Electroporation and genetic supply of Cas9 increase the generation efficiency of CRISPR/Cas9 knock-in alleles in C57BL/6J mouse zygotes

Abstract

CRISPR/Cas9 machinery delivered as ribonucleoprotein (RNP) to the zygote has become a standard tool for the development of genetically modified mouse models. In recent years, a number of reports have demonstrated the effective delivery of CRISPR/Cas9 machinery via zygote electroporation as an alternative to the conventional delivery method of microinjection. In this study, we have performed side-by-side comparisons of the two RNP delivery methods across multiple gene loci and conclude that electroporation compares very favourably with conventional pronuclear microinjection, and report an improvement in mutagenesis efficiency when delivering CRISPR via electroporation for the generation of simple knock-in alleles using single-stranded oligodeoxynucleotide (ssODN) repair templates. In addition, we show that the efficiency of knock-in mutagenesis can be further increased by electroporation of embryos derived from Cas9-expressing donor females. The maternal supply of Cas9 to the zygote avoids the necessity to deliver the relatively large Cas9 protein, and high efficiency generation of both indel and knock-in allele can be achieved by electroporation of small single-guide RNAs and ssODN repair templates alone. Furthermore, electroporation, compared to microinjection, results in a higher rate of embryo survival and development. The method thus has the potential to reduce the number of animals used in the production of genetically modified mouse models.

CRISPR-Cas9 effectors facilitate generation of single-sex litters and sex-specific phenotypes

“…In matings using X Cas9 Y fathers, Atm mutations were induced only in daughters, while in matings using XY Cas9 fathers, Atm mutations were induced only in sons. Although the Atm sgRNA was only transiently present, we could achieve targeting efficiencies equivalent to those expected from zygote electroporation 40 . The mean mutation efficiency was 99.3% (±0.5 s.d.)…”

“…Although multigene targeting using genomically integrated CRISPR-Cas9 has previously been achieved 15 , 19 , 41 , it has not been deployed efficiently in a sex-specific manner. Finally, using genetically loaded Cas9 in pre-implantation embryos is more efficient at generating targeted mutations than provision via injection or electroporation, and allows for better embryo survival 40 .…”

Highly reliable creation of floxed alleles by electroporating single-cell embryos

“…The limit of the number of embryos to be electroporated in a given day is determined by the availability of embryos and recipients rather than available time under the scope and skilled microinjectionists. Higher embryo survival and thus birth rates are consistently observed after electroporation compared to microinjection owed in large part to less physical damage to the embryo [ 28 , 29 , 31 ]. If combined with using HyperOVA [ 32 ] and IVF to produce fertilized eggs, it is possible to electroporate large numbers of eggs with RNPs plus ssODNs in one day to obtain over 100 live births.…”

Applications and challenges of CRISPR-Cas gene-editing to disease treatment in clinics

“…Furthermore, microinjection is difficult to operate, and requires injection skills for high accuracy of delivery, and the hydrodynamic delivery approach may be traumatic to tissues. 103 , 104 In addition, the deformation of the cell membrane is a potential mode of transmission in vitro . The rapid mechanical deformation of the cell membrane may lead to temporary membrane disturbance or holes that may be conducive to the passive diffusion of macromolecules into tumor cells to increase tumor viability.…”