Generation of albino C57BL/6J mice by CRISPR embryo editing of the mouse tyrosinase locus.

Abstract

After the arrival of the CRISPR/Cas9 genome editing technology, genetic engineering of model organisms has become much faster and more efficient. The development of genetically modified mouse models is also facilitated by the application of various CRISPR methodologies. Although the very first studies utilized pronuclear injection (PNI) of Cas9 mRNA and sgRNAs into the zygote stage embryos to create knockout and knockin mutations, the repertoire of techniques and collection of reagents for CRISPR editing has rapidly expanded. This presents researchers in the field with a versatility of choices for genetic engineering. However, there are not many comparative studies that analysed the efficacy of gene editing when Cas9 and sgRNA/ssDNA oligos were transferred to the embryos by different methodologies. Here, we aimed to compare two different methods, electroporation and PNI. One of the recent developments gaining wide use in mouse model research is the application of electroporation for the introduction of Cas9/sgRNA ribonucleoprotein complexes into zygote stage embryos. Here, we have used this technique to generate albino coat-coloured C57BL/6J mice by targeted inactivation of the mouse tyrosinase gene through indel or knockin mutations. We have also applied the PNI protocol with the same set of reagents, to compare the efficiency of the two techniques in generation of indel and knockin mutations. Although PNI results in signifi- cantly higher efficiency for knockin mutations, it requires specialized equipment setup and advanced training in embryo micromanipulation and microinjection. Therefore, for the generation of simple gene knockouts by indel mutations, electroporation can be used.