Tools for genome engineering in rodents
Transgenic mice have become powerful systems for understanding gene function and modeling genetic disorders. The classic genome engineering strategy in mice introduces intended mutations at a given endogenous locus in rodent embryonic stem cells (ESCs) via homologous recombination. The ESCs carrying the desired modifications are injected into recipient blastocysts to generate chimeric offspring, which are subsequently bred to establish a mutant animal line .
Genetically modified mice have also been produced by modern genome engineering techniques that directly introduce double-strand breaks at defined loci using programmable endonucleases, such as zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), or clustered regularly interspaced short palindromic repeats (CRISPR) nuclease [2–5]. More recently, the CRISPR-Cas9 system has been widely perceived as the preferred approach for rodent genome engineering, due to its efficiency and simplicity.
Microinjection of CRISPR components presents challenges
Delivery of pre-assembled Cas9 ribonucleoprotein (RNP) complexes facilitates highly efficient genome editing in multiple model organisms [6,7]. As the gold standard technique for delivery of nucleic acids into embryos, microinjection also is routinely used to introduce CRISPR components—such as Cas9 RNP complexes—into rodent embryos . However, conventional pronuclear microinjection is a labor-intensive, costly procedure that requires sophisticated equipment and highly skilled personnel. It is also time-consuming, as embryos need to be injected individually . These features pose substantial obstacles for researchers to readily adopt the CRISPR-Cas9 system to efficiently generate strains carrying mutations of interest.
Successful high throughput delivery with a novel electroporation method
However, electroporation can work as an alternative approach for delivering CRISPR RNPs into embryos. Drs Kaneko and Mashimo from the Universities of Kyoto and Osaka, Japan, described a newly optimized electroporation approach, “Technique for Animal Knockout system by Electroporation” (TAKE), which allows easy and rapid, high throughput generation of engineered animals . In this study, they used a novel electroporation platform, the NEPA21 electroporator (Nepa Gene Company,* Ltd, Japan), to enable a 4-step electroporation pulse .
Using their electroporation protocol, the researchers efficiently introduced CRISPR-Cas9 reagents into mouse and rat zygotes, without the need for special handling skills or weakening the zona pellucida, which is required for conventional embryo electroporation but can have negative impacts on subsequent embryo development. As shown in Table 1, Dr Kaneko tested TAKE in rat embryos and compared its performance to the conventional microinjection approach. With the use of Cas9 endonuclease and gRNA obtained from IDT, he achieved 100% desired gene knock-out (verified by Sanger sequencing) in the resulting 17 live offspring .
|Methods||Embryos delivered||offspring||edited offspring|
|Microinjection||40||13 (68%)||10 (77%)|
|TAKE||25||17 (68%)||17 (100%)|
Protocols for optimizing electroporation in your system
Outside Japan, the NEPA21 electroporator can be obtained through the Nepa Gene Company distributor network (www.nepagene.jp/e_about02.html). Here you can access protocols for mouse zygote electroporation and human-induced iPS cells using the NEPA21 Electroporator. These protocols serve as a starting point for using the Alt-R® CRISPR-Cas9 system in similar model organisms.*IDT has no interests or affiliation with Nepa Gene Company, the manufacturer of the electroporation system.