Search

Bacterial RNA-directed Cas9 endonuclease is a versatile tool for site-specific genome modification in eukaryotes. Co-microinjection of mouse embryos with Cas9 mRNA and single guide RNAs induces on-target and off-target mutations that are transmissible to offspring. However, Cas9 nickase can be used to efficiently mutate genes without detectable damage at known off-target sites. This method is applicable for genome editing of any model organism and minimizes confounding problems of off-target mutations. [ABSTRACT FROM AUTHOR]

Highlights: [•] Embryo co-microinjection of Cas9 mRNA and multiple sgRNAs leads to heritable multiple gene modifications. [•] One-step generating multiple immunodeficient mouse models with multiallelic mutations. [•] Optimal concentration of Cas9 mRNA and sgRNAs improved targeting efficiency. [•] Targeting one site with two adjacent sgRNAs increases Cas9:sgRNA-mediated targeting efficiency and bi-allelic mutations. [ABSTRACT FROM AUTHOR]

Co-injection of zygotes with Cas9 mRNA and sgRNA has been proven to be an efficient gene-editing strategy for genome modification of different species. Genetic engineering in pigs holds a great promise in biomedical research. By co-injection of one-cell stage embryos with Cas9 mRNA and Npc1l1 sgRNA, we achieved precise Npc1l1 targeting in Chinese Bama miniature pigs at the efficiency as high as 100%. Meanwhile, we carefully analyzed the Npc1l1 sgRNA:Cas9-mediated on- and off-target mutations in various somatic tissues and ovaries, and demonstrated that injection of zygotes with Cas9 mRNA and sgRNA is an efficient and reliable approach for generation of gene-modified pigs. [ABSTRACT FROM AUTHOR]