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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.

Monkeys serve as important model species for studying human diseases and developing therapeutic strategies, yet the application of monkeys in biomedical researches has been significantly hindered by the difficulties in producing animals genetically modified at the desired target sites. Here, we first applied the CRISPR/Cas9 system, a versatile tool for editing the genes of different organisms, to target monkey genomes. By coinjection of Cas9 mRNA and sgRNAs into one-cell-stage embryos, we successfully achieve precise gene targeting in cynomolgus monkeys. We also show that this system enables simultaneous disruption of two target genes (Ppar-γ and Rag1) in one step, and no off-target mutagenesis was detected by comprehensive analysis. Thus, coinjection of one-cell-stage embryos with Cas9 mRNA and sgRNAs is an efficient and reliable approach for gene-modified cynomolgus monkey generation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Recent genome-wide studies have revealed that the majority of the mouse genome is transcribed as non-coding RNAs (ncRNAs) and growing evidence supports the importance of ncRNAs in regulating gene expression and epigenetic processes. However, the low efficiency of conventional gene targeting strategies has hindered the functional study of ncRNAs in vivo, particularly in generating large fragment deletions of long non-coding RNAs (lncRNAs) with multiple expression variants. The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has recently been applied as an efficient tool for engineering site-specific mutations of protein-coding genes in the genome. In this study, we explored the potential of using the CRISPR/Cas9 system to generate large genomic deletions of lncRNAs in mice. We developed an efficient one-step strategy to target the maternally expressed lncRNA, Rian, on chromosome 12 in mice. We showed that paired sgRNAs can precisely generate large deletions up to 23kb and the deletion efficiency can be further improved up to 33% by combining multiple sgRNAs. The deletion successfully abolished the expression of Rian from the maternally inherited allele, validating the biological relevance of the mutations in studying an imprinted locus. Mutation of Rian has differential effects on expression of nearby genes in different somatic tissues. Taken together, we have established a robust one-step method to engineer large deletions to knockout lncRNA genes with the CRISPR/Cas9 system. Our work will facilitate future functional studies of other lncRNAs in vivo.

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]