Fig. 2
Eimeria genome editing using CRISPR/Cas system and its application. A DNA double-strand breaks (DSB) mediated by CRISPR/Cas system: Illustration of DSB principle induced by CRISPR/Cas system. B Two repair modes of DSB. a. Non-homologous end joining (NHEJ): Causes insertion/deletion mutations in the targeted locus. b. Homologous direct repair (HDR): Occurs with a DNA repair template, enabling single nucleotide correction/introduction or DNA sequence insertion. C Strategies for Eimeria genome editing each with three steps: Strategy Design and Plasmid/Repair Template Construction â‘ Begin by designing the editing strategy and constructing plasmids or repair templates. a. Strategy 1: Utilizes Cas9, sgRNA expression cassettes, circular plasmids, and repair template DNA. Wide applicability but limited by low efficacy in multi-plasmids co-transfection. b. Strategy 2: Employs a single repair template DNA with an sgRNA expression cassette. Applicable only to stable Cas9 expression strain. c. Strategy 3: RNP-Mediated Genome Editing: Wide applicability but lower efficacy in vitro. Transfection â‘¡ Optimize transfection conditions for efficient delivery of editing components into Eimeria cells. Selection and Propagation â‘¢ Enrich and propagate the modified Eimeria population. Utilize drug-mediated selection or other techniques to isolate edited recombinants. Transfected sporozoites or merozoites were inoculated via cloacal (suitable to E. tenella, E. mitis and E. necatrix) or intravenous injection (suitable to E. acervulina) and genome modified Eimeira oocyst were selected by fluorescent protein activated cell sorting (FACS) and/or drug(s) pressure. GOI: Gene of Interest; Selection Marker: Fluorescent protein and/or drug-resistance genes