Abstract
The CRISPR/Cas9 system has been developed as an easy-handle and multiplexable approach for engineering eukaryotic genomes by zygote microinjection of Cas9 and sgRNA, while preparing Cas9 for microinjection is laborious and introducing inconsistency into the experiment. Here, we describe a modified strategy for gene targeting through using oocyte-specific Cas9 transgenic mouse. With this mouse line, we successfully achieve precise gene targeting by injection of sgRNAs only into one-cell-stage embryos. Through comprehensive analysis, we also show allele complexity and off-target mutagenesis induced by this strategy is obviously lower than Cas9 mRNA/sgRNA injection. Thus, injection of sgRNAs into oocyte-specific Cas9 transgenic mouse embryo provides a convenient, efficient and reliable approach for mouse genome editing.
Highlights
Genetic modified mice are essential tools for scientists to gain the necessary understanding of gene function and diseases, and to discover improved methods to prevent, diagnose and treat diseases
Of the current generation of genome editing approaches, the most rapidly developing is a type of RNA-guided endonucleases known as Cas9 from the microbial adaptive immune system CRISPR [9, 10]
All the mice were housed in Individually Ventilated Cages (IVC) in a specific pathogen-free (SPF) animal facility credited by the Association for Assessment and Accreditation of Laboratory Animals Care (AAALAC) on a 12-hours light and 12-hours dark cycle
Summary
Genetic modified mice are essential tools for scientists to gain the necessary understanding of gene function and diseases, and to discover improved methods to prevent, diagnose and treat diseases. We documented that the successful generation of oocyte specific expression Cas9 transgenic mouse, and the site-specific gene modification can be efficiently achieved by injection of sgRNA only into the transgenic embryos at one-cell stage. Results and Discussion Generation of Oocyte-Specific Cas9 Transgenic Mouse To establish a simplified and consistent CRISPR/Cas9 system for mouse gene targeting, we sought out to generate an oocyte-specific Cas9 transgenic mouse as a tool strain.
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