Figure 1.Conventional gene targeting in ES cells versus CRISPR/Cas9‐mediated gene editing in zygotes
Generation of genetically engineered mouse models via ES cell targeting can take an average of 1 year and involves many intermediate steps. In contrast, CRISPR/Cas9 simplifies and accelerates the production of mice carrying the desired mutation (N1). This is achieved by eliminating the need to generate targeted ES cell line. In some cases, CRISPR/Cas9‐mediated embryo editing also produces homozygous mutants, which can add the additional benefit of preliminary phenotype assessment.
Table 1.The percentage cost savings and the timelines using the CRISPR/Cas9 method compared with traditional homologous recombination technology in murine ES cells
CRISPR (in zygotes)
conventional gene targeting
indel (small deletion/insertion)
precise editing (SNP)
conditional KO (LoxP, FRT)
% cost savings
These estimates include project initiation through the N1 stage, at which germline transmission has been achieved and a genetically modified mouse strain has been established. The major difference between the costs and timelines reflects the necessity of performing electroporation, selection, clone picking, and the subsequent screening in mES cells for traditional gene targeting. Downstream phases (injections and subsequent breeding) are similar with respect to the timelines. Note that for conventional targeting for which there are no murine ES cells for the desired background strain, the project timeline will increase by an additional 6–18 months, depending on the extent of backcrossing required to move the mutation to the desired background strain.