An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes

Electroporation of zygotes represents a rapid alternative to the elaborate pronuclear injection procedure for CRISPR/Cas9-mediated genome editing in mice. However, current protocols for electroporation either require the investment in specialized electroporators or corrosive pre-treatment of zygotes which compromises embryo viability. Here, we describe an easily adaptable approach for the introduction of specific mutations in C57BL/6N mice by electroporation of intact zygotes using a common electroporator with synthetic CRISPR/Cas9 components and minimal technical requirement. Direct comparison to conventional pronuclear injection demonstrates significantly reduced physical damage and thus improved embryo development with successful genome editing in up to 100% of living offspring. Hence, our novel approach for Easy Electroporation of Zygotes (EEZy) allows highly efficient generation of CRISPR/Cas9 transgenic mice while reducing the numbers of animals required.

Highly-efficient Cpf1-mediated gene targeting in mice following high concentration pronuclear injection

ABSTRACTCpf1 has emerged as an alternative to the Cas9 RNA-guided nuclease. Here we show that gene targeting rates in mice using Cpf1 can meet or even surpass Cas9 targeting rates (approaching 100% targeting) but require higher concentrations of mRNA and guide. We also demonstrate that co-injecting two guides with close targeting sites can result in synergistic genomic cutting, even if one of the guides has minimal cutting activity.