Improvements in Gene Editing Technology Boost Its Applications in Livestock

Abstract

Accelerated development of novel CRISPR/Cas9-based genome editing techniques provides a feasible approach to introduce a variety of precise modifications in the mammalian genome, including introduction of multiple edits simultaneously, efficient insertion of long DNA sequences into specific targeted loci as well as performing nucleotide transitions and transversions. Thus, the CRISPR/Cas9 tool has become the method of choice for introducing genome alterations in livestock species. The list of new CRISPR/Cas9-based genome editing tools is constantly expanding. Here, we discuss the methods developed to improve efficiency and specificity of gene editing tools as well as approaches that can be employed for gene regulation, base editing, and epigenetic modifications. Additionally, advantages and disadvantages of two primary methods used for the production of gene-edited farm animals: somatic cell nuclear transfer (SCNT or cloning) and zygote manipulations will be discussed. Furthermore, we will review agricultural and biomedical applications of gene editing technology.

Keywords: CRISPR/Cas9; agriculture; animal models; gene editing; livestock.

FIGURE 1

CRISPR/Cas9 structure. (A) X-ray structure of the Streptococcus pyogenes (Sp) CRISPR/Cas9 system (5F9R.pdb) in the pre-activated state (Jiang et al., 2016), created using Mol* (Sehnal et al., 2018). Cas9 (gray) is shown in molecular surface. The guide RNA (orange), the target DNA (dark blue), and non-target DNA (pink) strands are shown as cartoons. (B) A schematic CRISPR/Cas9 ribonucleoprotein structure formed by six domains: Rec I, Rec II, RuvC, HNH, Bridge Helix, and PAM Interacting domain, and guide RNA targeting DNA. The black arrow heads indicate the cut sites from each RuvC and HNH domains. The yellow/green nucleotides represent the PAM sequence.

FIGURE 2

Schematic summary of CRISPR/Cas9 gene editing using either zygote micromanipulation (electroporation or microinjection) or somatic cell nuclear transfer (SCNT) for generation of livestock animals for various applications.