Figure 1

CRISPR/Cas9 mediated target gene DSBs. A SpCas9 expression was concentrated in the nucleus. The expression of SpCas9, which fused with EYFP, was controlled by EtHis4 promoter and nls as the schematic of the plasmid. Wild-type (WT) E. tenella sporozoites were transfected with the circular plasmid and then cultured in vitro or inoculated into the chicks for in vivo propagation. The EYFP signal was located in the nucleus of the sporozoites, both after in vitro culture and in vivo propagation (sporozoites released from the sporulated oocyst was stained with DAPI and polyclonal antibodies against SAG13 as index of parasites nucleus and periphery). Bar = 5 μm. B CRISPR/Cas9 mediated eyfp disruption. The SpCas9 expression and sgRNA (specific to eyfp) production, which were controlled by His4 and EtU6 promoter, were constructed in one plasmid. EtER sporozoites were transfected with this circular plasmid and then inoculated into the chicks for in vivo propagation. The ratio of fluorescence positive oocysts was decreased in the progeny of mEtER after adverse selection by FACS and propagation. C Mismatch repair of eyfp on the genome of mEtER was analyzed by T7 endonuclease I assay. Specific bands of original (863 bp) and mismatch repair (red arrow, ~430 bp) was detected after the visualization of mEtER. The genomic DNA of EtER and WT serve as control. Scissor logo: homologous sequence to sgRNA on the genome.