Strategies to reduce genetic mosaicism following CRISPR-mediated genome edition in bovine embryos

I Lamas-Toranzo,O Ousinde,B Galiano-Cogolludo,F Cornudella-Ardiaca,J Cobos-Figueroa,P Bermejo-Álvarez

doi:10.1038/s41598-019-51366-8

Abstract

Genetic mosaicism is the presence of more than two alleles on an individual and it is commonly observed following CRISPR microinjection of zygotes. This phenomenon appears when DNA replication precedes CRISPR-mediated genome edition and it is undesirable because it reduces greatly the odds for direct KO generation by randomly generated indels. In this study, we have developed alternative protocols to reduce mosaicism rates following CRISPR-mediated genome edition in bovine. In a preliminary study we observed by EdU incorporation that DNA replication has already occurred at the conventional microinjection time (20 hpi). Aiming to reduce mosaicism appearance, we have developed three alternative microinjection protocols: early zygote microinjection (10 hpi RNA) or oocyte microinjection before fertilization with either RNA or Ribonucleoprotein delivery (0 hpi RNA or 0 hpi RNP). All three alternative microinjection protocols resulted in similar blastocyst and genome edition rates compared to the conventional 20 hpi group, whereas mosaicism rates were significantly reduced in all early delivery groups (~10–30% of edited embryos being mosaic depending on the loci) compared to conventional 20 hpi microinjection (100% mosaicism rate). These strategies constitute an efficient way to reduce the number of indels, increasing the odds for direct KO generation.

Highlights

Genome modification at specific loci allows the ablation or insertion of specific DNA sequences to unequivocally assess the role of a specific gene on a particular physiological process or to alter the phenotype of an animal for diverse purposes
CRISPR is directed to the beginning of the coding region of the target gene, where it will induce a double-strand break (DSB)
The DSB can be repaired by either homologous recombination (HR), which reconstitutes the target site allowing CRISPR recognition and thereby the generation of another DSB, or by non-homologous end joining repair (NHEJ), which often generates random insertions or deletions at the target site

Summary

Introduction

Genome modification at specific loci allows the ablation (knock-out, KO) or insertion (knock-in, KI) of specific DNA sequences to unequivocally assess the role of a specific gene on a particular physiological process or to alter the phenotype of an animal for diverse purposes. Technical limitations have largely restricted targeted genome modification in mammals to the mouse model These limitations derived from the extremely low efficiency of the only available technique for targeted mutagenesis, homologous recombination (HR)[1], which impedes its direct application on embryos. CRISPR technology allows KO generation in a single-step by microinjection at the zygote stage[6] For this purpose, CRISPR is directed to the beginning of the coding region of the target gene, where it will induce a double-strand break (DSB). The DSB can be repaired by either HR, which reconstitutes the target site allowing CRISPR recognition and thereby the generation of another DSB, or by non-homologous end joining repair (NHEJ), which often generates random insertions or deletions (indels) at the target site Those indels constitute a stable mutation, as they impede target recognition by CRISPR, and can produce KO alleles, as those indels not multiple of three disrupt the open reading frame (ORF) of the target gene, leading to a truncated protein. We report strategies to reduce genetic mosaicism following CRISPR edition of bovine embryos based on early delivery of CRISPR components

Methods

Results

Conclusion