Improved efficiency of genome editing by constitutive expression of Cas9 endonuclease in genetically-modified mice.

Despite its convenience and precision, CRISPR-based gene editing approaches still suffer from off-target effects and low efficiencies, which are partially rooted in Cas9, the nuclease component of the CRISPR/Cas9 system. In this study, we showed how mouse genome editing efficiency can be improved by constitutive and inheritable expression of Cas9 nuclease. For this goal, a transgenic mouse line expressing the Cas9 protein (Cas9-mouse) was generated. For in vitro assessment of gene editing efficiency, the Cas9-mice were crossed with the EGFP-mice to obtain mouse embryonic fibroblasts (MEF) expressing both EGFP and Cas9 (MEFCas9-EGFP). Transfection of these cells with in vitro transcribed (IVT) EGFP sgRNA or phU6-EGFPsgRNA plasmid led to robust decrease of Mean Fluorescent Intensity (MFI) to 8500 ± 1025 a.u. and 13,200 ± 1006 a.u. respectively. However, in the control group, in which the MEFEGFP cells were transfected with a pX330-EGFPsgRNA plasmid, the measured MFI was 16,800 ± 2254 a.u. For in vivo assessment, the Cas9-zygotes at two pronuclei stage (2PN) were microinjected with a phU6-HhexsgRNA vector and the gene mutation efficiency was compared with the wild-type (WT) zygotes microinjected with a pX330-HhexsgRNA plasmid. The analysis of born mice showed that while the injection of Cas9-zygotes resulted in 43.75% Hhex gene mutated mice, it was just 15.79% for the WT zygotes. In conclusion, the inheritable and constitutive expression of Cas9 in mice provides an efficient platform for gene editing, which can facilitate the production of genetically-modified cells and animals.

Pipeline for the generation of gene knockout mice using dual sgRNA CRISPR/Cas9-mediated gene editing.

Animal models possess undeniable utility for progress on biomedical research projects and developmental and disease studies. Transgenic mouse models recreating specific disease phenotypes associated with ß-hemoglobinopathies have been developed previously. However, traditional methods for gene targeting in mouse using embryonic stem cells (ESCs) are laborious and time consuming. Recently, CRISPR has been developed to facilitate and improve genomic modifications in mouse or isogenic cell lines. Applying CRISPR to gene modification eliminates the time consuming steps of traditional approach including selection of targeted ESC clones and production of chimeric mouse. This study shows that microinjection of a plasmid DNA encoding Cas9 protein along with dual sgRNAs specific to Hbb-bs gene (hemoglobin, beta adult s chain) enables breaking target sequences at exons 2 and 3 positions. The injections led to a knockout allele with efficiency around 10% for deletion of exons 2 and 3 and 20% for indel mutation.