CRISPR/Cas9 is the new state-of-the-art approach for genome editing in mammalian systems, and mice remain the organism of choice for understanding human biology and for obtaining preclinical therapeutic data. Scientists at The Jackson Laboratory are not only using this method with great success, but are actively developing methods to further improve throughput efficiency. In a recent article in Genetics (Qin et al. 2015) JAX researchers demonstrate that electroporation can increase delivery throughput of Cas9 mRNA, sgRNA, and donor nucleotides to mouse embryos. The new method allows for more efficient generation of mice with a range of genetic alterations.
In order to perform genome editing with the CRISPR/Cas9 system, multiple components must be delivered to live, fertilized mouse zygotes. These components include: Cas9 mRNA, sgRNA (single guide RNA), and, sometimes, a donor oligonucleotide (the third component is not required to create small deletions). The method most commonly used to introduce these components into the zygote is microinjection into the pronucleus of a zygote. This method requires specialized microinjection equipment, highly skilled technicians, and is very labor intensive and time consuming – individual zygotes must be injected one at a time. A method that would allow delivery of the CRISPR/Cas9 components to multiple zygotes simultaneously would eliminate the technical and temporal bottlenecks associated with microinjection and improve the throughput of creating CRISPR/Cas9-mutated mice.
For years, electroporation has been commonly used to introduce DNA constructs into a wide range of cell types, but had not been optimized for CRISPR/Cas9 genome editing in live animals. Using a multistep approach, researchers at JAX have now adapted and optimized the technique to deliver the CRISPR/Cas9 components to multiple zygotes simultaneously.
The first requirement for delivering CRISPR/Cas9 reagents to the zygote is their efficient passage through the zona pellucida, a glycoprotein layer that surrounds the fertilized oocyte. The JAX team weakened this layer by exposing the embryos for 10 seconds to acidic Tyrode’s solution. Another important step in developing the new electroporation protocol was identifying a medium that would permit the zygote to survive the electroporation procedure. Ultimately, the JAX team found that preparing embryos in Opti-MEM media and the CRISPR/Cas9 components in Tris-EDTA (TE), and combining them in a 1:1 ratio was optimal in supporting both zygote survival and successful development in culture to the blastocyst stage. The next challenge was to determine the optimal voltage and pulse frequency needed to successfully deliver the CRISPR/Cas9 components, while maintaining a high embryo survival rate. Multiple voltages and pulse interval combinations were tested, and a treatment strategy of 30v for 100ms was adopted following observation of a 92% embryo survival rate.
Having established the conditions necessary for the zygotes to be amenable to and to survive the electroporation procedure, the next step was to test the new protocol using reagents to introduce specific allele modifications. Two alleles previously modified by CRISPR/Cas9 and well documented in the literature are Tet1 and Tet2. The JAX researchers attempted to modify Tet1 and Tet2 by targeting, respectively, naturally occurring SacI and EcoRV restriction sites to create gene knockouts. A key variable that the JAX team needed to establish was the ratio of Cas9 mRNA to sgRNA necessary to create the desired mutations. The Tet1 allele was tested at 40/20 ng/µl and 100/50 ng/µl. No modifications were observed in Tet1 zygotes when the Cas9 mRNA and sgRNA were combined at a 40/20 ng/µl ratio. When the reagents were combined at a 100/50 ng/µl ratio, 3 of 15 zygotes with the Sac1 site mutation were produced. The Tet2 allele was tested using a Cas9 mRNA/sgRNA ratio of 400/200 ng/µl and 4 of 9 zygotes contained mutations in the targeted EcoRV site. The genetic modifications were verified by restriction fragment length polymorphism (RFLP) and direct sequencing. This data confirmed that ZEN could be used to deliver components to correctly edit the genomes of live zygotes, and demonstrated the importance of the Cas9 mRNA to sgRNA quantity and ratio in ZEN mutagenesis efficiency.
The next very important question in applying ZEN to CRISPR/Cas9 was whether live mice containing designed genetic modifications could be generated at high efficiency following transfer of the ZEN-treated embryos into pseudopregnant females. When Tet2-specific Cas9 mRNA and sgRNAs were delivered to embryos at 600/300 ng/µl, 18 of 39 electroporated and transferred embryos (~ 46%) were born as live mice. Fourteen of these mice were evaluated, and 8 (~ 57%) were found to carry the desired Tet2 modification. Lower concentrations of Cas9 mRNA and sgRNA ratios (e.g. 200/100 and 400/200 ng/µl) produced lower percentages of founders with the appropriate Tet2 mutations. In a separate experiment, the Tet2 allele was used to test whether a precise genome modification using an oligonucleotide could be achieved (Homology Directed Repair, or HDR). Here, a 126 base length oligonucleotide was included that contained a sequence designed to change two nucleotides in a Tet2 EcoRV restriction site to convert it into an EcoRI site. All 11 mice analyzed contained mutations in EcoRV, and 3 of them (27%) carried the converted EcoRI restriction site by HDR. Finally, 10 other genes were tested for modification by ZEN. In 5 out of 10 of these loci, the JAX team successfully created live mice with edited gene sequences. It is also important to note that ZEN was applicable to both B6D2F2/J and C57BL/6NJ embryos. Together, these results indicate broad and robust applications for the technique.