Electroporation-Mediated Genome Editing of Livestock Zygotes

Abstract

The introduction of genome editing reagents into mammalian zygotes has traditionally been accomplished by cytoplasmic or pronuclear microinjection. This time-consuming procedure requires expensive equipment and a high level of skill. Electroporation of zygotes offers a simplified and more streamlined approach to transfect mammalian zygotes. There are a number of studies examining the parameters used in electroporation of mouse and rat zygotes. Here, we review the electroporation conditions, timing, and success rates that have been reported for mice and rats, in addition to the few reports about livestock zygotes, specifically pigs and cattle. The introduction of editing reagents at, or soon after, fertilization can help reduce the rate of mosaicism, the presence of two of more genotypes in the cells of an individual; as can the introduction of nuclease proteins rather than mRNA encoding nucleases. Mosaicism is particularly problematic in large livestock species with long generation intervals as it can take years to obtain non-mosaic, homozygous offspring through breeding. Gene knockouts accomplished via the non-homologous end joining pathway have been more widely reported and successfully accomplished using electroporation than have gene knock-ins. Delivering large DNA plasmids into the zygote is hindered by the zona pellucida (ZP), and the majority of gene knock-ins accomplished by electroporation have been using short single stranded DNA (ssDNA) repair templates, typically less than 1 kb. The most promising approach to deliver larger donor repair templates of up to 4.9 kb along with genome editing reagents into zygotes, without using cytoplasmic injection, is to use recombinant adeno-associated viruses (rAAVs) in combination with electroporation. However, similar to other methods used to deliver clustered regularly interspaced palindromic repeat (CRISPR) genome-editing reagents, this approach is also associated with high levels of mosaicism. Recent developments complementing germline ablated individuals with edited germline-competent cells offer an approach to avoid mosaicism in the germline of genome edited founder lines. Even with electroporation-mediated delivery of genome editing reagents to mammalian zygotes, there remain additional chokepoints in the genome editing pipeline that currently hinder the scalable production of non-mosaic genome edited livestock.

Optimized Cas9:sgRNA delivery efficiently generates biallelic MSTN knockout sheep without affecting meat quality

“…However, negative correlations with embryo development rates were observed, which is consistent with previous studies [ 41 , 42 ]. This observation highlights the importance of determining the optimal editing efficiencies and embryo development rates during optimizing the delivery of the CRISPR/Cas9 system [ 43 ]. Embryo development rates (8–16-cell stage) of groups #1, #2, #4, #5, #7, and #8 were approximately 50% (Fig.…”

LincRNA#1 knockout alone does not affect polled phenotype in cattle heterozygous for the celtic POLLED allele

“…Microinjection is time consuming, even if done by a skilled individual. A better approach would be to edit all embryos simultaneously in a shorter amount of time using recently developed electroporation techniques 29 . The optimization of electroporation methods to simultaneously introduce editing reagents into hundreds of livestock zygotes may increase the obtainment of genome edited biallelic KO embryos with low mosaicism rates.…”

Multidrug-Resistant Microbial Therapy Using Antimicrobial Peptides and the CRISPR/Cas9 System

“…However, the harmfulness of those strategies restricts their suitability to change zygotes or for ex vivo experiments. 107 , 108 Electroporation is an extensively used technique to deliver proteins and nucleic acids into mammalian cells. 108 The permeability of the cell membrane is temporarily increased during electroporation, permitting proteins or nucleic acids to enter the cells.…”

“… 107 , 108 Electroporation is an extensively used technique to deliver proteins and nucleic acids into mammalian cells. 108 The permeability of the cell membrane is temporarily increased during electroporation, permitting proteins or nucleic acids to enter the cells. Electroporation is appropriate for all sorts of CRISPR-Cas9 systems, such as plasmid-based CRISPR-Cas9 systems, to deliver the mixture of Cas9 and the sgRNA or the Cas9/sgRNA RNP.…”