Title: Abstracts of the 18th Transgenic Technology Meeting (TT2023)
Abstract: CRISPR-Cas9 technology has revolutionized our ability to create genetically modified animals.Many animal models require the need to insert (knock-in) preconstructed DNA templates called repair templates.DNA repair templates along with CRISPR-Cas9 reagents can be introduced into embryos by pronuclear injection (PNI), electroporation (EP), or delivery via adeno-associated virus with electroporation (AAV ?EP).Currently, no published literature compares the efficiency of these delivery techniques as it relates to DNA insertions via CRISPR mediated genome editing in rats.We used a 400-base pair (bp) repair template consisting of homology arms flanking a floxed short artificial intron designed to target exon 2 of the Crh gene.Superovulated Sprague Dawley (SD) female rats mated to SD stud males were used to generate zygotes.Zygotes were randomly assigned into four groups: culture only control, PNI, EP, and AAV ?EP.After manipulation, embryos were cultured to the blastocyst stage and submitted for Next Generation Sequencing (NGS) to detect evidence of genome editing.Embryo survival after one day in culture was significantly less following PNI, 58% (101/175), compared to the culture only control, 98% (109/111).Cleavage rates and development to a 4-cell stage did not differ between embryos that survived 24 h in culture.Knock-in rates for manipulated embryos were 67% (12/18) for PNI, 0% (0/35) for EP, and 63% (22/35) for AAV ?EP.We conclude that PNI decreases embryo survivability but not development, and that EP and AAV ?EP do not decrease embryo survival or development.Using a 400 bp DNA repair template, we found knock-in rates were similar with PNI and AAV ?EP while the template failed to be inserted into the genome with EP only.