Super-Mendelian inheritance mediated by CRISPR/Cas9 in the female mouse germline

Abstract

A gene drive biases the transmission of a particular allele of a gene such that it is inherited at a greater frequency than by random assortment. Recently, a highly efficient gene drive was developed in insects, which leverages the sequence-targeted DNA cleavage activity of CRISPR/Cas9 and endogenous homology directed repair mechanisms to convert heterozygous genotypes to homozygosity. If implemented in laboratory rodents, this powerful system would enable the rapid assembly of genotypes that involve multiple genes (e.g., to model multigenic human diseases). Such complex genetic models are currently precluded by time, cost, and a requirement for a large number of animals to obtain a few individuals of the desired genotype. However, the efficiency of a CRISPR/Cas9 gene drive system in mammals has not yet been determined. Here, we utilize an active genetic \"CopyCat\" element embedded in the mouse Tyrosinase gene to detect genotype conversions after Cas9 activity in the embryo and in the germline. Although Cas9 efficiently induces double strand DNA breaks in the early embryo and is therefore highly mutagenic, these breaks are not resolved by homology directed repair. However, when Cas9 expression is limited to the developing female germline, resulting double strand breaks are resolved by homology directed repair that copies the CopyCat allele from the donor to the receiver chromosome and leads to its super-Mendelian inheritance. These results demonstrate that the CRISPR/Cas9 gene drive mechanism can be implemented to simplify complex genetic crosses in laboratory mice and also contribute valuable data to the ongoing debate about applications to combat invasive rodent populations in island communities.