The CRISPR-Cas9 System in Entamoeba histolytica Trophozoites: ehcp112 Gene Knockout and Effects on Other Genes in the V1 Virulence Locus

Gene editing enables a better understanding of protein functions. The genome of the protozoan parasite <i>Entamoeba histolytica</i> contains a 4500 bp DNA fragment comprising the <i>ehcp112</i>, <i>ehadh</i>, and <i>ehrabb</i> genes, which together form the V1 virulence locus. Studying these genes has been challenging due to the lack of suitable methodologies. Here, we report the first <i>in vitro</i> and <i>in vivo</i> knockout in <i>E. histolytica</i> (<i>ehcp112</i> gene) using a modified CRISPR-Cas9 strategy and explore its effects on the other V1 locus genes. Confocal and transmission electron microscopy were used to detect the RNP pathway formed by the Cas9 enzyme and the crRNA-tracrRNA complex, from their entry into the trophozoites until their arrival at the nucleus and heterochromatin. Scanning electron microscopy revealed that the mutant cells (EhCP112-KO) were smaller, with fewer pseudopodia and plasma membrane depressions. DNA sequencing and RT-qPCR assays identified a four-base deletion in the <i>ehcp112</i> gene in the mutant trophozoites. Western blot assays of EhCP112-KO extracts revealed the absence of the EhCP112 protein. While the EhCP112-KO lysates digested gelatin more efficiently than the HM1:IMSS extracts, their secreted products showed poor enzymatic activity. The <i>ehcp112</i> knockout also affected the transcription of the <i>ehadh</i> and <i>ehrabb</i> genes, probably due to their genomic position. In conclusion, the implementation of the CRISPR-Cas9 strategy in <i>E. histolytica</i> evidenced the coordinated expression of the <i>ehcp112</i> gene and the other members of the V1 locus.