Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse

Abstract

The CRISPR/Cas9 system is a powerful tool for elucidating the roles of genes in a wide variety of organisms including mice. To obtain genetically modified embryos or mice by this method, Cas9 mRNA and sgRNA are usually introduced into zygotes by microinjection or electroporation. However, most mutants generated with this method are genetically mosaic, composed of several types of cells carrying different mutations, which complicates phenotype analysis in founder embryos or mice. To simplify the analysis and to elucidate the roles of genes involved in developmental processes, a method for producing non-mosaic mutants is needed. Here, we established a method for generating non-mosaic mouse mutant embryos. We introduced Cas9 protein and sgRNA into in vitro fertilized (IVF) zygotes by electroporation, which enabled the genome editing to occur before the first replication of the mouse genome. As a result, all of the cells in the mutant carried the same set of mutations. This method solves the problem of mosaicism/allele complexity in founder mutant embryos or mice generated by the CRIPSR/Cas9 system.

Responsible innovation in human germline gene editing: Background document to the recommendations of ESHG and ESHRE

“…For example, microinjection of the genomic tool machinery into zygotes only (not embryos) is a technically demanding and laborintensive procedure which can hamper subsequent embryo development. As an alternative, a simple electroporation-based strategy to deliver Cas9/sgRNA ribonucleoproteins into mouse zygotes was proposed with 100% efficiency for in vivo genome editing and no decrease in embryo viability .. A similar technology was recently used by Hashimoto et al (2016), claiming that electroporation of the Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic embryos in the mouse. The key to success was performing the electroporation into the fertilized zygotes very early, enabling the genomic editing to occur before the first replication (S-phase) of the mouse genomic DNA.…”

Relationship between somatic mosaicism of Pax6 mutation and variable developmental eye abnormalities—an analysis of CRISPR genome-edited mouse embryos

“…A recent paper shows that using electroporation, Cas9 protein with sgRNA, and early pronuclear zygotes is a most effective way to generate non-mosaic mutants in the mouse 13 . Our results indicated genetic mosaicism ranging from two to eight including wild-type (Tables S3, S4 and S7).…”

“…Our results indicated genetic mosaicism ranging from two to eight including wild-type (Tables S3, S4 and S7). This could be interpreted as that before E0.8 the translational machinery is not completely activated in the zygotes 13 , and therefore Cas9 is not effective at the time of microinjection, but nuclease activity of Cas9 might drag on as cleavage proceed. Thus, balancing of the concentration of Cas9/sgRNA injected must be useful to regulate genetic mosaicism, which awaits a further study.…”

“…In the genome-edited founder mice, varying degrees of mosaicism in mutations of targeted genes have been reported 11–13 . Here, we used the CRISPR/Cas system to generate Pax6 -deficient mice.…”

Guide RNA engineering for versatile Cas9 functionality

“…In vitro -transcribed (IVT) sgRNA can be microinjected into embryos along with mRNA encoding SpCas9 ORF (67), or IVT sgRNAs can be transfected with purified SpCas9 protein (68). Additionally, chemical synthesis of sgRNAs and chemically modified sgRNA nucleotides transfected into human primary cells with either SpCas9 mRNA or purified protein has shown to enhance target specificity (69).…”