• Asian-Australasian Journal of Animal Sciences
• /
• 제29권3호
• /
• pp.413-418
• /
• 2016

Myostatin (MSTN) is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Gene knockout of MSTN can result in increasing muscle mass in sheep. The objectives were to investigate whether myostatin gene can be edited in sheep by transcription activator-like effector nucleases (TALENs) in tandem with single-stranded DNA oligonucleotides (ssODNs). We designed a pair of TALENs to target a highly conserved sequence in the coding region of the sheep MSTN gene. The activity of the TALENs was verified by using luciferase single-strand annealing reporter assay in HEK 293T cell line. Co-transfection of TALENs and ssODNs oligonucleotides induced precise gene editing of myostatin gene in sheep primary fibroblasts. MSTN gene-edited cells were successfully used as nuclear donors for generating cloned embryos. TALENs combined with ssDNA oligonucleotides provide a useful approach for precise gene modification in livestock animals.

• Journal of Genetic Medicine
• /
• 제3권1호
• /
• pp.15-19
• /
• 1999

We have investigated the genetic variation in the human apo B mRNA editing protein (apobec-1) gene. Exon 3 of the apobec-1 gene was amplified by polymerase chain reaction. After detection of an additional band by single strand conformational polymorphism (SSCP) analysis, sequencing of the SSCP-shift sample revealed a single-base mutation. The mutation was a CGG transversion at codon 80 resulting in a lleRMet substitution. This substitution was confirmed by restriction fragment length polymorphism analysis since a Pvull site is abolished by the substitution. Population and family studies confirmed that the inheritance of the genotypes for apobec-1 gene polymorphism is controlled by two codominant alleles (P1 and P2). A significant difference in plasma triglyceride was detected among the different apobec-1 genotypes in the CAD patients (P<0.05). Our study could provide the basis for elucidating the interaction between genetic variation of the apobec-1 gene and disorders related to lipid metabolism.

• Molecules and Cells
• /
• 제40권11호
• /
• pp.823-827
• /
• 2017

Genome editing using programmable nucleases such as CRISPR/Cas9 or Cpf1 has emerged as powerful tools for gene knock-out or knock-in in various organisms. While most genetic diseases are caused by point mutations, these genome-editing approaches are inefficient in inducing single-nucleotide substitutions. Recently, Cas9-linked cytidine deaminases, named base editors (BEs), have been shown to convert cytidine to uridine efficiently, leading to targeted single-base pair substitutions in human cells and organisms. Here, we first report on the generation of Xenopus laevis mutants with targeted single-base pair substitutions using this RNA-guided programmable deaminase. Injection of base editor 3 (BE3) ribonucleoprotein targeting the tyrosinase (tyr) gene in early embryos can induce site-specific base conversions with the rates of up to 20.5%, resulting in oculocutaneous albinism phenotypes without off-target mutations. We further test this base-editing system by targeting the tp53 gene with the result that the expected single-base pair substitutions are observed at the target site. Collectively, these data establish that the programmable deaminases are efficient tools for creating targeted point mutations for human disease modeling in Xenopus.

• BMB Reports
• /
• 제57권1호
• /
• pp.66-70
• /
• 2024

Prime editors (PEs), which are CRISPR-Cas9 nickase (H840A)-reverse transcriptase fusion proteins programmed with prime editing guide RNAs (pegRNAs), can not only edit bases but also install transversions, insertions, or deletions without both donor DNA and double-strand breaks at the target DNA. As the demand for in-locus tagging is increasing, to reflect gene expression dynamics influenced by endogenous genomic contexts, we demonstrated that PEs can be used to introduce the hemagglutinin (HA) epitope tag to a target gene locus, enabling molecular and biochemical studies using in-locus tagged plants. To promote genome-wide in-locus tagging, we also implemented a publicly available database that designs pegRNAs for in-locus tagging of all the Arabidopsis genes.

• Animal Bioscience
• /
• 제36권2_spc호
• /
• pp.333-338
• /
• 2023

Genetic modification enables modification of target genes or genome structure in livestock and experimental animals. These technologies have not only advanced bioscience but also improved agricultural productivity. To introduce a foreign transgene, the piggyBac transposon element/transposase system could be used for production of transgenic animals and specific target protein-expressing animal cells. In addition, the clustered regularly interspaced short palindromic repeat-CRISPR associated protein 9 (CRISPR-Cas9) system have been utilized to generate chickens with knockout of G0/G1 switch gene 2 (G0S2) and myostatin, which are related to lipid deposition and muscle growth, respectively. These experimental chickens could be the invaluable genetic resources to investigate the regulatory pathways and mechanisms of improvement of economic traits such as fat quantity and growth. The gene-edited animals could also be applicable to the livestock industry.

• Clinical and Experimental Pediatrics
• /
• 제64권6호
• /
• pp.269-279
• /
• 2021

Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR-Cas9) is an ancient prokaryotic defense system that precisely cuts foreign genomic DNA under the control of a small number of guide RNAs. The CRISPR-Cas9 system facilitates efficient double-stranded DNA cleavage that has been recently adopted for genome editing to create or correct inherited genetic mutations causing disease. Congenital heart disease (CHD) is generally caused by genetic mutations such as base substitutions, deletions, and insertions, which result in diverse developmental defects and remains a leading cause of birth defects. Pediatric CHD patients exhibit a spectrum of cardiac abnormalities such as septal defects, valvular defects, and abnormal chamber development. CHD onset occurs during the prenatal period and often results in early lethality during childhood. Because CRISPR-Cas9-based genome editing technology has gained considerable attention for its potential to prevent and treat diseases, we will review the CRISPR-Cas9 system as a genome editing tool and focus on its therapeutic application for CHD.

• International Journal of Industrial Entomology and Biomaterials
• /
• 제37권1호
• /
• pp.21-28
• /
• 2018

CRISPR/Cas9 gene editing system is an efficient method to mutation in a sequence specific manner. Here we report the direct transfection of the Cas9 nuclease and gene specific guide RNA can be used in BM-N cell line derived from Bombyx mori ovarian tissue to enfeeble function of endogenous gene in vitro. We have used gene editing system to negative regulation components of major signaling cascade, the Toll pathway, which controls B. mori resistance to microbe infections, such as fungi and gram positive bacteria. We demonstrate that the $I{\kappa}B-like$ protein Cactus may controls the activation of transcription factors such as Rel A and Rel B. The direct transfection of Cas9 nuclease and Cactus-specific guide-RNA complex may be used in BM-N cells to disrupt the function of endogenous genes in vitro. A mutation frequency of 30-40% was observed in the transfected cells, and various mutations caused the target region. Moreover, RT-PCR analysis revealed that Cactus gene was down regulated after these mutations. More importantly, mutation of BmCactus stimulated expression of lysozyme, moricin, and lebocin genes. These results suggest that the CRISPR/Cas9 systems are expected to efficiently induce site-specific mutations and it was possible to produce antimicrobial peptide through the gene editing.

• International Journal of Industrial Entomology and Biomaterials
• /
• 제39권1호
• /
• pp.14-21
• /
• 2019

Genome editing by CRISPR/Cas9, a third-generation gene scissor in molecular breeding at the genome level, is attracting much attention as one of the breeding techniques of the future. In this study, genetic and phenotypic analysis was used to examine the responsiveness of the Bakokjam variety of the silkworm Bombyx mori to molecular breeding using CRISPR/Cas9 in editing the white egg 2 (w-2) gene. The nucleotide sequence of the w-2 gene was analyzed and three different guide RNAs (gRNA) were prepared. The synthesized gRNA was combined with Cas9 protein and then analyzed by T7 endonuclease I after introduction into the Bm-N silkworm cell line. To edit the silkworm gene, W1N and W2P gRNA and Cas9 complexes were microinjected into silkworm embryos. Based on the results of microinjection, the hatching rate was 16-24% and the incidence of mutation was 33-37%. The gene mutation was verified in the heterozygous F1 generation, but no phenotypic change was observed. In F2 homozygotes generated by F1 self-crosses, a mutant phenotype was observed. These results suggest that silkworm molecular breeding using the CRISPR/Cas9 system is possible and will be a very effective way to shorten the time required than the traditional breeding process.

• Animal Bioscience
• /
• 제36권2_spc호
• /
• pp.339-349
• /
• 2023

Gene editing (GE) offers a new breeding technique (NBT) of sustainable value to animal agriculture. There are 3 GE working sites covering 5 feasible pathways to generate GE pigs along with the crucial intervals of GE/genotyping, microinjection/electroporation, induced pluripotent stem cells, somatic cell nuclear transfer, cryopreservation, and nonsurgical embryo transfer. The extension of NBT in the new era of pig breeding depends on the synergistic effect of GE and reproductive biotechnologies; the outcome relies not only on scientific due diligence and operational excellence but also on the feasibility of application on farms to improve sustainability.

• 한국환경농학회지
• /
• 제40권3호
• /
• pp.179-185
• /
• 2021

BACKGROUND: Tomato genome editing using CRISPR-Cas9 is being actively conducted in recent days, and lots of plant researches have been aiming to develop high valued crops by editing target genes without inserting foreign genes. Many researchers have been involved in the manipulation of the crop ripening process because fruit ripening is an important fruit phenotype for increasing fruit shelf life, taste, and texture of crops. This paper intends to evaluate target sgRNA to edit the two ripening-related genes encoding pectate lyase (PL) and phytoene synthase (Psy) with the CRISPR-Cas9 system. METHODS AND RESULTS: The CRISPR-Cas9 expression vector was cloned to target the PL (Solyc03g111690), Psy1 (Solyc03g031860), and Psy2 (Solyc02g081330) genes, which are the ripening genes of tomatoes. Tomatoes injected with Agrobacterium containing the CRISPR-Cas9 expression vector were further cultured for 5 days and used to check gene editing efficiency. As a result of the target gene sequence analysis by the next generation sequencing method, gene editing efficiency was calculated, and the efficient target location was selected for the PL and Psy genes. CONCLUSION: Therefore, this study was aimed to establish target sgRNA data that could have higher efficiency of the CRISPR-Cas9 system to obtain the delayed ripening phenotype of tomato. The developed method and sgRNA information is expected to be utilized in the development of various crops to manage its ripening processes.