80
different genetic loci, mostly in exons, using the same cellular assay. We found that
78 of these sgRNAs (38%) have activity below the minimum threshold, including
14 (7%) with zero activity (Fig. 4.1b). This suggests that even though the majority
of sgRNAs show some editing activity in cultured cells, more than a third of the
sgRNAs do not pass the activity threshold. sgRNAs below this threshold are unlikely
to show efficient editing in mouse zygotes. Due to the prevalence of low-efficiency
sgRNAs, it is critical to utilize available resources to assess a sgRNA’s editing activ-
ity before costly zygote injection.
4.6 Design of the CRISPR/Cas9-Mediated Targeting
Given that a sgRNA’s activity is the key to the successful genome targeting in mouse
embryos, a substantial amount of research efforts have been devoted to optimize the
system and increase efficiency. Below we describe the strategies that have been
Fig. 4.1 Identification of the minimum sgRNA activity required for genome editing in mouse
embryos. (a) sgRNAs targeting 29 different loci, mostly in exons, in the mouse genome were
cloned into the pX458 vector (addgene #48138) that contains U6 promoter-driven sgRNA and
ubiquitously expressed SpCas9 and GFP. Individual sgRNA vectors were transfected into mK4
cells in 24-well plates using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.). Cells were
examined for GFP expression under a fluorescence microscope to ensure equivalent transfection
efficiency between wells. Two days after transfection, cells were harvested for DNA extraction,
and the T7E1 mismatch cleavage assay was performed. The editing efficiency was measured
according to band intensity on the gel, relative to that of Tet2 sgRNA. Values represent an average
of two replicates for each sgRNA. Then, the same set of sgRNAs were in vitro transcribed and
purified. 50 ng/μL sgRNA and 100 ng/μL Cas9 mRNA (and 100 ng/μL single-strand DNA oligo
for some samples) were injected into fertilized mouse zygotes by a piezo-driven cytoplasmic injec-
tion. Injected zygotes were transferred to pseudopregnant females immediately. Pups were born
and genotyped by PCR and Sanger sequencing. The editing efficiency in animals are expected to
be underestimated because small indels could be overlooked. In addition, deleting the entire func-
tion of the essential genes causes embryonic lethality, favoring the pups with the wild-type alleles
to survive. The dashed line indicates the recommended activity threshold. (b) A survey of the edit-
ing activity of 204 sgRNAs targeting 74 different loci in the mouse genome was performed in the
same cellular assay. The activity threshold was set at 85% of Tet2 sgRNA activity
C.L. Yuan and Y.-C. Hu