• Asian-Australasian Journal of Animal Sciences
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• v.33 no.2
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• pp.360-372
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• 2020

Objective: Specific genomic sites can be recognized and permanently modified by genome editing. The discovery of endonucleases has advanced genome editing in pigs, attenuating xenograft rejection and cross-species disease transmission. However, off-target mutagenesis caused by these nucleases is a major barrier to putative clinical applications. Furthermore, off-target mutagenesis by genome editing has not yet been addressed in pigs. Methods: Here, we generated genetically inheritable α-1,3-galactosyltransferase (GGTA1) knockout Yucatan miniature pigs by combining transcription activator-like effector nuclease (TALEN) and nuclear transfer. For precise estimation of genomic mutations induced by TALEN in GGTA1 knockout pigs, we obtained the whole-genome sequence of the donor cells for use as an internal control genome. Results: In-depth whole-genome sequencing analysis demonstrated that TALEN-mediated GGTA1 knockout pigs had a comparable mutation rate to homologous recombination-treated pigs and wild-type strain controls. RNA sequencing analysis associated with genomic mutations revealed that TALEN-induced off-target mutations had no discernable effect on RNA transcript abundance. Conclusion: Therefore, TALEN appears to be a precise and safe tool for generating genomeedited pigs, and the TALEN-mediated GGTA1 knockout Yucatan miniature pigs produced in this study can serve as a safe and effective organ and tissue resource for clinical applications.

• Journal of Life Science
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• v.29 no.5
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• pp.537-544
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• 2019

Genome editing technology employing gene scissors has generated interest in molecular breeding in various fields, and the development of the third-generation gene scissors of the clustered, regularly interspaced short palindromic repeat (CRISPR) system has accelerated the field of molecular breeding through genome editing. In this study, we analyzed the possibility of silkworm molecular breeding using gene scissors by genomic and phenotypic analysis after editing the biogenesis of lysosome-related organelles (BmBLOS) gene of Bakokjam using the CRISPR/Cas9 system. Three types of guide RNAs (gRNA) were synthesized based on the BmBLOS gene sequence of Bakokjam. Complexes of the prepared gRNA and Cas9 protein were formed and introduced into Bombyx mori BM-N cells by electroporation. Analysis of the gene editing efficiency by T7 endonuclease I analysis revealed that the B4N gRNA showed the best efficiency. The silkworm genome was edited by microinjecting the Cas9/B4N gRNA complex into silkworm early embryos and raising the silkworms after hatching. The hatching rate was as low as 18%, but the incidence of mutation was over 40%. In addition, phenotypic changes were observed in about 70% of the G0 generation silkworms. Sequence analysis showed that the BmBLOS gene appeared to be a heterozygote carrying the wild-type and mutation in most individuals, and the genotype of the BmBLOS gene was also different in all individuals. These results suggest that although the possibility of silkworm molecular breeding using the CRISPR/Cas9 system would be very high, continued research on breeding and screening methods will be necessary to improve gene editing efficiency and to obtain homozygotes.

• Journal of Microbiology and Biotechnology
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• v.30 no.10
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• pp.1583-1591
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• 2020

CRISPR/Cpf1 has emerged as a new CRISPR-based genome editing tool because, in comparison with CRIPSR/Cas9, it has a different T-rich PAM sequence to expand the target DNA sequence. Single-base editing in the microbial genome can be facilitated by oligonucleotide-directed mutagenesis (ODM) followed by negative selection with the CRISPR/Cpf1 system. However, single point mutations aided by Cpf1 negative selection have been rarely reported in Corynebacterium glutamicum. This study aimed to introduce an amber stop codon in crtEb encoding lycopene hydratase, through ODM and Cpf1-mediated negative selection; deficiency of this enzyme causes pink coloration due to lycopene accumulation in C. glutamicum. Consequently, on using double-, triple-, and quadruple-base-mutagenic oligonucleotides, 91.5-95.3% pink cells were obtained among the total live C. glutamicum cells. However, among the negatively selected live cells, 0.6% pink cells were obtained using single-base-mutagenic oligonucleotides, indicating that very few single-base mutations were introduced, possibly owing to mismatch tolerance. This led to the consideration of various target-mismatched crRNAs to prevent the death of single-base-edited cells. Consequently, we obtained 99.7% pink colonies after CRISPR/Cpf1-mediated negative selection using an appropriate single-mismatched crRNA. Furthermore, Sanger sequencing revealed that single-base mutations were successfully edited in the 99.7% of pink cells, while only two of nine among 0.6% of pink cells were correctly edited. The results indicate that the target-mismatched Cpf1 negative selection can assist in efficient and accurate single-base genome editing methods in C. glutamicum.

• Molecules and Cells
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• v.40 no.11
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• pp.823-827
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• 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.

• Korean Journal of Environmental Agriculture
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• v.40 no.3
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• pp.186-193
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• 2021

BACKGROUND: Before generating transgenic plant using the CRISPR-Cas9 system, the efficiency test of sgRNAs is recommended to reduce the time and effort for plant transformation and regeneration process. The efficiency of the sgRNA can be measured through the transient expression of sgRNA and Cas9 gene in tomato cotyledon; however, we found that the calculated efficiency showed a large variation. It is necessary to increase the precision of the experiment to obtain reliable sgRNA efficiency data from transient expression. METHODS AND RESULTS: The cotyledon of 11^th, 15^th, 19^th, and 23^rd-day-old tomato (Solanum lycopersicum cv. Micro-Tom) were used for expressing CRISPR-Cas9 transiently. The agrobacterium harboring sgRNA for targeting ALS2 gene of tomato was injected through the stomata of leaf adaxial side and the genomic DNA was extracted in 5 days after injection. The target gene edition was identified by amplifying DNA fragment of target region and analyzing with Illumina sequencing method. The target gene editing efficiency was calculated by counting base deletion and insertion events from total target sequence read. CONCLUSION: The CRISPR-Cas9 editing efficiency varied with tomato cotyledon age. The highest efficiency was observed at the 19-day-old cotyledons. Both the median and mean were the highest at this stage and the sample variability was also minimized. We found that the transgene of CRISPR-Cas9 system was strongly correlated with plant leaf development and suggested the optimum cotyledon leaf age for Agrobacterium-mediated transfection in tomato.

• Korean Journal of Plant Taxonomy
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• v.52 no.2
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• pp.114-117
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• 2022

Lindera angustifolia is mainly distributed in the temperate climate zone of China but shows an extraordinary distribution, disjunctively isolated on the western coastal islands of Korea. We therefore present the complete chloroplast genome of Korean L. angustifolia. The complete plastome was 152,836 bp in length, with an overall GC content of 39.2%. A large single copy (93,726 bp) and a small single copy (18,946 bp) of the genome were separated by a pair of inverted repeats (20,082 bp). The genome consists of 125 genes, including 81 protein-coding, eight ribosomal RNA, and 36 transfer RNA genes. While five RNA editing genes (psbL, rpl2, ndhB×2, and ndhD) were identified in L. angustifolia from China, the "ndhD" gene was not recognized as an RNA editing site in the corresponding Korean individual. A phylogenetic analysis revealed that Korean L. angustifolia is most closely related to the Chinese L. angustifolia with strong bootstrap support, forming a sister group of L. glauca.

• Journal of Plant Biotechnology
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• v.44 no.2
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• pp.107-114
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• 2017

Implementation of crop improvement programs relies on genetic diversity. To overcome the limited occurrence of natural mutations, researchers and breeders applied diverse methods, ranging from conventional crossing to classical bio-technologies. Earlier generations of knockout and gain-of-function technologies often result in incomplete gene disruption or random insertions of transgenes into plant genomes. The newly developed editing tool, CRISPR/Cas9 system, not only provides a powerful platform to efficiently modify target traits, but also broadens the scope and prospects of genome editing. Customized Cas9/guide RNA (gRNA) systems suitable for efficient genomic modification of mammalian cells or plants have been reported. Following successful demonstration of this technology in mammalian cells, CRISPR/Cas9 was successfully adapted in plants, and accumulating evidence of its feasibility has been reported in model plants and major crops. Recently, a modified version of CRISPR/Cas9 with added novel functions has been developed that enables programmable direct irreversible conversion of a target DNA base. In this review, we summarized the milestone applications of CRISPR/Cas9 in plants with a focus on major crops. We also present the implications of an improved version of this technology in the current plant breeding programs.

• Molecules and Cells
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• v.39 no.3
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• pp.179-185
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• 2016

Posttranscriptional regulation of RNA metabolism, including RNA processing, intron splicing, editing, RNA export, and decay, is increasingly regarded as an essential step for fine-tuning the regulation of gene expression in eukaryotes. RNA-binding proteins (RBPs) are central regulatory factors controlling posttranscriptional RNA metabolism during plant growth, development, and stress responses. Although functional roles of diverse RBPs in living organisms have been determined during the last decades, our understanding of the functional roles of RBPs in plants is lagging far behind our understanding of those in other organisms, including animals, bacteria, and viruses. However, recent functional analysis of multiple RBP family members involved in plant RNA metabolism and elucidation of the mechanistic roles of RBPs shed light on the cellular roles of diverse RBPs in growth, development, and stress responses of plants. In this review, we will discuss recent studies demonstrating the emerging roles of multiple RBP family members that play essential roles in RNA metabolism during plant growth, development, and stress responses.

• Journal of Genetic Medicine
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• v.3 no.1
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• pp.15-19
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• 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.

• Proceedings of the Zoological Society Korea Conference
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• 1996.04a
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• pp.53.1-53
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• 1996

No Abstract, See Full Text