85Fig. 4.3 Use of optimized sgRNA scaffold increases editing activity in some cases without a
damaging effect. The sgRNA target sequences were cloned into both original pX458 and opti-
mized pX458M vectors, and transfections were done side-by-side in mK4 cells. Editing activity
was assessed by the T7E1 assay relative to that of Tet2 sgRNA. Values here are means ± standard
deviation from two independent experiments. P two-tailed Student’s t-test
Fig. 4.4 Use of Cas9 protein enhances editing activity, compared to Cas9 mRNA. sgRNA mixed
with either Cas9 mRNA or Cas9 protein was injected into fertilized mouse zygotes, and injected
zygotes were transferred into pseudopregnant females for continued development. Editing activity
in live born pups was assessed by genotyping PCR and Sanger sequencing
Table 4.1 Percentage of
embryo survival and birth
rate following pizeo-driven
cytoplasmic injection
StrainEmbryo survival
after injectionBirth rate of
transferred embryos
C57BL/6 90.1 ± 5.5% 32.1 ± 13.2%
B6D2F2 86.5 ± 7.6% 30.8 ± 10.9%
FVB/N 89.0 ± 8.0% 34.9 ± 9.2%
Data were collected from the service we performed between
June 2014 and September 20154 A Transgenic Core Facility’s Experience in Genome Editing Revolution