Floxing by Electroporating Single-Cell Embryos with Two CRISPR RNPs and Two ssODNs

Floxed alleles and Cre drivers are two components of most conditional knockout mouse models, which are not only important for studying a given gene in a tissue-specific manner, but also useful for functional analysis of various sized genomic regions. With the increased demand for floxed mouse models in biomedical research, reliable and economical creation of floxed alleles is clearly highly valuable yet remains challenging. Here we provide technical details on the method consisting of electroporating single-cell embryos with CRISPR RNPs and ssODNs, next-generation sequencing (NGS)-based genotyping, an in vitro Cre assay (recombination followed by PCR) for loxP phasing determination, and optional second round targeting of an indel in cis with one loxP insertion in embryos obtained via in vitro fertilization (IVF). As importantly, we present protocols for validation of gRNAs and ssODNs before electroporation of embryos, to confirm phasing of loxP and the indel to be retargeted in individual blastocysts and an alternative strategy to insert loxP sites sequentially. Together, we hope to help researchers reliably obtain floxed alleles in a predictable and timely manner.