Gene editing was confirmed by PCR, and further by gene sequencing. Results The amplified K13 and NUP116 homologous regions(K13 full-length 557 bp, NUP116 full-length 569 bp) and tandem sgRNA were incorporated into the vector pL6cs to obtain the expression vector pL6-K13-NUP116, which was then successfully transfected into P. falciparum. Positive targets were selected by culturing in the presence of drugs. On day 28 of culture, P. falciparum parasites were seen under a microscope. PCR revealed successful mutations of K13 and NUP116, and sequencing results further confirmed these mutations at target sites. Conclusion The tandem-sgRNA expression vector based on CRISPR/Cas9 gene editing technique can be used to edit different genes of P. falciparum simultaneously.

Key words: Plasmodium falciparum, CRISPR/Cas9, Gene editing