Quantitative comparison of CRISPR-Cas9-mediated mutation efficiency between mice and MEFs using digital PCR assays

Abstract

The creation of mutant mice has been invaluable for advancing biomedical science, but is too time- and resource-intensive for investigating the full range of mutations and polymorphisms. Cell culture models are therefore an invaluable complement to mouse models, especially for cell-autonomous pathways like the circadian clock. In this study, we quantitatively assessed the use of CRISPR to create cell models in MEFs as compared to mouse models. We generated two point mutations in the clock genes Per1 and Per2 in mice and in MEFs using the same sgRNAs and repair templates for HDR and quantified the frequency of the mutations by digital PCR. The frequency was about an order of magnitude higher in mouse zygotes compared to that in MEFs. However, the mutation frequency in MEFs was still high enough for clonal isolation by simple screening of a few dozen individual cells. The Per mutant cells that we generated provide important new insights into the role of the PAS domain in regulating PER phosphorylation, a key aspect of the circadian clock mechanism. Accurate quantification of the mutation frequency in bulk MEF populations provides a critical basis for optimizing CRISPR protocols and time/resource planning for generating cell models for further studies.