Background: Floxed (flanked by loxP) alleles are a crucial portion of conditional knockout mouse models. However, an efficient and reliable strategy to flox genomic regions of any desired size is still lacking.
Results: Here, we demonstrate that the method combining electroporation of fertilized eggs with gRNA/Cas9 complexes and single-stranded oligodeoxynucleotides (ssODNs), assessing phasing of loxP insertions in founders using an in vitro Cre assay and an optional, highly specific and efficient second-round targeting ensures the generation of floxed F1 animals in roughly five months for a wide range of sequence lengths (448 bp to 160 kb reported here).
Conclusions: Floxed alleles can be reliably obtained in a predictable timeline using the improved method of electroporation of two gRNA/Cas9 ribonucleoprotein particles (RNPs) and two ssODNs.
Keywords: CRISPR; Conditional knockout mice; Floxing; Functional genomics; Gene editing; Mouse models.
© 2022. The Author(s).