Targeted gene knock-in by CRISPR/Cas ribonucleoproteins in porcine zygotes

Ki-Eun Park,Anne Powell,Shelley E S Sandmaier,Chan-Mi Kim,David M Donovan,Alan Mileham,Bhanu P Telugu

doi:10.1038/srep42458

Abstract

The domestic pig is an important “dual purpose” animal model for agricultural and biomedical applications. There is an emerging consensus in the biomedical community for the use of large animal models such as pigs to either serve as an alternative, or complement investigations from the mouse. However, the use of pig has not proven popular due to technical difficulties and time required in generating models with desired genetic modifications. In this regard, the ability to directly modify the genome in the zygote and generate edited animals is highly desirable. This report demonstrates for the first time, the generation of gene targeted animals by direct injection of Cas9 ribonucleoprotein complex and short stretches of DNA sequences into porcine zygotes. The Cas9 protein from Streptococcus pyogenes was pre-complexed with a single guide RNA targeting downstream of the ubiquitously expressed COL1A gene, and co-injected with a single-stranded repair template into porcine zygotes. Using this approach a line of pigs that carry pseudo attP sites within the COL1A locus to enable phiC31 integrase mediated introduction of transgenes has been generated. This new route for genome engineering in pigs via zygote injection should greatly enhance applications in both agriculture and biomedicine.

Highlights

The domestic pig is an important agricultural and biomedical model species
The CRISPR/Cas system has evolved in archaea and eubacteria as an RNA-based adaptive immunity system to detect and cleave invading viruses and plasmids[7,8]
SpCas[9] is a fusion construct with green fluorescent protein (GFP) and nuclear localization signals (NLS) that allows for tracking the expression of full length Cas[9] protein and its translocation into the nucleus of the zygote

Summary

Introduction

The domestic pig is an important agricultural and biomedical model species. A growing global human population, an increasing demand for animal protein in human diets, and a population that is living longer, necessitate a renewed focus on developing technologies in “dual purpose” animals such as pigs that can serve both agriculture and biomedicine. The Streptococcus pyogenes (Sp) Type II CRISPR/Cas system has been adapted to successfully modify somatic cells and generate modified pigs[9,10,11,12,13]. In these studies, a mammalian codon-optimized Cas[9] nuclease along with a synthetic single-guide RNA (sgRNA)[14] has been used to generate genetically modified pigs[9,10,11,12,13]. We sought to go a step further and demonstrate that it is possible to knock-in short DNA sequences (or transgenes) into target loci by injection into in vivo derived zygotes and to generate live offspring bearing targeted mutations using the CRISPR/Cas system. It is only necessary to inject the CRISPRs and targeting oligonucleotides into the cytoplasm in order to generate targeted offspring

Objectives

Methods

Results

Conclusion