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 β-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. •Simple and flexible design of the CRISPR/Cas9 system to make it as a versatile tool suitable for the genome engineering.•Using an all-in-one plasmid vector containing dual sgRNA cassettes against the target gene.•Increased the probability of indel mutations in the target gene.