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.

Figure 1

Experimental design of the three-way comparison study. Schematic experimental design to assess the total mutagenesis and knock-in rate induced in embryos or live pups obtained from wild-type C57BL/6J zygotes or maternally supplied Cas9 C57BL/6J zygotes after microinjection or electroporation of CRISPR reagents. ssODN⁺: optional inclusion of ssODN in the electroporation mix.

Figure 2

Proof-of-principle and conditions optimization experiments. (a) Table summarizing editing of Gene A after pronuclear microinjection (PNI) or electroporation (EP) of CRISPR reagents into wild-type C57BL/6J embryos with various concentrations of Cas9 protein. (b) Total mutagenesis rate at Gene A locus. (c) Rate of homology directed repair at Gene A locus. The values presented in table (a) are total numbers across all replicates, whereas the numbers from individual replicate experiments are shown in (b) and (c).

Figure 3

Comparison of pronuclear microinjection versus electroporation (live pups). (a) Table summarizing editing efficiency at 5 gene loci in live pups after pronuclear microinjection (PNI) or electroporation (EP) of CRISPR reagents into wild-type C57BL/6J zygotes. (b) Total mutagenesis rate at 4 gene loci in live pups obtained from pronuclear microinjection (PNI) or electroporation (EP WT) of CRISPR reagents into wild-type C57BL/6J zygotes. (c) Rate of homology directed repair at 3 gene loci. Colours representing individual target loci are consistent across the figures (Supplementary Table 4).

Figure 4

Comparison of electroporation with exogenously or maternally supplied Cas9 in blastocyst. (a) Table summarizing editing efficiency at target sites for 6 gene loci in embryos cultured to the blastocyst stage after electroporation (EP) of CRISPR reagents into zygotes harvested from wild-type C57BL/6J females (exogenous Cas9) or Cas9-expressing females (maternal Cas9). (b) Total mutagenesis rate at 6 gene loci for electroporation delivery using either exogenous (EP WT) or maternally supplied Cas9 (EP Cas9). (c) Rate of homology directed repair at 4 gene loci. Colours representing individual target loci are consistent across the figures (Supplementary Table 5).

Figure 5

Comparison of electroporation with exogenously or maternally supplied Cas9 in live pups. (a) Table summarizing editing efficiency at 4 gene loci in live pups after electroporation (EP) of CRISPR reagents into embryos harvested from wild-type C57BL/6J females (exogenous Cas9) or Cas9-expressing females (maternal Cas9). (b) Total mutagenesis rate at 3 gene loci for electroporation delivery with either exogenous (EP WT) or maternally supplied Cas9 (WP Cas9). (c) Efficiency of homology directed repair at 3 gene loci. Colours representing individual target loci are consistent across the figures (Supplementary Table 4).

Figure 6

Overall mutagenesis rates for the three delivery methods across all genes tested. Dot plot showing the range of efficiencies for total mutagenesis (upper panel) and knock-in allele production by homology directed repair (lower panel), combining data from both live pup generation and in vitro cultured blastocysts, across all targeted gene loci. CRISPR/Cas9 reagents have been delivered either by pronuclear microinjection of C57BL/6J embryos (PNI), by electroporation of C57BL/6J zygotes (EP WT) or by electroporation of zygotes derived from Cas9-expressing donor females (EP Cas9). The mean is shown by the horizontal line, and the box indicates plus / minus the standard deviation. *p < 0.05.

Figure 7

Production efficiencies following pronuclear microinjection versus electroporation. (a) Table summarizing embryo and pup survival and production rates following the introduction of CRISPR/Cas9 reagents either by pronuclear microinjection of C57BL/6J zygotes (PNI) or electroporation of wild-type C57BL/6J zygotes (EP WT) or zygotes derived from Cas9-expressing females (EP Cas9). Comparison bar charts showing the rate of 2-cell development relative to the total number of zygotes harvested (b), the rate of embryo transfer recipient females that became pregnant (c), the rate of pups born, adjusted for non-pregnant transfers (d) and the rate of pups born relative to the total number of embryos harvested (e). *p < 0.05, **p < 0.01,***p < 0.001 versus microinjection by a two sided Fisher's exact test.