Modified EP method for genome editing in two-cell mouse embryos
Our modified EP system for genome editing in two-cell-stage mouse embryos is shown in Fig. 1. There is a concern that the two blastomeres of a two-cell-stage embryo may fuse together when conventional EP is performed on a two-cell-stage embryo32,34,35. These fused embryos cannot develop to term as they become tetraploid. First, we examined how the EP procedure affected the development in two-cell-stage embryos (Fig. 2). The basic EP system utilized the same conditions as the instrument, with the medium and settings being 20 V for 3 ms (on)/97 ms (off), repeated 5 times in fertilized eggs, which we adopted as the initial condition36. The two-cell embryo groups were prepared with a changed orientation of the electrode (Pre- EP in Fig. 1d and Types A and B in Fig. 2a). Subsequently, they were electroporated using 1–3 sets (20 V for 3 ms (on)/97 ms (off) 5 times per set) (Fig. 2a–c). The EP solvent was a 1:1 mixture of Opti-MEM and 75% PBS media without CRISPR/Cas9 components. Consequently, for type A groups, no fusion of blastomeres in two-cell-stage embryos was observed (Fig. 2b). The development rate into blastocysts remained high but decreased with the number of EP sets (93–73%) (Fig. 2d). In contrast, for the type B groups, the fusion of blastomeres in two-cell-stage embryos was observed (Fig. 2c) and increased with the number of EP sets (Fig. 2f). The rate of blastocysts developed from non-fused in two-cell-stage embryos was high (83–100%), but the rate of blastocysts per total two-cell-stage embryos decreased to 57–60% (Fig. 2e). Thus, these results indicate that blastomere fusion can be avoided by orienting the axis of the contact surface of two blastomeres horizontally to the electrodes and that the EP of two-cell-stage embryos does not affect embryo development (Fig. 2a, Type A).
Overview of the electroporation (EP) system and procedure at two-cell mouse embryos. (a) The EP system consists of a stereo microscope (Olympus SZX16) and an electroporator (BEX CUY21-EDIT II), which were previously used with mouse fertilized eggs36. (b) The EP chamber with platinum electrodes (BEX #LF501PT1-10) was used for this EP system. (c) Microscopic view of a two-cell mouse embryo positioned within the 1-mm gap between the platinum electrodes. (d) Flow chart outlining the experimental procedure for evaluating EP-based CRISPRCas genome editing in two-cell mouse embryos. Pay attention to the contact surface of the two blastomeres of the two-cell-stage embryo, which is positioned perpendicular to the electrodes during the EP stage.
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Examination of EP to avoid blastomere fusion in a two-cell mouse BDF1×B6 embryo. (a) Two groups, type A and B, were subjected to electroporation (EP). The two-cell embryos were prewashed in EP media and then placed in EP media containing CRISPR reagents, as shown in Fig. 1d, before being manually positioned in type A and B lines for EP. (b) The two-cell embryos electroporated in the type A group did not exhibit any fused embryos. The scale bar indicates 50 μm. (c) In the type B group, two-cell embryos subjected to EP resulted in the observation of several fused embryos (denoted using asterisks). The scale bar also indicates 50 μm. (d) (e) Blastocysts developed from the two-cell embryos electroporated in the type A and B group are shown. The scale bar indicates 50 μm. (f) The table summarizes the rates of fused embryos and blastocyst development in two-cell embryos electroporated one to three times in both the type A and B groups.
Next, we examined the blastocyst formation rate in two-cell embryos subjected to varying EP voltage and several repetitions of EP. The experiments were repeated three to five times. The blastocyst formation rate of inbred B6 and BDF1×B6 hybrid strains decreased with an increase in voltage ranging from 15 to 25 V and an increase in EP repetitions (Fig. S1a). The decrease in the blastocyst formation rate of B6 strains was more significant than that in BDF1×B6 hybrid strains with a significant difference at 25 V and 10 repeats of EP condition (B6 42 ± 6% vs. Hybrid 80 ± 8%). Finally, we investigated the relationship between EP voltage and the uptake of mRNA in two-cell embryos (Fig. S1b–c). We prepared EP solutions containing 200 ng/mL EGFP A95 mRNA and placed them between two electrodes on a plate; two-cell embryos of the BDF1×B6 hybrid strain were introduced into the drop, prepared with a changed orientation concerning the electrode (Fig. 2a), and immediately subjected to in vitro EP at 15, 20, and 25 V with five repetitions of 97/3 ms. The experiments were repeated twice, and the average fluorescence intensity of EGFP A95 in resulting four to eight cell embryos cultivated in vitro was measured using a fluorescence microscope. The results show that the uptake of EGFP A95 mRNA by two-cell embryos increased with an increase in EP voltage ranging from 15 to 25 V) (Fig. S1b).
Based on the above results (Fig. 2 and S1), we adopted the following conditions for the experiments below: Type A orientation of two-cell-stage embryos; a 1:1 mixture of media; 1 set of 20 V for 3 ms (on)/97 ms (off) five repeats for the two-cell-stage embryo EP in this study.
Efficiency comparisons and indel mutation analysis in fresh and thawed two-cell embryos and fertilized eggs in genome-edited mice
Frozen-thawed two-cell-stage embryos are potentially useful in genome editing, representing a valuable resource and promising significant benefits. Therefore, to examine the genome editing efficiency of two-cell-stage embryos and to confirm that there was no difference in efficiency between fresh and thawed two-cell-stage embryos, we attempted to create genome-edited mice using our modified EP method with both fresh and frozen-thawed two-cell-stage embryos. We designed and synthesized guide RNAs (gRNAs) targeting base sequences on three genes (Tyr, Adm, and Ramp1, Table S4). Fresh and frozen-thawed two-cell-stage embryos were prepared via in vitro fertilization (IVF) of BDF1×B6 hybrid mice. EP was performed under the following conditions: a 1:1 mixture of media, 20 V with a 3 ms (on)/97 ms (off) duration repeated five times, and the Type A orientation of the two-cell-stage embryo (Figs. 1 and 2a). The concentration of gRNA and Cas9 proteins was 200 ng/µL and 50 ng/ µL, respectively.
The summarized results are presented in Table 1. As anticipated, none of the experiments exhibited fusion of blastomeres in the two-cell-stage embryos. To generate Tyr indel mutated mice, we included fertilized eggs prepared via IVF in BDF1×B6 hybrid mice as a comparative reference. We found that 93% of indel mutated mice were obtained from fresh two-cell-stage embryos, while 81% were obtained from frozen-thawed two-cell-stage embryos; 100% of indel mutated mice were obtained from fertilized eggs. No significant differences were observed among the three groups. Furthermore, the average number of indels per genome in indel-mutated mice obtained from fresh two-cell-stage embryos, frozen-thawed two-cell-stage embryos, and fertilized eggs were 3.6 ± 0.5, 4.2 ± 0.7, and 4.4 ± 0.8, respectively (no significant differences). The coat color of indel mutated mice obtained from the three types of embryos was predominantly white (Fig. S2), indicating null Tyr mutations. We also checked for off-target effects induced by Tyr-gRNA in 42 Tyr indel mice (25 and 17 obtained from fresh and frozen-thawed two-cell embryos, respectively, Table 1). No mutations with off-target probability were observed in the three target regions listed (Table S1). Furthermore, for generating Adm and Ramp1 indel mutated mice, the number and rate of indel mutations obtained from fresh two-cell-stage embryos and frozen-thawed two-cell-stage embryos were not significantly different from those obtained for the generation of Tyr indel mutated mice (Table 1).
We demonstrated that CRISPR/Cas9 genome-edited mice can be generated from two-cell-stage embryos using our modified EP method. Our results indicate no difference in genome editing efficiency between fresh and frozen-thawed two-cell-stage embryos.
Validation of the utility of EP in two-cell-stage embryos for indel mutagenesis in different genes and mouse strains
To assess the broad applicability of our modified EP method for genome editing in two-cell-stage embryos, we investigated indel mutations in six genes (Adm2, Ddx58, Klf5, Gt(ROSA)26Sor, Trl4, and Trl9) (Tables S2), in addition to the previously studied Tyr, Adm, and Ramp1. The guide RNA sequences targeting the six genes are provided in Table S4. Here, we employed CRISPR/Cas12a to examine the indel mutation capability of the Adm2 gene. The synthesized guide RNA concentration was 200 ng/µL, and both Cas9 and Cas12a proteins were used at 50 ng/µL. We utilized freshly prepared embryos or thawed embryos from BDF1xB6 hybrid mice at the two-cell stage. EP was carried out under identical conditions as described previously (Fig. 1; Table 1), and embryos were allowed to develop to the blastocyst stage in vitro. These blastocysts were used for indel mutation analysis. Notably, fusion of blastomeres in two-cell-stage embryos was not observed, and the blastocyst formation rate after EP ranged from 69 to 90%. The indel mutation rates in the blastocysts were highly efficient, with 100% success for five genes using CRISPR/Cas9 and 1 gene using CRISPR/Cas12a.
Furthermore, we investigated whether the conditions of this EP method affect the efficiency of genome editing and embryonic development rates in two-cell-stage embryos from various mouse strains, including B6, ICR, 129/Sv. Two-cell stage embryos derived from each strain were subjected to EP with Ramp1-gRNA under the same conditions as before (Fig. 1; Table 1), and blastocysts developed from electroporated two-cell-stage embryos in vitro were analyzed for the indel mutation capability. The results are summarized in Table S3. As expected, no fused blastomeres were observed in two-cell-stage embryos from each strain, and the blastocyst formation rate was not different from that of untreated embryos. The Ramp1-Indel mutation rate in blastocysts was 100% in all strains.
Thus, these findings confirm that the conditions of the modified EP method presented in Fig. 1 can be widely applied to genome editing in two-cell-stage embryos.
Efficient ssODN-knock-in editing through modified EP of two-cell stage mouse embryos
We investigated the efficiency of ssODN-KI genome editing using modified EP in two-cell-stage embryos on four genes, Adm, Klf5, Or7a36, and Ramp3 (Fig. 3 and S3). The sequences of gRNAs on the four genes and those of ssODN are summarized in Tables S4 and S5. First, for the KI of the loxP sequence to the Adm gene (Fig. 3a), 108 two-cell stage embryos were electroporated with AM-gRNA-D (200 ng/µl), Cas9 protein (50 ng/µl), and Adm + loxP ssODNs-D (400 ng/µl) (Fig. 3b). No fused blastomeres were observed. These embryos were transferred to pseudopregnant mice, and 38 offspring were obtained. The KI rate of the loxP sequence was evaluated based on the presence of the EcoRI site in the PCR amplification product of the target sequence region (Fig. 3c) and Sanger sequencing (Fig. 3d). Of the 38 offspring, 20 were loxP KI offspring (KI rate 53%), including 15 (KI rate 39%) with loxP/+ and 5 (KI rate 20%) with loxP/loxP. We also investigated off-target effects induced by AM-gRNA-D in the 20 loxPKI mice obtained from fresh and thawed two-cell embryos. No mutations were observed in three regions with off-target probabilities (Table S1).
Efficiency of single-stranded oligodeoxynucleotide (ssODN) knock-in genome editing in two-cell mouse BDF1×B6 embryos. (a) Table summarizes the efficiency of ssODN knock-in in two-cell embryos. (b) Illustration of AM-gRNA-D targeting a location following exon 3 of the murine Adm gene. The sequence recognized by the AM-gRNA-D is highlighted in blue, and the protospacer adjacent motif sequence is highlighted in red. Arrows indicate the positions of the PCR primers (refer to Table S5). All Adm + loxP ssODN sequences are shown in Table S5. (c) Representative full-length image of agarose gel electrophoresis of PCR products amplified from the genomic DNA of pups (nos. 1–14) obtained from the electroporated two-cell embryos. Top panel: results of the restriction fragment length polymorphism assay using the EcoRI enzyme. Bottom panel: PCR products amplified from the target region of the Adm gene. Pups 1, 3, 6, 10, and 13 were heterozygous for the loxP-KI editing. Pups 5 and 8 were homozygous for the loxP-KI editing. M, lambda HindIII + 100-bp ladder markers. (d) Sequences of 5′ and 3′ junction sites in pup 5. Sequence chromatograms show the correct KI of Adm + loxP ssODN-D (as described in Table S5) into the target region of the Adm gene.
Furthermore, we investigated the KI efficiency of Flagx3 for the Klf5 and Or7a36 genes and loxP sequences for the Ramp3 genes, respectively (Fig. S3). To this end, two-cell-stage embryos were electroporated with Klf5 + Flagx3 ssODNs (400 ng/µL) and Ramp3-gRNA (200 ng/µL), Cas9 protein (50 ng/µL), and Ramp3 + loxP ssODNs (400 ng/µL) (Fig. S3 and Table S5). No fused blastomeres were observed. The treated two-cell-stage embryos were cultivated into blastocysts. The ssODN KI rate was evaluated through the RFLP assay using ClaI (Klf5) or EcoRI (Ramp3) enzyme in the PCR amplification product of the target sequence region using crude DNA solution from the blastocyst as a template. For Or7a36, the increase in size of the PCR products was used. Klf5 ssODN-treated two-cell-stage embryos showed a blastocyst formation rate of 80% and a ssODN KI rate of 63%. The Klf5-gRNA used was confirmed to have a genome editing efficiency of approximately 44% in offspring derived from fertilized eggs36. Or7a36 ssODN-treated two-cell-stage embryos showed a blastocyst formation rate of 75% and a ssODN KI rate of 39%. Ramp3 ssODN-treated two-cell-stage embryos showed a blastocyst formation rate of 90% and a ssODN KI rate of 4%. The Ramp3-gRNA used demonstrated a low KI rate of 5% in fertilized eggs, along with a blastocyst formation rate of 85%. Thus, the low KI rate of Ramp3-gRNA was not altered by the EP of two-cell-stage embryos.
Based on the above results, we demonstrated that CRISPR/Cas9-mediated ssODN KI genome editing of two-cell-stage embryos via EP is feasible and achieves efficiency equivalent to that of fertilized eggs.
Efficiency of KI editing of dsDNA and ssDNA fragments via modified EP in mouse two-cell-stage embryos
We investigated the efficiency of DNA fragment-KI genome editing using modified EP in two-cell-stage embryos on 2 genes, Ramp2, and Gt(ROSA)26Sor (Fig. 4, S4, and Table S6). To this end, two-cell embryos were electroporated with 1.23 kb dsDNA or 1.23 knt ssDNA, along with Ramp2-gRNA and Cas9 protein (Fig. 4a). As a control, fertilized eggs were treated similarly. The development rates of blastocysts derived from electroporated two-cell-stage embryos and fertilized eggs with 1.23 kb dsDNA and 1.23 knt ssDNA were 78%, 80%, 67%, and 73%, respectively (Fig. 4b). The KI rates of these blastocysts were 19%, 32%, 14%, and 27%, respectively (no significant differences). Subsequently, similar experiments were conducted using 1.80 kb dsDNA and 1.80 knt ssDNA. Consequently, the KI rates were 0%, 0%, 0%, and 0%, respectively, while the development rates of these blastocysts were 64%, 62%, 51%, and 53%, respectively.
Efficiency of knock-in genome editing using various sizes of dsDNA and ssDNA fragments through modified EP of two-cell-stage mouse BDF1×B6 embryos. (a) Left illustration of Ramp2-gRNA targeting the intron of the murine Ramp2 gene. The sequence recognized by the Ramp2-gRNA is highlighted in blue, and the protospacer adjacent motif sequence is highlighted in red. Arrows indicate the positions of the PCR primers (Table S5). Both 1.23 kb dsDNA and 1.23 knt ssDNA, as well as both 1.80 kb dsDNA and 1.80 knt ssDNA, were utilized. They consist of both 5′ and 3′ Ramp2 homology arms, and EGFP cDNA (Table S6). Right illustration of R26-gRNA targeting the intron of the murine Gt(ROSA)26Sor gene . Both 0.66 kb dsDNA and 0.66 knt ssDNA, as well as both 1.36 kb dsDNA and 1.36 knt ssDNA, were utilized (Table S6). (b) (c) Table summarizes the efficiency of various sizes of DNA fragments about knock-in genome editing in the Ramp2 and Gt(ROSA)26Sor locus in fertilized eggs and two-cell embryos. They were electroporated with 100 ng/µL of dsDNA or 50 ng/µL of ssDNA along with 200 ng/µL of guide RNA and 50 ng/µL of Cas9 protein. Each experiment was conducted at least three times.
Next, two-cell embryos and fertilized eggs were electroporated with 0.66 kb dsDNA or 0.66 knt ssDNA, along with R26 -gRNA and Cas9 protein (Fig. 4c). The results showed that the KI rates were 17%, 17%, 14%, and 10%, respectively (no significant differences) with the development rates of these blastocysts being 76%, 73%, 78%, and 80%, respectively (Fig. 4c). Alternatively, the results obtained from similar experiments were conducted using 1.36 kb dsDNA and 1.36 knt ssDNA, the KI rates being 0%, 20%, 0%, and 11%, respectively, with the development rates of these blastocysts at 80%, 87%, 68%, and 73%, respectively.
Based on these results, we observed that ssDNA generally leads to a higher KI editing rate compared to dsDNA. When the size of the KI DNA exceeded 1.80 kb for dsDNA or 1.80 kb for ssDNA, the efficiency of KI editing decreased. Additionally, using two-cell embryos tended to result in a higher KI editing rate compared to using fertilized eggs.
Direct generation of floxed adm mice via sequential EP of fertilized egg and then in two-cell-stage embryos
Genome editing of fertilized eggs through EP has been well-studied21,22,23. If EP-based genome editing in two-cell embryos can be easily implemented, it will pave the way for sequential genome editing using fertilized eggs and two-cell embryos, increasing opportunities for genome editing32. To assess this challenge (Fig. 5a), we designed and synthesized 2 gRNAs, Adm-gRNA-A and Adm-gRNA-D targeting the Adm gene (Table S4), and 2 loxP sequences, Adm-loxP ssODN-A and Adm-loxP ssODN-D for KI on the Adm gene (Table S5). The EP was performed under the same conditions described, except that the voltage was reduced from 20 to 15. During the EP of the two-cell-stage embryos, Type A orientation was performed (Figs. 1 and 2a). The results are summarized in Fig. 5b. First, 60 fertilized eggs were subjected to EP with AM-gRNA-D (200 ng/µL), Cas9 protein (50 ng/µL), and Adm + loxP ssODNs-D (400 ng/µL). Second, the following day, the 60 two-cell stage embryos derived from the electroporated fertilized eggs were subjected to a second round of electroporation with AM-gRNA-A (200 ng/µL), Cas9 protein (50 ng/µL), and Adm + loxP ssODNs-A (400 ng/µL). The resulting embryos were then transferred into pseudopregnant ICR females. We examined the fetuses at 15.5 days post-coitum because knockout for Adm results in fetus lethality37. Consequently, we obtained 13 fetuses from a total of 60 two-cell stage embryos and observed no lethal fetuses. To evaluate the KI rate of Adm loxP ssODN-A and D sequences, we performed an RFLP assay using EcoRI enzyme on the PCR product of the target sequence region (Fig. S5) amplified by genomic DNA isolated from the limbs of the 13 fetuses. A 46% KI rate of loxP ssODN-D on the Adm gene was found in the fertilized eggs. The KI rate of loxP ssODN-A on the gene was 54% in the two-cell-stage embryos. Based on the analysis of In vitro Cre assay (Fig. 5c and d), the floxed KI rate of the Adm gene was determined to be 15% (2 of 13 individuals). These results demonstrate that the modified EP of genome editing at the two-cell stage contributes to the overall success of genome editing in both fertilized eggs and two-cell-stage embryos.
Sequential electroporation (EP) of fertilized egg and two-cell-stage BDF1×B6 embryos for directly generating floxed Adm mice. (a) Schematic illustration of the experimental procedure based on the sequential EP method32. (b) Table summarizes the result of sequential EP of fertilized egg stage and then two-cell-stage embryos. (c) In vitro Cre and PCR analysis for identifying a founder mouse with floxed Adm allele. (d) Agarose gel electrophoresis of PCR products amplified from the in vitro Cre-treated genomic DNA of the fetus (no. 1, 3, 9, 11, 12, 13, as shown in Fig. S5). Fetus no. 1, 3, 9, and 11 are positive for both loxP sites on the Adm locus. Fetus no. 12 and 13 have only loxP-A and -D on the Adm locus, respectively, and were used for PCR control. This result indicates that fetuses 3 and 9 are founder mice with the floxed Adm allele. M, Lambda HindIII + 100-bp ladder markers. (e) Sequence chromatograms of PCR products from fetuses 3 and 9, as detected in (d).
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- Source: https://www.nature.com/articles/s41598-024-81198-0