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Precision genetic engineering accelerates the genetic improvement of livestock for agriculture and biomedicine. We have recently reported our success in producing gene-modified goats using the CRISPR/Cas9 system through microinjection of Cas9 mRNA and sgRNAs targeting the MSTN and FGF5 genes in goat embryos. By investigating the influence of gene modification on the phenotypes of Cas9-mediated goats, we herein demonstrate that the utility of this approach involving the disruption of FGF5 results in increased number of second hair follicles and enhanced fiber length in Cas9-mediated goats, suggesting more cashmere will be produced. The effects of genome modifications were characterized using H&E and immunohistochemistry staining, quantitative PCR, and western blotting techniques. These results indicated that the gene modifications induced by the disruption of FGF5 had occurred at the morphological and genetic levels. We further show that the knockout alleles were likely capable of germline transmission, which is essential for goat population expansion. These results provide sufficient evidences of the merit of using the CRISPR/Cas9 approach for the generation of gene-modified goats displaying the corresponding mutant phenotypes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The bacterial clustered regularly interspaced short palindromic repeats ( CRISPR)/ CRISPR-associated 9 (Cas9) system is a versatile RNA-guided mammalian genome modification system. One-step generation of mouse genome targeting has been achieved by co-microinjection of one-cell stage embryos with Cas9 m RNA and small/single guide (sg) RNA. Many studies have focused on enhancing the efficiency of this system. In the present study, we report that simultaneous use of dual sg RNAs to target an individual gene significantly improved the Cas9-mediated genome targeting with a bi-allelic modification efficiency of up to 78%. We further observed that the target gene modifications were characterized by efficient germline transmission and site-dependent off-target effects, and also that the apolipoprotein E gene knockout-mediated defects in blood biochemical parameters were recapitulated by CRISPR/Cas9-mediated heritable gene modification. Our results provide a dual sg RNAs strategy to facilitate CRISPR/Cas9-mediated mouse genome targeting. [ABSTRACT FROM AUTHOR]