CRISPR/Cas9 ribonucleoprotein (RNP)-mediated gene editing has recently been applied to Ganoderma lucidum as a promising tool for functional genomics and strain improvement. However, the multinuclear nature of this basidiomycete can result in genetic mosaicism, raising concerns about the long-term stability of edited strains. In this study, we report the occurrence of revertant phenotypes in CRISPR/Cas9-edited transformants of G. lucidum. Although the edited colonies initially exhibited the expected phenotype, repeated subculturing led to the reappearance of wild-type phenotypes. PCR and sequencing analyses revealed the coexistence of edited and non-edited nuclei, and the progressive loss of edited genotypes over successive generations. These findings demonstrate that multinuclearity is a key factor contributing to the instability of CRISPR/Cas9-based edits in G. lucidum. This brief report provides the first direct documentation of revertant occurrence in edited G. lucidum strains and underscores the need for rigorous selection strategies and novel approaches to secure stable homokaryotic transformants in mushroom genetic engineering.

The use of genetically modified animals for human diseases contributes to the understanding of the pathogenesis of various diseases and aids in the search for effective treatments. Consequently, there is an emerging trend of establishing appropriate genetic animal models capable of recapitulating the crucial phenotypes of human diseases to facilitate investigations about the pathogenesis and effective treatments. Recently, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) technology demonstrated its successful application in genetically modifying the genomes of diverse species. Furthermore, CRISPR/ Cas technology has been established as a tool for producing genetic animal models that more faithfully mimic human diseases. In this review, we have focused on the application of CRISPR/Cas technology in developing innovative genetic animal models for the study of human diseases, specifically for the investigation of disease pathogenesis and treatment. We have also discussed the current technical challenges of CRISPR technology, such as off-target effects, and reviewed the emerging strategies developed to enhance its precision. Finally, we have provided an overview of the considerable potential of advanced tools derived from the CRISPR/dead Cas9 (dCas9) system, such as base and prime editors, and explore their future applications in creating more sophisticated disease models.

This study evaluated the effects of the cells alive system (CAS) freezing method and different thawing methods (refrigeration and running water) on the quality of aged Hanwoo shoulder clods. Thawing time was significantly influenced by the thawing method, with running water thawing taking 3 h and refrigeration thawing exceeding 20 h. CAS freezing yielded significantly higher lightness (L*) values during running water thawing than it did during general freezing (p<0.05), likely owing to reduced water loss. CAS-frozen meat exhibited lower volatile basic nitrogen levels and thawing loss (2.05%–2.11%) compared with that of general frozen meat (3.17% –3.79%), indicating superior freshness and reduced quality deterioration (p<0.05). Among the textural properties, springiness was highest in frozen and running water-thawed CAS meat (10.5 mm). In contrast, general freezing combined with refrigeration thawing caused the greatest quality degradation, as reflected by the lowest pH (5.45) and water holding capacity (86.36%), and highest cooking loss (32.98%). These results suggest that CAS freezing combined with running water thawing minimizes changes in quality and enhances springiness, making it an optimal approach for the production of high-quality aged meat.

Despite their historical use, studies on the genetic functions of mushrooms and varietal improvement via biomolecular techniques are limited compared to other organisms. Recent advancements in CRISPR/Cas9 have enabled precise genetic modifications in mushrooms, with RNP-based systems offering high editing efficiency without foreign gene insertion. In this study, we optimized gene-editing conditions for Ganoderma lucidum (Yongji 2) by utilizing RNP/nanoparticle complexes to enhance efficiency. The optimal conditions included a 0.2 M sorbitol buffer (pH 7.0) and a protoplast-to-complex ratio of 10:1. Among eight gRNAs designed for the catA gene, three were identified with high activity, and PEG-mediated transformation resulted in successful gene edits, primarily involving 1 bp deletions. The editing efficiency reached 7–8%, demonstrating that nanoparticle-supported RNP systems are effective for marker-free gene editing in mushrooms. These findings highlight a promising approach for advancing genetic research and varietal improvement in G. lucidum and other mushroom species.

최근 전세계적으로 기능성 성분이 강화된 유전자교정 작 물의 생산 및 소비가 증가하고 있다. 하지만 유전자교정 작물의 유전독성에 관한 소비자의 우려도 증가하고 있어 과학적인 자료 확보 및 정보 공유에 대한 인프라가 필요한 실정이다. 본 연구에서는 CRISPR/Cas9 시스템을 활용한 유 전자교정 토마토 동결건조물(LTT)이 DNA나 염색체에 직접 적인 손상을 일으키고 형태적 또는 기능적 이상을 유발하 는지 여부를 확인하기 위해 유전독성 평가를 수행하였다. 이를 위하여 미생물 복귀돌연변이시험, 염색체이상시험, 골수세포를 이용한 체내 소핵시험을 국제적으로 표준화된 OECD Guidelines에 따라 시험을 진행하였다. 복귀돌연변이 시험에서 LTT는 S9의 존재 여부와 관계없이 Salmonella typhimurium 균주 TA98, TA100, TA1535 및 TA1537, 그리 고 Escherichia coli WP2 uvrA에서 복귀돌연변이를 유발하 지 않았다. LTT는 CHL 세포의 수적이상 중기상과 구조적 이상 중기상 등의 염색체 이상을 유발하지 않았다. 또한, LTT는 다염성 적혈구에서 소핵화된 다염성 적혈구의 빈도 를 증가시키지 않았다. 이러한 연구를 통해 CRISPR/Cas9 시스템을 활용한 유전자교정 토마토의 안전성을 검증하고, 향후 CRISPR/Cas9 시스템을 활용한 유전자교정 작물의 유 전독성을 평가하는 기초 자료로 사용될 수 있을 것이다.

This perspective article delves into the evolving landscape of non-viral vectors for efficient CRISPR delivery, addressing the challenges associated with viral vectors and highlighting the potential of carbon-based nanomaterials as promising alternatives. The article underscores the importance of design strategies in enhancing the interactions between CRISPR components and carbon-based nanomaterials. Various design approaches are explored, including the incorporation of modified nanoparticles between carbonic layers and the creation of unique morphologies to facilitate optimal CRISPR interactions. Specific case studies are presented to exemplify the effectiveness of carbon-based nanomaterials in CRISPR delivery. This perspective sheds light on the dynamic field of non-viral CRISPR delivery vectors, emphasizing the significance of design strategies and showcasing the promising outcomes achieved through the utilization of carbon-based nanomaterials. The provided insights contribute to the ongoing efforts to develop efficient and safe methods for gene delivery and therapy.

Rapid and accurate detection of pathogenic bacteria is crucial for various applications, including public health and food safety. However, existing bacteria detection techniques have several drawbacks as they are inconvenient and require time-consuming procedures and complex machinery. Recently, the precision and versatility of CRISPR/Cas system has been leveraged to design biosensors that offer a more efficient and accurate approach to bacterial detection compared to the existing techniques. Significant research has been focused on developing biosensors based on the CRISPR/Cas system which has shown promise in efficiently detecting pathogenic bacteria or virus. In this review, we present a biosensor based on the CRISPR/Cas system that has been specifically developed to overcome these limitations and detect different pathogenic bacteria effectively including Vibrio parahaemolyticus, Salmonella, E. coli O157:H7, and Listeria monocytogenes. This biosensor takes advantage of the CRISPR/Cas system's precision and versatility for more efficiently accurately detecting bacteria compared to the previous techniques. The biosensor has potential to enhance public health and ensure food safety as the biosensor’s design can revolutionize method of detecting pathogenic bacteria. It provides a rapid and reliable method for identifying harmful bacteria and it can aid in early intervention and preventive measures, mitigating the risk of bacterial outbreaks and their associated consequences. Further research and development in this area will lead to development of even more advanced biosensors capable of detecting an even broader range of bacterial pathogens, thereby significantly benefiting various industries and helping in safeguard human health

배스(Micropterus salmoides)는 수생태계에서 최상위단계에 위치하는 생태계교란 어종으로 심각한 담수생태계의 불균형을 초래하고 있다. 배스의 퇴치 및 관리를 위한 다양한 시도를 하고 있지만 효과적인 방안은 없는 상황이므로 배스의 고유한 특성에 기반한 개체군 감소의 효율성을 극대화할 수 있는 방식을 모색하였다. 본 연구에서는 배스의 Transcriptom 분석으로 Unigene contigs는 182,887개, 그리고 정자-난자 인식 단백질인 IZUMO1과 Zona pellucida sperm-binding protein의 유전자에서 CRISPR/Cas9 system을 적용할 최종 Target sequence는 12종을 산출하였다. 각 Target sequence를 인식할 수 있는 12종의 sgRNA를 합성한 후 후속 연구에 사용할 12종의 Cas9-sgRNA ribonucleoprotein (RNP) complex를 제작하였다. 본 연구에서는 차세대염기서열 분석법으로 정자-난자 인식 단백질을 암호화하는 유전자를 탐색하였고, CRISPR/Cas9 system으로 유전자를 편집하여 번식행동은 하지만 수정란을 형성하지 못하는 생식세포를 생산하는 불임개체를 유도하기 위한 조성물 개발 과정을 확립하였다. 그리고 배스와 동일한 수계에 있는 고유 생물종의 서식에는 영향을 미치지 않는 생태교란종 관리 방안으로서의 유용성을 검증하기 위한 후속 연구의 귀중한 기초 자료를 확보하는데 기여했다고 판단된다.

새롭게 부상하는 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated protein) 9 유전자 편집 기술은 장기 이식(organ transplantation)과 같은 생의학 연구(biomedical research)와 동물 산업에 대한 전통적인 접근 방식을 빠르게 변화시키고 있다. 돼지 생식 및 호흡기 증후군 바이러스(porcine reproductive and respiratory syndrome virus; PRRSV)와 전염성 위장염 바이러스 (transmissible gastroenteritis coronavirus; TGEV)는 돼지 산업에 막대한 경제적 손실을 초래하는 치명적인 바이러스이다. 바이러스의 숙주 수용체 단백질 CD163과 pAPN에 대한 이중 유전자 녹아웃(double knock-out; DKO) 돼지는 PRRSV와 TEGV에 내성을 나타내었으며, 정상(wild-type; WT) 돼지와 비교할 때 성장과 생식 특성의 차이가 없었다. 이러한 결과는 경제 동물 돼지에 CRISPR-Cas9 매개 유전자 편집 기술을 적용하여 바이러스 저항성 유전자 변형에 의한 품종 개량이 달성될 수 있다는 것을 보여주며, 질병 저항성 돼지 생산을 위한 육종 시작점을 제공한다. 종간 배반포 보완(interspecies blastocyst complementation)은 이종 만능 줄기세포 유도체(xenogenic pluripotent stem cell derivatives)의 장기 특이적 생산(organ-specific enrichment)을 가능하게 한다. CRISPR-Cas9 매개 접합자 유전자 편집(CRISPR-Cas9-mediated zygote gene editing)을 이용하여 췌장 생성(pancreatogenesis), 신장 생성(nephrogenesis), 간 생성(hepatogenesis) 및 혈관 생성(vasculogenesis)이 불가능 생쥐 숙주를 만들었으며, 이러한 숙주와 배반포 보완 플랫폼을 결합하여 키메라를 만들었다. 또한 돼지와 소 같은 유제류(ungulate)의 섬유아세포(fibroblasts)를 이용하여 CRISPR-Cas9 매개 유전자 편집과 체세포 핵 치환(somatic cell nuclear transfer) 과정을 거쳐 복제 배아(genome-edited cloned embryos) 를 생산하였다. 복제 배아의 1차 배양 섬유아세포(primary cultured fibroblasts)를 재복제하여 배반포 보완을 위한 숙주 배아로 이용하였다. CRISPR-Cas9 유전자 편집 기술과 종간 배반포 보완 플랫폼 전략의 조합은 유전자 변형 돼지를 생산하는 데 유용하다. 본 논문에서는 CRISPR/Cas9 유전자 편집 기술과 배반포 보완 플랫폼, 질병 저항성(disease resistance) 돼지, 이종장기이식(xenotransplantation) 목적의 키메라 생산을 소개하고자 한다.

We investigate the effects of Yb2O3 and calcium aluminosilicate (CAS) glass as sintering additives on the sintering behavior of AlN. The AlN specimens are sintered at temperatures between 1700oC and 1900oC for 2 h in a nitrogen atmosphere. When the Yb2O3 content is low (within 3 wt.%), an isolated shape of secondary phase is observed at the AlN grain boundary. In contrast, when 3 wt.% Yb2O3 and 1 wt.% CAS glass are added, a continuous secondary phase is formed at the AlN grain boundary. The thermal conductivity decreases when the CAS glass is added, but the sintering density does not decrease. In particular, when 10 wt.% Yb2O3 and 1 wt.% CAS glass are added to AlN, the flexural strength is the highest, at 463 MPa. These results are considered to be influenced by changes in the microstructure of the secondary phase of AlN.