• Journal of Microbiology and Biotechnology
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• v.33 no.9
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• pp.1228-1237
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• 2023

The CRISPR-Cas system has emerged as the most efficient genome editing technique for a wide range of cells. Delivery of the Cas9-sgRNA ribonucleoprotein complex (Cas9 RNP) has gained popularity. The objective of this study was to develop a quantitative polymerase chain reaction (qPCR)-based assay to quantify the double-strand break reaction mediated by Cas9 RNP. To accomplish this, the dextransucrase gene (dsr) from Leuconostoc citreum was selected as the target DNA. The Cas9 protein was produced using recombinant Escherichia coli BL21, and two sgRNAs were synthesized through in vitro transcription to facilitate binding with the dsr gene. Under optimized in vitro conditions, the 2.6 kb dsr DNA was specifically cleaved into 1.1 and 1.5 kb fragments by both Cas9-sgRNA365 and Cas9-sgRNA433. By monitoring changes in dsr concentration using qPCR, the endonuclease activities of the two Cas9 RNPs were measured, and their efficiencies were compared. Specifically, the specific activities of dsr365RNP and dsr433RNP were 28.74 and 34.48 (unit/㎍ RNP), respectively. The versatility of this method was also verified using different target genes, uracil phosphoribosyl transferase (upp) gene, of Bifidobacterium bifidum and specific sgRNAs. The assay method was also utilized to determine the impact of high electrical field on Cas9 RNP activity during an efficient electroporation process. Overall, the results demonstrated that the qPCR-based method is an effective tool for measuring the endonuclease activity of Cas9 RNP.

• Korean Journal of Environment and Ecology
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• v.35 no.5
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• pp.503-524
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• 2021

A largemouth bass (Micropterus salmoides) is an ecosystem disturbance fish species at the highest rank in the aquatic ecosystem, causing a serious imbalance in freshwater ecosystems. Although various attempts have been made to eradicate and control largemouth bass, no effective measures were found. Therefore, it is necessary to find an approach to maximize the effective population reduction based on the unique characteristics of largemouth bass. This study used the transcriptome analysis to derive 182,887 unigene contigs and select 12 types of final target sequences for applying the CRISPR/Cas9 system in the genes of IZUMO1 and Zona pellucida sperm-binding protein, which are proteins involved in sperm-egg recognition. After synthesizing 12 types of sgRNA capable of recognizing each target sequence, 12 types of Cas9-sgRNA ribonucleoprotein (RNP) complexes to be used in subsequent studies were prepared. This study searched the protein-coding gene of sperm-egg through the Next Generation Sequencing (NGS) and edited genes through the CRISPR/Cas9 system to induce infertile individuals that produced reproductive cells but could not form fertilized eggs. Through such a series of processes, it successfully established a composition development process for largemouth bass. It is judged that this study contributed to securing the valuable basic data for follow-up studies to verify its effect for the management of ecological disturbances without affecting the habitat of other endemic species in the same water system with the largemouth bass.

• Journal of Mushroom
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• v.20 no.3
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• pp.178-182
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• 2022

Despite the long history of mushroom use, studies examining the genetic function of mushrooms and the development of new varieties via bio-molecular methods are significantly lacking compared to those examining other organisms. However, owing to recent developments, attempts have been made to use a novel gene-editing technique involving CRISPR/Cas9 technology and genetic scissors in mushroom studies. In particular, research is actively being conducted to utilize ribonucleoprotein particles (RNPs) that can be genetically edited with high efficiency without foreign gene insertion for ease of selection. However, RNPs are too large for Cas9 protein to pass through the cell membrane of the protoplasmic reticulum. Furthermore, guide RNA is unstable and can be easily decomposed, which remarkably affects gene editing efficiency. In this study, nanoparticles were used to mitigate the shortcomings of RNP-based gene editing techniques and to obtain transformants stably. We used Lentinula edodes (shiitake mushroom) Sanjo705-13 monokaryon strain, which has been successfully used in previous genome editing experiments. To identify a suitable osmotic buffer for the isolation of protoplast, 0.6 M and 1.2 M sucrose, mannitol, sorbitol, and KCl were treated, respectively. In addition, with various nanoparticle-forming materials, experiments were conducted to confirm genome editing efficiency via the formation of nanoparticles with calcium phosphate (CaP), which can be bound to Cas9 protein without any additional amino acid modification. RNPs/NP complex was successfully formed and protected nuclease activity with nucleotide sequence specificity.