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

Abstract

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/6 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.

Fig 1. Characterization of EEZy for specific genome editing in intact C57Bl/6N zygotes.

(A) Representative RFLP analysis for evaluation of the HDR efficiency in Nphs2-targeted blastocysts upon EEZy using Opti-MEM with and without additives. PCR controls from untreated blastocysts (WT) and without DNA template (H₂O) and RFLP controls from untreated blastocysts (WT) are depicted. (B) Quantification of the genotypes of Nphs2-targeted blastocysts upon EEZy using Opti-MEM with and without additives from three independent experiments. (C) Assessment of embryo development from (B) as percentage of developed blastocysts from zygotes after EEZy. (D) Quantification of RFLP analysis from Nphs2-targeted blastocysts upon EEZy using sgRNA or pgRNA from three independent experiments and (E) assessment of percentage of developed blastocysts from these zygotes. As depicted zygotes in (D) and (E) are either derived from natural mating or IVF. (F) Assessment of developed blastocysts after PNI using pgRNA or sgRNA targeting Nphs2 from four independent experiments. (G) Quantification of Sanger sequencing of biopsies from Tmem218 transgenic mice generated by EEZy. Data of a total of 13 mice displaying either solely the desired mutation (HDR), a mixture of the desired mutation and INDELs or only INDELs are depicted. Data are means ± standard deviation. *p < 0.05, ns = non-significant. N = total number of embryos analyzed.

Fig 2. Electroporation of zygotes incompatible with pronuclear injection.

(A) Average ratio of harvested zygotes with polar bodies from natural mating with visible pronuclei (+) and without visible pronuclei (-) from 47 independent experiments. Total number of embryos analyzed was 6368 and 3314 for zygotes with and without visible pronuclei, respectively. (B) Representative RFLP analysis for evaluation of HDR efficiency in Nphs2-targeted blastocysts upon EEZy of zygotes without visible pronuclei. PCR controls from untreated blastocysts (WT) and without DNA template (H₂O) and RFLP controls from untreated blastocysts (WT) are depicted. (C) Quantification of the genotype from three independent experiments using zygotes without visible pronuclei are compared to EEZy of all zygotes from the experiments depicted in Fig 1B and 1D, right columns. (D) Percentage of developed blastocysts from zygotes after EEZy from (C). Box plot represents median, boxes equal 25 to 75 percentiles, whiskers include all values. Remaining data are means ± standard deviation. ***p < 0.001, ns = non-significant. N = total number of embryos analyzed.

Fig 3. Embryo toxicity of EEZy as compared to pronuclear injection.

(A) Assessment of embryo development as percentage of developed blastocysts from zygotes after EEZy. Untreated zygotes are compared to zygotes electroporated with Opti-MEM (Mock) or CRISPR/Cas9 components targeting the Gt(ROSA)26Sor locus (CRISPR). Data represent three independent experiments. (B) Quantification of viable Nphs2-targeted zygotes from four experiments immediately after PNI compared to electroporated zygotes and (C) the correspondingly developed blastocysts. (D) Developed blastocyst calculated from the number of zygotes before transgenesis in (B). (E) Quantification of RFLP genotyping from four independent experiments for HDR efficiency in blastocysts from zygotes after PNI compared to electroporated zygotes from the experiments depicted in Fig 1B and 1D, right columns. Data are means ± standard deviation. *p < 0.05, **p < 0.01, ns = non-significant. N = total number of embryos analyzed.