Redefining mouse transgenesis with CRISPR/Cas9 genome editing technology

Abstract

The generation of genetically modified alleles in mice using conventional transgenesis technologies is a long and inefficient process. A new study shows that the in situ delivery of CRISPR/Cas9 reagents into pregnant mice results in a high efficiency of editing, and enables the rapid generation of both simple and complex alleles.

Fig. 1

a Generation of knockout and knockin alleles using embryonic stem (ES) cell technology in mice. A cloning procedure is undertaken to insert the construct into a plasmid vector as a template to replace the endogenous locus. This template could be a drug-selection cassette only (knockout) or an exon flanked with two loxP sites, or a more complex feature (knockin). These vectors contain a positive and negative selection cassette. The plasmid is then electroporated into the ES cells and then drug selected in vitro. After verification that the sequence is correctly inserted, the cells are microinjected into a blastocyst, before being surgically transferred into pseudopregnant females. The chimeric progenies will be genotyped to ensure the expected construct is correctly inserted into the genome by homologous recombination. b Generation of complex alleles using improved-genome editing via oviductal nucleic acid delivery (i-GONAD) technology. One or two single guide RNAs (sgRNA) are designed to either disrupt a critical exon (knockout) or remove an entire exon for replacement with a repair template (knockin). The sgRNAs are synthesized, or in vitro transcribed, and then complexed with the tracrRNA and then Cas9 protein to form a ribonucleoprotein (RNP) complex. The RNPs are in situ electroporated with a long single-stranded oligonucleotide repair template (ssODN) into the oviduct of a pregnant female. The progenies are genotyped to ascertain successful editing of the gene of interest