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Cas9-binding duplex. The sgRNAs in the optimized vector, named pX458M,
increase the editing efficiency in some cases and does not elicit a damaging
effect when compared side-by-side with the those in the unmodified pX458 vec-
tor (Fig. 4.3). The sgRNA with the optimized scaffold has been used routinely in
our service.- Cas9 protein vs. Cas9 mRNA. Cas9 can be delivered in either DNA, mRNA, or
protein form. When a DNA expression plasmid is used, it is expected to have a
delayed expression because it requires transcription, and one-cell-stage mouse
embryos are transcriptionally silent until the entry of the S phase [ 50 , 51 ], lead-
ing to a high rate of mosaicism. In addition, the DNA vector can be randomly
integrated into the genome of a subset of embryos, and the prolonged expression
in embryos before its degradation potentially increases the off-target effects. On
the other hand, Cas9 mRNA and protein are not associated with these concerns,
therefore these two forms of delivery are preferred for zygote injection. When
using Cas9 protein, it should be incubated with sgRNAs at 37 °C for a short time
before injection to allow for the formation of ribonucleoproteins (RNPs). We
typically use Cas9 protein because it displays a higher targeting efficiency than
Cas9 mRNA (Fig. 4.4), in line with other reports [ 38 , 41 , 52 , 53 ].- Selection of injection methods. The editing reagents are commonly delivered into
the fertilized zygotes using a standard pronuclear microinjection technique with
some modifications. For instance, the needle can be slightly delayed on pulling
out of the zygote, so that the editing materials can deposit in the cytoplasm, in
addition to the pronucleus. The technique can also be used for the cytoplasmic
injection without penetrating the pronucleus. Although the overall targeting effi-
ciency between pronuclear and cytoplasmic injections is comparable, the latter
method gives a higher rate of live born pups [ 54 , 55 ]. In our practice, we use a
piezo-driven cytoplasmic microinjection technique for the projects that do not
involve a donor plasmid, such as making knockout mice and knock-in mice with
single-strand donor oligos. The advantage of this technique is that it provides
superior zygote survival and birth rates following injection (Table 4.1). As a
result of these high survival rates, we only need 40–60 zygotes for each project.
For a project requiring a donor plasmid, such as generation of mice with Cre or
fluorescent protein knock-in or conditional allele, pronuclear injection is pre-
ferred. Although the number of pups yielded from this injection method is low,
we normally obtain a higher KI efficiency compared to the cytoplasmic injec-
tion. We reason that the pronuclear injection increases the concentration of DNA
donors in the pronucleus where the homologous recombination takes place.
Electroporation poses an attractive alternative delivery method that is simple and
high-throughput, and it does not require highly technical training to operate as
opposed to the microinjection [ 56 – 59 ]. However, it is currently limited to gen-
eration of animal models with gene deletions or knock-in using single-strand
donor oligos.C.L. Yuan and Y.-C. Hu