One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering

Abstract

SUMMARY The type II bacterial CRISPR/Cas system is a novel genome engineering technology with the ease of multiplexed gene targeting. Here we created reporter and conditional mutant mice by co-injection of zygotes with Cas9 mRNA, different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2 and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances.

Chromosome engineering in zygotes with CRISPR/Cas9

“…The CRISPR/Cas9 system is a very efficient and comparatively fast method to generate genome edited mice with small modifications such as insertion–deletions of a few nucleotides and the introduction of short tags via direct injection in zygotes (Wang et al ., 2013; Yang et al ., 2013, 2014). Here we extend this technology to very large chromosomal rearrangements, facilitating the facile generation of mouse models with this important class of structural variants.…”

Generating rats with conditional alleles using CRISPR/Cas9

“…In comparison with ZFNs or TALENs, CRISPR/ Cas9 provides a simpler way to edit the eukaryotic genome even in a multiplex manner [4][5]. Recently, mice carrying conditional alleles have been generated by using CRISPR/Cas9 system [7]. Here, we extend the application of the CRISPR/Cas9 system and report an effective strategy to generate rats with conditional alleles.…”

“…DNA sequencing of the PCR products confirmed the correct targeting (data not shown). More- Considering that 2 sgRNAs can efficiently delete the intervening region [7] Figure S4B and S4C). The circular donor vector for each gene contains 2 mloxP sites (each site locates 3 bp away from the corresponding PAM), the floxed exon1 and 2 homology arms ( Figure 1A and 1C).…”

“…A mixture of Cas9 mRNA (25 ng/µl) and sgRNA (10 ng/µl) was pooled with circular donor vectors (4 ng/µl), and microinjected into one-cell stage fertilized eggs of Sprague Dawley (SD) rat (Supplementary information, Table S2). The circular donor vector was used to minimize random integrations [2,7].…”

Caution required for handling genome editing technology

“…The nucleases may fail to induce a biallelic modification in diploid organisms, thereby resulting in an organism with a monoallelic modification [2]. Furthermore, the microinjection of the nuclease mRNAs into zygotes may induce not only germline modifications, but also mosaic modifications in which wild type cells, including germline, and genetically modified cells coexist in the resultant organisms [3].…”