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, 66 (1), 41-48

Suppression of Mosaic Mutation by Co-Delivery of CRISPR Associated Protein 9 and Three-Prime Repair Exonuclease 2 Into Porcine Zygotes via Electroporation

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Suppression of Mosaic Mutation by Co-Delivery of CRISPR Associated Protein 9 and Three-Prime Repair Exonuclease 2 Into Porcine Zygotes via Electroporation

Shiro Yamashita et al. J Reprod Dev.

Abstract

Gene-modified animals, including pigs, can be generated efficiently by introducing CRISPR associated protein 9 (CRISPR/Cas9) into zygotes. However, in many cases, these zygotes tend to become mosaic mutants with various different mutant cell types, making it difficult to analyze the phenotype of gene-modified founder animals. To reduce the mosaic mutations, we introduced three-prime repair exonuclease 2 (Trex2), an exonuclease that improves gene editing efficiency, into porcine zygotes along with CRISPR/Cas9 via electroporation. Although the rate of porcine blastocyst formation decreased due to electroporation (25.9 ± 4.6% vs. 41.2 ± 2.0%), co-delivery of murine Trex2 (mTrex2) mRNA with CRISPR/Cas9 did not affect it any further (25.9 ± 4.6% vs. 31.0 ± 4.6%). In addition, there was no significant difference in the diameter of blastocysts carrying CRISPR/Cas9 (164.7 ± 10.2 μm), and those with CRISPR/Cas9 + mTrex2 (151.9 ± 5.1 μm) as compared to those from the control group (178.9 ± 9.0 μm). These results revealed that mTrex2 did not affect the development of pre-implantation embryo. We also found bi-allelic, as well as mono-allelic, non-mosaic homozygous mutations in the blastocysts. Most importantly, co-delivery of mTrex2 mRNA with CRISPR/Cas9 increased non-mosaic mutant blastocysts (29.3 ± 4.5%) and reduced mosaic mutant blastocysts (70.7 ± 4.5%) as compared to CRISPR/Cas9 alone (5.6 ± 6.4% and 92.6 ± 8.6%, respectively). These data suggest that the co-delivery of CRISPR/Cas9 and mTrex2 is a useful method to suppress mosaic mutation.

Keywords: CRISPR associated protein 9 (CRISPR/Cas9); Electroporation; Gene editing; Mosaic mutants; Three-prime repair exonuclease 2 (Trex2) exonuclease.

Figures

Fig. 1.
Fig. 1.
Analysis of gene modification conditions using TIDE software. The bar in deep pink indicates mutant allele, that in light pink indicates wild-type allele, and that in black indicates noise. A. Wild-type blastocysts. B. Partially gene-modified blastocysts carrying wild-type allele. C. Completely gene-modified blastocysts with no wild-type allele.
Fig. 2.
Fig. 2.
Comparison of blastocyst morphology on day 6 after in vitro fertilization (IVF). Each scale bar indicates 200 μm.
Fig. 3.
Fig. 3.
Analysis of mosaic mutant blastocysts using TIDE software and electrophoresis. Bar in deep pink indicates mutant allele, that in light pink indicates wild-type allele, and the black one indicates noise. A: Blastocysts with more than three alleles. B: Blastocysts with two different alleles at rates of more than 25%. C: Blastocysts with R2 < 0.9 because of indels larger than 50 bp. D: Electrophoretic analysis of blastocysts with indels larger than 50 bp.
Fig. 4.
Fig. 4.
Analysis of non-mosaic homozygous mutant blastocysts using TIDE software. The bar in deep pink indicates mutation allele and that in black indicates noise. A: Non-mosaic homozygous mutant blastocysts with two different mutation alleles. B: Non-mosaic homozygous mutant blastocysts carrying alleles with the same sequence mutation. C: CRISPR/Cas9 targeting sequence and mutant sequence of non-mosaic homozygous mutant blastocysts. Nucleotides in blue show the target sequence, those in red indicate the PAM sequence, and those in green are indel mutations.

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