Suppression of mosaic mutation by co-delivery of CRISPR associated protein 9 and three-prime repair exonuclease 2 into porcine zygotes via electroporation

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 preimplantation 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.

Effect of Aphidicolin, a Reversible Inhibitor of Eukaryotic Nuclear DNA Replication, on the Production of Genetically Modified Porcine Embryos by CRISPR/Cas9

“…Mosaicism is the main problem related to the production of genetically edited animals when gene editing is performed in embryos and not in somatic cells before performing SCNT [37]. Different strategies have been carried out to try to reduce mosaicism, all of them based on time factors, to try to generate INDELs before the first DNA replication [14,38,39]. Among these strategies, the use of modified Cas9 protein with ubiquitin-proteasomal degradation signals to reduce the half-life of the RNP, has been employed [39].…”

Production of genome-edited mice by visualization of nucleases introduced into the embryos using electroporation

“…Many kinds of knockout and knock-in mice and rats have already been produced by TAKE method using ZFN, TALEN, CRISPR-Cas system [ 12 , 13 , 14 , 15 , 16 , 17 ], and other nucleases [ 18 ]. Furthermore, this method has widely been applied for the production of genome-edited strains in other animals [ 19 , 20 ]. Although this method is easy and simple to operate, it is difficult to confirm the introduction of nucleases into embryos and energization during operation.…”

Electroporation-Mediated Genome Editing of Livestock Zygotes

“…The paper used Cas9 protein for both procedures and also noted that mutation efficiency and bi-allelic mutation rate were higher when one cell embryos were microinjected (Le et al, 2021). Additional attempts to further reduce mosaicism have included substituting Cas9 protein for Cas9 mRNA, speeding up the editing process, degrading Cas9 sooner, in vivo germline editing, and co-transfection with other reagents such as a three-prime repair exonuclease to improve gene editing efficiency (Chapman et al, 2015;Hashimoto et al, 2016;Tu et al, 2017;Yamashita et al, 2020).…”

“…The authors claim to have increased the production of non-mosaic blastocysts by 70.7% when Trex2 was co-transfected with Cas9. Unfortunately, Trex2 is a known inhibitor of HDR which may result in problems if attempting to generate non-mosaic knock-in animals (Yamashita et al, 2020).…”