Abstract
Functional genomics in a mammalian model such as mice is fundamental for understanding human biology. The CRISPR/Cas9 system dramatically changed the tempo of obtaining genetic mouse models due to high efficiency. However, experimental evidence for the establishment of sgRNA knock-in animals and analyses of their value in functional genomics are still not sufficient, particularly in mammalian models. In this study, we demonstrate that the establishment of sgRNA knock-in mice is feasible, and more importantly, crosses between sgRNA knock-in mice and the Cas9 constitutively expressing mice result in complete deletion of the target gene. Such sgRNA knock-in provides an alternative approach for in vivo genetic modification and can be useful in multiple circumstances, such as maintenance of genetically modified animals, which are difficult to breed as homozygotes, and cross of such mice to diverse genomic backgrounds to obtain genetically modified animals.
Highlights
Mouse models are among the most frequently used model animals in understanding human biology
To test the feasibility of single-guide RNA (sgRNA) knock-in at the Rosa26 locus in mice, we obtained a targeting vector that served as a homologydirected repair (HDR) template, including two independent sgRNAs targeting Gfi1, an essential gene for neutrophil development (Ordoñez-Rueda et al, 2012)
To determine whether the two U6-sgRNAs were expressed or not in the early developmental stage, approximately 700 two-cell embryos derived from in vitro fertilization (IVF) using sperms of Rosa26U6-sgRNA-Gfi1 homozygous mice and WT B6 oocytes were subjected to RNA preparation and real-time qPCR for evaluating the expression of sgRNA in comparison to embryos derived from both WT B6 sperms and oocytes (Figure 1C)
Summary
Mouse models are among the most frequently used model animals in understanding human biology. Genetic deletion of essential genes, which are highly represented in mammalian genomes, can lead to the lethality of animals or physiological abnormality such as immune deficiency. As Cas nuclease may function to cleave double-strand DNA when coupled to sgRNA, it is reasonable to cross the wellestablished Cas9-expressing line such as Rosa26-Cas knock-in mice (Platt et al, 2014) to the sgRNA-expressing knock-in mice. Both Cas9- and sgRNA-expressing mice can be maintained with the intact genome, without deficiency in the gene of interest, allowing for rapid expansion of the colony
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