Advances in Genome Editing and Genome Engineering Toolbox for Improving Livestock Health and Productivity

Abstract

Introduction An ever-growing world population with increased income causes significant rises in the demand for livestock products such as dairy and meat. From 2020 to 2050, global demand for livestock products is projected to increase by 8%, and per capita protein requirements will rise by 38% (Komarek et al., 2021). In addition, livestock production is under increasing pressure to provide high-quality animal protein while reducing production costs, disease losses, environmental impacts, and animal-welfare concerns. Traditional breeding strategies for sustained genetic improvement in growth, milk yield, and fertility rely on natural genetic variation, which can limit the development of new traits. In addition, it is constrained by long generation intervals and inadvertently introduces unfavourable traits while selecting for beneficial ones (Mueller & Van Eenennaam, 2022). Sequence-specific nucleases or genome editors provide a transformative solution by rapidly creating traits that are difficult or nearly impossible to achieve through conventional breeding. It enables a precise, efficient, and rapid platform to alter, introduce, remove, or modify traits of agricultural importance (Fahrenkrug et al., 2010). Genome editing has been shown to enhance genetic gain in the livestock field by improving productivity, disease resistance, trait enhancement, and animal welfare (Mueller & Van Eenennaam, 2022). Despite these promising broad long-term applications, the widespread adoption of gene-edited livestock needs to be improved. In recent years, technologies such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR/Cas9, base editors, and prime editors have expanded the livestock genome-engineering toolbox. Zinc-Finger Nucleases (ZFNs) Mediated Genome Editing This represents the 1st type of genome editor, consisting of zinc-finger proteins built from Cys-Cys-His-His zinc-finger domains, rationally designed to recognize specific trinucleotide sequences of the genome. Notably, zinc finger triplets (ZFPs) only provide sequence specificity, thus linking to the FokI nuclease, which is a type IIs nuclease causing nonspecific DNA cleavage, to make the chimeric protein known as zinc finger nucleases (ZFNs). Zinc finger triplets (ZFPs) are fused with the FokI nuclease, which is a type IIs nuclease, to make zinc finger nucleases (Chandrasegaran & Smith, 1999). As FokI only functions as an obligate dimer, using the paired ZFP-FokI constructs that bind to the opposite strands of target DNA in proximity enables DNA cleavage activity (Bitinaite et al., 1998) (Figure 1). ZFNs have been successfully used in livestock to improve mastitis resistance in dairy cattle and reduce milk β-lactoglobulin allergen in goats (Liu et al., 2013; Xiong et al., 2013). Despite these advances in their applicability in livestock, the widespread use of ZFNs has been hampered by the complexity of zinc finger protein design and optimization, complex modular assembly, the nonavailability of ZFPs for each genomic target, relatively low editing efficiency, and constraints related to intellectual property rights (Gupta & Musunuru, 2014). Transcription Activator-like Effector Nucleases (TALEN) Mediated Genome Editing This represents the 2nd type of genome editor and is a class of transcription activator-like effector proteins, found in the plant pathogen Xanthomonas Oryza, with a novel DNA-binding domain, termed TALE repeats. The TALE repeats comprise tandem arrays with 10 to 30 repeats that recognize and bind extended DNA sequences (Bogdanove & Voytas, 2011). Each repeat is 33 to 35 amino acids in length, with two adjacent amino acids, termed the repeat-variable di-residue (RVD), conferring specificity for one of the four DNA base pairs (Boch et al., 2009). Within each RVD, the 12th and 13th amino acids are highly variable and play a key role in base recognition. For example, NI binds to adenine (A), NG to thymine (T), HD to cytosine (C), and NN to guanine (G). Unlike ZFNs, there is a one-to-one correspondence between the repeats and the base pairs in the target DNA sequences. The advantage of recognizing each RVD to one nucleotide, literally any DNA sequence can be targeted by assembling appropriate RVDs in a defined order. Similar to ZFNs, TALE repeats fuse with FokI nuclease to form the TALEN. However, two pairs of TALEN pair made with a spacer sequence in such a way that the two TALE repeats maintain specificity for the targeted DNA sequence, whereas the FokI nuclease sits opposite each other to dimerise and initiate the site-specific double-strand break (Wefers et al., 2012, 2013) (Figure 1). TALENs have been successfully used in mice to generate various disease models (Panda et al., 2013). Similarly, TALENs have been used in livestock for improving disease-resistant traits towards tuberculosis (Wu et al., 2015) and generation of pooled cattle (Carlson et al., 2016). Compared to ZFNs, TALENs offer higher targeting specificity and are easier to design due to their one-to-one correspondence between RVD and DNA bases. However, construction of TALE arrays remains labour-intensive, requires highly specialised molecular biology expertise, and limits delivery efficiency. CRISPR/Cas9 Mediated Genome Editing This represents the 3rd type of genome editor, found in bacteria and archaea as an adaptive immune system, enabling them to defend against foreign pathogen elements such as bacteriophages and plasmids (Horvath & Barrangou, 2010). The widely used CRISPR/Cas9 genome editing system is derived from the Type II CRISPR system of Streptococcus pyogenes (spCas9). The 20-base-pair spacer sequence is needed, along with the proto spacer adjunct motif (PAM) is needed for target site recognition. In addition, various Cas9 proteins recognise various PAM sequences; for example, spCas9 typically recognizes 5′-NGG, while Cas12a (Cpf1) recognizes 5′-TTTV (where V represents A, C, or G) (Sander & Joung, 2014; Han et al., 2024). For genome editing applications, crRNA pairs with a trans-activating crRNA (tra-crRNA) to form a duplex, known as sgRNA, which attracts Cas9 and introduces DSBs three base pairs downstream of the PAM site through the coordinated Cas9 activity of its two nuclease domains, i.e., HNH and RuvC (Jinek et al., 2012). Due to its simplicity, efficiency, and broad applicability, CRISPR/Cas9 has become the preferred method for gene knockout, knock-in, and high-throughput genome screening in diverse organisms. However, spCas9 has several limitations, such as the NGG PAM sequence, which limits its application in AT-rich genomic regions; the large 1368 aa sequence limits its delivery; and promoting the unintended off-target activity and genomic instability (Fu et al., 2013). Mechanism of Genome Editing Gene editing is an advanced genome engineering tool that enables precise genetic modification. The basis of gene editing tools, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system, is to introduce double-strand breaks (DSBs) at designated genomic loci, which eventually promote non-homologous end joining (NHEJ) or homologous recombination, two primary DNA repair pathways (Gupta & Musunuru, 2014). NHEJ is a predominant, rapidly acting, template-independent repair mechanism that introduces indels leading to a frameshift and thus an allele that achieves gene knockout. In contrast, HDR is a rare, template-dependent repair mechanism that uses exogenous DNA templates with homologous arms to achieve a gene knock-in allele (Figure 1). Base Editing and Prime Editing It referred as 4th generation of genome editing technology, working independently of DSBs and donor templates, posing enhanced precision and reduced off-target effects, thereby improving safety and expanding the scope of genome editing. To overcome the DSB-mediated unintended off-target activities and cellular toxicity of spCas9, base editors (BEs) and prime editors (PEs) have been developed to cause a single-strand break, representing a safer and promising tool in the genome editing field. (Chen & Liu, 2023; Gaudelli et al., 2017). Base editors are two types: cytosine base editing (CBE) and adenine base editing (ABE). CBE is achieved by fusing a cytidine deaminase (e.g., rat APOBEC1) to a Cas9 nickase (nCas9) and an uracil DNA glycosylase inhibitor (UGI). Upon delivery of these complexes, cytosine is deaminated to uracil, whereas UGI inhibits uracil DNA glycosylase (UDG) from excising uracil, allowing it to be interpreted as thymine during DNA replication or repair, resulting in a C-to-T substitution. Similarly, ABE uses adenine deaminase fused to nCas9, where adenine deaminase converts T to inosine (I); I is recognized as G, and through DNA repair, I is ultimately converted to G (Gaudelli et al., 2017). Base editors have been used to introduce stop codons via three consecutive cytosines (c.59-61CCC > TTT) to thymine conversion to disrupt beta-lactoglobulin expression in dairy cows (Ding et al., 2025) and modification of myostatin in Hu sheep (Wang et al., 2025).s Prime editors consist of nCas9 and reverse transcriptase, and prime editing guide RNA (pegRNA), which comprises a primer binding site (PBS) and a reverse transcription template (RTT) at its 3’ end. Upon target identification by pegRNA, nCas9 induces a nick in the non-target DNA strand, which then hybridises with the PBS region of pegRNA, and reverse transcriptase uses the RTT sequence to synthesize a DNA strand encoding the desired edit. This results in a 3’ DNA flap, which replaces the original sequence through flap resolution and DNA repair. Subsequently, the edited strand is then used as a template to correct the opposite strand, achieving precise, bidirectional editing without inducing DSBs (Chen & Liu, 2023). Notably, prime editors have been used for the efficient modification of the pig genome (Qi et al., 2023). Delivery Method