Revolutionary advancements, such as the reduction in DNA sequencing costs and genome editing, have transformed biotechnology, fostering progress in manipulating biomolecules, engineering cells, and computational biology. Agriculture and food production have significantly benefited from tools like high-throughput microarrays, accelerating the selection of desired traits. Genetic engineering, especially utilizing genome editing, facilitates precise alterations in plants and animals, harnessing microbiomes and fostering lab-grown meat production to alleviate environmental pressures. The emergence of new biotechnologies, notably genome editing, underscores the necessity for regulatory frameworks governing LM (living modified) organisms. Global regulations overseeing genetically engineered or genome-edited (GE) organisms, encompassing animals, exhibit considerable diversity. Nonetheless, prevailing international regulatory trends typically exclude genomeedited plants and animals, employing novel biotechnological techniques, from GMO/ LMO classification if they lack foreign genes and originate through natural mutations or traditional breeding programs. This comprehensive review scrutinizes ongoing risk and safety assessment cases, such as genome-edited beef cattle and fish in the USA and Japan. Furthermore, it investigates the limitations of existing regulations related to genome editing in Korea and evaluates newly proposed legislation, offering insights into the future trajectory of regulatory frameworks.

The Transgenic livestock can be useful for the production of disease-resistant animals, pigs for xenotranplantation, animal bioreactor for therapeutic recombinant proteins and disease model animals. Previously, conventional methods without using artificial nuclease-dependent DNA cleavage system were used to produce such transgenic livestock, but their efficiency is known to be low. In the last decade, the development of artificial nucleases such as zinc-finger necleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas has led to more efficient production of knock-out and knock-in transgenic livestock. However, production of knock-in livestock is poor. In mouse, genetically modified mice are produced by co-injecting a pair of knock-in vector, which is a donor DNA, with a artificial nuclease in a pronuclear fertilized egg, but not in livestock. Gene targeting efficiency has been increased with the use of artificial nucleases, but the knock-in efficiency is still low in livestock. In many research now, somatic cell nuclear transfer (SCNT) methods used after selection of cell transfected with artificial nuclease for production of transgenic livestock. In particular, it is necessary to develop a system capable of producing transgenic livestock more efficiently by co-injection of artificial nuclease and knock-in vectors into fertilized eggs.

A specific serotonin receptor (Se-5HTR) has been identified in the beet armyworm, Spodoptera exigua and classified into 5-HT7 type. Se-5HTR expression was up-regulated in hemocytes and fat body in response to immune challenge. As being a GPCR, this receptor is presumably coupled with intracellular trimeric Gαs protein activating cAMP-dependent protein kinase (PKA) pathway to regulate several cellular functions. RNA interference (RNAi) of Se-5HTR as well as its downstream signal proteins exhibited significant suppression in cellular immune responses including nodulation and phagocytosis. Application of inhibitors to the signaling cascade suppressed the immune responses as well. To validate the Se-5HTR involvement in mediating cellular immunity, 5-HTR knock-out mutants were developed using CRISPR-Cas9 technique and suffered significant developmental anomalies.

CRISPR/Cas9-based genome editing technology fast replaces the previous methods that require protein engineering such as Zinc Finger Nucleases (ZFNs) and TALE nucleases (TALENs). Conventional genome editing of plant cells using CRISPR/Cas9 technology largely depends on Agrobacterium-mediated transformation of the plant cells and subsequent regeneration of whole plants from the edited cells. During this process, unwanted foreign DNAs including the antibiotics gene and fragments of the T-DNA can be introduced into plant genome. Insertion of these unwanted DNA causes lots of regulatory restrictions when commercializing the LMO products. To step aside these issues, we designed DNA-free ribonucleoprotein-based method and regenerated whole plants from the successfully engineered cells. We will share our discovery on the successful implement of this technology in lettuce protoplasts.

Genome editing that allows targeted mutagenesis in higher eukaryotic cells and organisms is broadly useful in biology, biotechnology, and medicine. We have developed zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and Cas9 RNA-guided engineered nucleases (RGENs), derived from the type II CRISPR/Cas prokaryotic adaptive immune system, to cleave chromosomal DNA in a targeted manner, producing DNA double-strand breaks in cells, the repair of which via endogenous systems gives rise to targeted genome modifications. The Cas9 protein, when complexed with small guide RNAs (sgRNAs), recognizes and cleaves target DNA sequences complementary to the guide RNAs in vivo, inducing targeted genome modifications at high frequencies in cultured cells and whole organisms. Despite broad interest in RNA-guided genome editing, RGENs are limited by off-target mutations. Here, we show that off-target effects of RGENs can be reduced below the detection limits of deep sequencing by choosing unique target sequences in the genome and modifying both guide RNA and Cas9. Furthermore, we deliver purified recombinant Cas9 protein complexed with sgRNAs (RGEN ribonucleoproteins (RNPs)) to animal embryos and cultured human cells including hard-to-transfect pluripotent stem cells to achieve highly efficient RNA-guided genome editing in cells and whole organisms. RGEN RNPs cleave chromosomal DNA almost immediately after delivery and are degraded rapidly in cells, reducing off-target effects and mosaicism.

Reliable and precise techniques for targeting modification of plant genomes have been explored in plant breeding communities. Initiated in the animal genome first, now the genome editing tool using a nuclease has been reported in some plant species including Arabidopsis, Maize, Tobacco, and other model systems. When the artificial nuclease is introduced into a plant cell and breaks the genomic sites randomly, endogenously operating DNA-repair mechanisms including non-homologous end joining(NHEJ) or homologous recombination(HR) are anticipated, leading to insertion of foreign DNA or deletion of the target locus, which collectively allows changes in plant traits of interest. Traditionally custom designed for induction of double-strand DNA break(DSB) at a predetermined locus was based on zinc-finger nuclease which contains nonspecific cleavage domains with target specificities of DNA binding zinc finger domains(three to four). The binding domains containing more than 20 DNA bases with high affinity to the target gene enable recognition of the locus efficiently. From this project, we focus on a petunia chalcone synthase(CHS) as a model system. The engineered nuclease will target the CHS gene, which is expected to be modified either constitutely or transiently. The derived transformed plants will be genetically or phenotypicly screened, along with molecular confirmation analysis by using various tools. We eventually extend the tools to various crop species and target genes, which makes the brand-new breeding technique more reliable and robust.