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Abstract
Mosaicism is the most important limitation for one-step gene editing in embryos by CRISPR/Cas9 because cuts and repairs sometimes take place after the first DNA replication of the zygote. To try to minimize the risk of mosaicism, in this study a reversible DNA replication inhibitor was used after the release of CRISPR/Cas9 in the cell. There is no previous information on the use of aphidicolin in porcine embryos, so the reversible inhibition of DNA replication and the effect on embryo development of different concentrations of this drug was first evaluated. The effect of incubation with aphidicolin was tested with CRISPR/Cas9 at different concentrations and different delivery methodologies. As a result, the reversible inhibition of DNA replication was observed, and it was concentration dependent. An optimal concentration of 0.5 μM was established and used for subsequent experiments. Following the use of this drug with CRISPR/Cas9, a halving of mosaicism was observed together with a detrimental effect on embryo development. In conclusion, the use of reversible inhibition of DNA replication offers a way to reduce mosaicism. Nevertheless, due to the reduction in embryo development, it would be necessary to reach a balance for its use to be feasible.
Highlights
Accepted: 12 February 2022The production of genetically edited animals became a reality in the 1980s when the first transgenic mice were obtained by recombinant plasmid pronuclear microinjection [1].In 1985, the generation of the first genetically edited pigs by random insertion of foreignDNA was reported [2,3].Subsequently, the development and application of programmable endonucleases—such as zinc finger nucleases (ZFNs) [4], transcription activator-like effector nucleases (TALENs) [5], and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPRassociated protein (Cas) [6–8]—led to a revolution in the production of genetically modified animals
The development and application of programmable endonucleases—. Such as zinc finger nucleases (ZFNs) [4], transcription activator-like effector nucleases (TALENs) [5], and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPRassociated protein (Cas) [6–8]—led to a revolution in the production of genetically modified animals. The mechanism of these endonucleases consists in the generation of double-strand breaks in target DNA which can be repaired by two different mechanisms: non-homologous end joining (NHEJ), which allows the generation of insertions-deletions (INDELs) in the DNA sequence that can produce a knock-out (KO) allele, or homology-directed repair (HDR), which can allow the replacement of the wildtype (WT) sequence with a desired sequence that is introduced via a foreign DNA fragment, generating a KO or knock-in (KI)
DNA replication was evaluated in all groups. (B) Effect of aphidicolin on porcine embryo development
Summary
The development and application of programmable endonucleases— Such as zinc finger nucleases (ZFNs) [4], transcription activator-like effector nucleases (TALENs) [5], and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPRassociated protein (Cas) [6–8]—led to a revolution in the production of genetically modified animals. The mechanism of these endonucleases consists in the generation of double-strand breaks in target DNA which can be repaired by two different mechanisms: non-homologous end joining (NHEJ), which allows the generation of insertions-deletions (INDELs) in the DNA sequence that can produce a knock-out (KO) allele, or homology-directed repair (HDR), which can allow the replacement of the wildtype (WT) sequence with a desired sequence that is introduced via a foreign DNA fragment, generating a KO or knock-in (KI) allele [9].
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