Publications by authors named "Sangsu Bae"

58 Publications

In vivo gene editing via homology-independent targeted integration for adrenoleukodystrophy treatment.

Mol Ther 2021 May 28. Epub 2021 May 28.

Department and Research Institute of Rehabilitation Medicine, Yonsei University College of Medicine, Seoul 03722, South Korea;; Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul 03722, South Korea;; Graduate Program of Nano Science and Technology, Yonsei University, Seoul 03722, South Korea;; Rehabilitation Institute of Neuromuscular Disease, Yonsei University College of Medicine, Seoul 03722, Korea. Electronic address:

Adrenoleukodystrophy (ALD) is caused by various pathogenic mutations in the X-linked ABCD1 gene, which lead to metabolically abnormal accumulations of very long-chain fatty acids in many organs. However, curative treatment of ALD has not yet been achieved. To treat ALD, we applied two different gene editing strategies, base editing and homology-independent targeted insertion (HITI), in ALD patient-derived fibroblasts. Next, we performed in vivo HITI-mediated gene editing using AAV9 vectors delivered via intravenous administration in ALD model mice. We found that the ABCD1 mRNA level was significantly increased in HITI-treated mice and the plasma levels of C24:0-LysoPC and C26:0-LysoPC, sensitive diagnostic markers for ALD, were significantly reduced. These results suggest that HITI-mediated mutant gene rescue could be a promising therapeutic strategy for human ALD treatment.
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http://dx.doi.org/10.1016/j.ymthe.2021.05.022DOI Listing
May 2021

genome editing in single mammalian brain neurons through CRISPR-Cas9 and cytosine base editors.

Comput Struct Biotechnol J 2021 25;19:2477-2485. Epub 2021 Apr 25.

Department of Chemistry and Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul 04763, South Korea.

Gene manipulation is a useful approach for understanding functions of genes and is important for investigating basic mechanisms of brain function on the level of single neurons and circuits. Despite the development and the wide range of applications of CRISPR-Cas9 and base editors (BEs), their implementation for an analysis of individual neurons remained limited. In fact, conventional gene manipulations are generally achieved only on the population level. Here, we combined either CRISPR-Cas9 or BEs with the targeted single-cell electroporation technique as a proof-of-concept test for gene manipulation in single neurons . Our assay consisted of CRISPR-Cas9- or BEs-induced gene knockout in single Purkinje cells in the cerebellum. Our results demonstrate the feasibility of both gene editing and base editing in single cells in the intact brain, providing a tool through which molecular perturbations of individual neurons can be used for analysis of circuits and, ultimately, behaviors.
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http://dx.doi.org/10.1016/j.csbj.2021.04.051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113754PMC
April 2021

Adenine base editing and prime editing of chemically derived hepatic progenitors rescue genetic liver disease.

Cell Stem Cell 2021 Apr 26. Epub 2021 Apr 26.

Department of Surgery, Hanyang University College of Medicine, Seoul 04763, Republic of Korea; HY Indang Center of Regenerative Medicine and Stem Cell Research, Hanyang University, Seoul 04763, Republic of Korea; Department of HY-KIST Bio-convergence, Hanyang University, Seoul 04763, Republic of Korea. Electronic address:

DNA base editors and prime editing technology enable therapeutic in situ correction of disease-causing alleles. These techniques could have broad applications for ex vivo editing of cells prior to transplantation in a range of diseases, but it is critical that the target population is efficiently modified and engrafts into the host. Chemically derived hepatic progenitors (CdHs) are a multipotent population capable of robust engraftment and hepatocyte differentiation. Here we reprogrammed hepatocytes from a mouse model of hereditary tyrosinemia type 1 (HT1) into expandable CdHs and successfully corrected the disease-causing mutation using both adenine base editors (ABEs) and prime editors (PEs). ABE- and PE-corrected CdHs repopulated the liver with fumarylacetoacetate hydrolase-positive cells and dramatically increased survival of mutant HT1 mice. These results demonstrate the feasibility of precise gene editing in transplantable cell populations for potential treatment of genetic liver disease.
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http://dx.doi.org/10.1016/j.stem.2021.04.010DOI Listing
April 2021

PE-Designer and PE-Analyzer: web-based design and analysis tools for CRISPR prime editing.

Nucleic Acids Res 2021 May 3. Epub 2021 May 3.

Department of Chemistry and Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul, South Korea.

Prime editing technology is capable of generating targeted insertions, deletions, and base conversions. However, the process of designing prime editing guide RNAs (pegRNAs), which contain a primer binding site and a reverse-transcription template at the 3' end, is more complex than that for the single guide RNAs used with CRISPR nucleases or base editors. Furthermore, the assessment of high-throughput sequencing data after prime editors (PEs) have been employed should consider the unique feature of PEs; thus, pre-existing assessment tools cannot directly be adopted for PEs. Here, we present two user-friendly web-based tools for PEs, named PE-Designer and PE-Analyzer. PE-Designer, a dedicated tool for pegRNA selection, provides all possible target sequences, pegRNA extension sequences, and nicking guide RNA sequences together with useful information, and displays the results in an interactive image. PE-Analyzer, a dedicated tool for PE outcome analysis, accepts high-throughput sequencing data, summarizes mutation-related information in a table, and provides interactive graphs. PE-Analyzer was mainly written using JavaScript so that it can analyze several data sets without requiring that huge sequencing data (>100MB) be uploaded to the server, reducing analysis time and increasing personal security. PE-Designer and PE-Analyzer are freely available at http://www.rgenome.net/pe-designer/ and http://www.rgenome.net/pe-analyzer/ without a login process.
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http://dx.doi.org/10.1093/nar/gkab319DOI Listing
May 2021

Efficient Human Cell Coexpression System and Its Application to the Production of Multiple Coronavirus Antigens.

Adv Biol (Weinh) 2021 04 11;5(4):e2000154. Epub 2021 Feb 11.

Department of Chemistry, Hanyang University, Seoul, 04763, South Korea.

Coproduction of multiple proteins at high levels in a single human cell line would be extremely useful for basic research and medical applications. Here, a novel strategy for the stable expression of multiple proteins by integrating the genes into defined transcriptional hotspots in the human genome is presented. As a proof-of-concept, it is shown that EYFP is expressed at similar levels from hotspots and that the EYFP expression increases proportionally with the copy number. It is confirmed that three different fluorescent proteins, encoded by genes integrated at different loci, can be coexpressed at high levels. Further, a stable cell line is generated, producing antigens from different human coronaviruses: MERS-CoV and HCoV-OC43. Antibodies raised against these antigens, which contain human N-glycosylation, show neutralizing activities against both viruses, suggesting that the coexpression system provides a quick and predictable way to produce multiple coronavirus antigens, such as the recent 2019 novel human coronavirus.
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http://dx.doi.org/10.1002/adbi.202000154DOI Listing
April 2021

CReVIS-Seq: A highly accurate and multiplexable method for genome-wide mapping of lentiviral integration sites.

Mol Ther Methods Clin Dev 2021 Mar 11;20:792-800. Epub 2021 Jan 11.

Department of Genetics, College of Medicine, Hanyang University, Seoul 04763, South Korea.

Lentiviruses have been widely used as a means of transferring exogenous DNAs into human cells to treat various genetic diseases. Lentiviral vectors are fundamentally integrated into the host genome, but their integration sites are generally unpredictable, which may increase the uncertainty for their use in therapeutics. To determine the viral integration sites in the host genome, several PCR-based methods have been developed. However, the sensitivities of the PCR-based methods are highly dependent on the primer sequences, and optimized primer design is required for individual target sites. In order to address this issue, we developed an alternative method for genome-wide mapping of viral insertion sites, named CReVIS-seq (CRISPR-enhanced Viral Integration Site Sequencing). The method is based on the sequential steps: fragmentation of genomic DNAs, circularization, cleavage of target sequence in a CRISPR guide RNA-specific manner, high-throughput sequencing of the linearized DNA fragments in an unbiased manner, and identification of viral insertion sites via sequence analysis. By design, CReVIS-seq is not affected by biases that could be introduced during the target enrichment step via PCR amplification using site specific primers. Furthermore, we found that multiplexed CReVIS-seq, using collections of different single-guide RNAs (sgRNAs), enables simultaneous identification of multiple target sites and structural variations (i.e., circularized viral genome), in both single cell clones and heterogeneous cell populations.
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http://dx.doi.org/10.1016/j.omtm.2020.10.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7961857PMC
March 2021

Web-Based Base Editing Toolkits: BE-Designer and BE-Analyzer.

Methods Mol Biol 2021 ;2189:81-88

Department of Chemistry, Hanyang University, Seoul, South Korea.

The CRISPR-Cas system is broadly used for genome editing because of its convenience and relatively low cost. However, the use of CRISPR nucleases to induce specific nucleotide changes in target DNA requires complex procedures and additional donor DNAs. Furthermore, CRISPR nuclease-mediated DNA cleavage at target sites frequently causes large deletions or genomic rearrangements. In contrast, base editors that consist of catalytically dead Cas9 (dCas9) or Cas9 nickase (nCas9) connected to a cytidine or a guanine deaminase can correct point mutations in the absence of additional donor DNA and without generating double-strand breaks (DSBs) in the target region. To design target sites and assess mutation ratios for cytosine and adenine base editors (CBEs and ABEs), we have developed web tools, named BE-Designer and BE-Analyzer. These tools are easy to use (such that tasks are accomplished by clicking on relevant buttons) and do not require a deep knowledge of bioinformatics.
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http://dx.doi.org/10.1007/978-1-0716-0822-7_7DOI Listing
March 2021

Transcriptomic and physiological analysis of OsCAO1 knockout lines using the CRISPR/Cas9 system in rice.

Plant Cell Rep 2021 Jun 27;40(6):1013-1024. Epub 2020 Sep 27.

Division of Horticultural Biotechnology, Hankyong National University, Anseong, 17579, South Korea.

Key Message: The altered rice leaf color based on the knockout of CAO1 gene generated using CRISPR/Cas9 technology plays important roles in chlorophyll degradation and ROS scavenging to regulate both natural and induced senescence in rice. Rice chlorophyllide a oxygenase (OsCAO1), identified as the chlorophyll b synthesis under light condition, plays a critical role in regulating rice plant photosynthesis. In this study, the development of edited lines with pale green leaves by knockout of OsCAO1 gene known as a chlorophyll synthesis process is reported. Eighty-one genetically edited lines out of 181 T plants were generated through CRISPR/Cas9 system. The edited lines have short narrow flag leaves and pale green leaves compared with wild-type 'Dongjin' plants (WT). Additionally, edited lines have lower chlorophyll b and carotenoid contents both at seedling and mature stages. A transcriptome analysis identified 580 up-regulated and 206 downregulated genes in the edited lines. The differentially expressed genes (DEGs) involved in chlorophyll biosynthesis, magnesium chelatase subunit (CHLH), and glutamate-1-semialdehyde2, 1-aminomutase (GSA) metabolism decreased significantly. Meanwhile, the gel consistency (GC) levels of rice grains, chalkiness ratios and chalkiness degrees (CD) decreased in the edited lines. Thus, knockout of OsCAO1 influenced growth period, leaf development and grain quality characters of rice. Overall, the result suggests that OsCAO1 also plays important roles in chlorophyll degradation and ROS scavenging to regulate both natural and induced rice senescence.
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http://dx.doi.org/10.1007/s00299-020-02607-yDOI Listing
June 2021

Simultaneous targeting of duplicated genes in Petunia protoplasts for flower color modification via CRISPR-Cas9 ribonucleoproteins.

Plant Cell Rep 2021 Jun 21;40(6):1037-1045. Epub 2020 Sep 21.

Department of Horticulture & Department of Smart Agriculture systems, Chungnam National University, Daejeon, 34134, South Korea.

Key Message: We obtained a complete mutant line of Petunia having mutations in both F3H genes via Cas9-ribonucleoproteins delivery, which exhibited a pale purplish pink flower color. The CRISPR-Cas system is now revolutionizing agriculture by allowing researchers to generate various desired mutations in plants at will. In particular, DNA-free genome editing via Cas9-ribonucleoproteins (RNPs) delivery has many advantages in plants; it does not require codon optimization or specific promoters for expression in plant cells; furthermore, it can bypass GMO regulations in some countries. Here, we have performed site-specific mutagenesis in Petunia to engineer flower color modifications. We determined that the commercial Petunia cultivar 'Madness Midnight' has two F3H coding genes and designed one guide RNA that targets both F3H genes at once. Among 67 T plants regenerated from Cas9-RNP transfected protoplasts, we obtained seven mutant lines that contain mutations in either F3HA or F3HB gene and one complete mutant line having mutations in both F3H genes without any selectable markers. It is noteworthy that only the f3ha f3hb exhibited a clearly modified, pale purplish pink flower color (RHS 69D), whereas the others, including the single copy gene knock-out plants, displayed purple violet (RHS 93A) flowers similar to the wild-type Petunia. To the best of our knowledge, we demonstrated a precedent of ornamental crop engineering by DNA-free CRISPR method for the first time, which will greatly accelerate a transition from a laboratory to a farmer's field.
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http://dx.doi.org/10.1007/s00299-020-02593-1DOI Listing
June 2021

Knockout of Gene ( Encoding bHLH Transcription Factor Using CRISPR/Cas9 System Confers Male Sterility Phenotype in Tomato.

Plants (Basel) 2020 Sep 11;9(9). Epub 2020 Sep 11.

Division of Horticultural Biotechnology, Hankyong National University, Anseong 17579, Korea.

The utilization of male sterility into hybrid seed production reduces its cost and ensures high purity of tomato varieties because it does not produce pollen and has exserted stigmas. Here, we report on the generation of gene edited lines into male sterility phenotype by knockout of gene ( encoding the bHLH transcription factor that regulates meiosis and cell death of the tapetum during microsporogenesis in the tomato. Twenty-eight gene edited lines out of 60 transgenic plants were selected. Of these, eleven different mutation types at the target site of the gene were selected through deep sequencing analysis. These mutations were confirmed to be transmitted to subsequent generations. The null lines without the transferred DNA (T-DNA) were obtained by segregation in the T and T generations. In addition, we showed that the cr-ms10-1-4 mutant line exhibited dysfunctional meiosis and abnormal tapetum during flower development, resulting in no pollen production. RT-PCR analysis showed that the most genes associated with pollen and tapetum development in tomatoes had lower expression in the cr-ms10-1-4 mutant line compared to wild type. We demonstrate that modification of the gene via CRISPR/Cas9-mediated genome editing results in male sterility of tomato plants. Our results suggest an alternative approach to generating male sterility in crops.
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http://dx.doi.org/10.3390/plants9091189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7570381PMC
September 2020

Web-Based CRISPR Toolkits: Cas-OFFinder, Cas-Designer, and Cas-Analyzer.

Methods Mol Biol 2021 ;2162:23-33

Department of Chemistry, Hanyang University, Seoul, South Korea.

The CRISPR-Cas system facilitates highly efficient genome editing; thus, it has been applied in many research fields such as biological science, medicine, and gene therapy. However, CRISPR nucleases can cleave off-target sites as well as on-target sites, causing unwanted mutations. Furthermore, after CRISPR treatments are delivered into cells or organisms, it is important to estimate the resulting mutation rates and to determine the patterns of mutations, but these tasks can be difficult. To address these issues, we have developed a tool for identifying potential off-target sites (Cas-OFFinder), a tool for designing CRISPR targets (Cas-Designer), and an assessment tool (Cas-Analyzer). These programs are all implemented on our website so that researchers can easily design CRISPR guide RNAs and assess the resulting mutations by simply clicking on the appropriate buttons; no login process is required.
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http://dx.doi.org/10.1007/978-1-0716-0687-2_2DOI Listing
March 2021

Safe scarless cassette-free selection of genome-edited human pluripotent stem cells using temporary drug resistance.

Biomaterials 2020 12 1;262:120295. Epub 2020 Sep 1.

College of Pharmacy, Seoul National University, Seoul, South Korea. Electronic address:

An efficient gene-editing technique for use in human pluripotent stem cells (hPSCs) has great potential value in regenerative medicine, as well as in drug discovery based on isogenic human disease models. However, the extremely low efficiency of gene editing in hPSCs remains as a major technical hurdle. Previously, we demonstrated that YM155, a survivin inhibitor developed as an anti-cancer drug, induces highly selective cell death in undifferentiated hPSCs. In this study, we demonstrated that the high cytotoxicity of YM155 in hPSCs, which is mediated by selective cellular uptake of the drug, is due to the high expression of SLC35F2 in these cells. Knockout of SLC35F2 with CRISPR-Cas9, or depletion with siRNAs, made the hPSCs highly resistant to YM155. Simultaneous editing of a gene of interest and transient knockdown of SLC35F2 following YM155 treatment enabled the survival of genome-edited hPSCs as a result of temporary YM155 resistance, thereby achieving an enriched selection of clonal populations with gene knockout or knock-in. This precise and efficient genome editing approach took as little as 3 weeks and required no cell sorting or the introduction of additional genes, to be a more feasible approach for gene editing in hPSCs due to its simplicity.
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http://dx.doi.org/10.1016/j.biomaterials.2020.120295DOI Listing
December 2020

Current Status and Challenges of DNA Base Editing Tools.

Mol Ther 2020 09 23;28(9):1938-1952. Epub 2020 Jul 23.

Department of Chemistry and Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul 04763, South Korea. Electronic address:

CRISPR-mediated DNA base editors, which include cytosine base editors (CBEs) and adenine base editors (ABEs), are promising tools that can induce point mutations at desired sites in a targeted manner to correct or disrupt gene expression. Their high editing efficiency, coupled with their ability to generate a targeted mutation without generating a DNA double-strand break (DSB) or requiring a donor DNA template, suggests that DNA base editors will be useful for treating genetic diseases, among other applications. However, this hope has recently been challenged by the discovery of DNA base editor shortcomings, including off-target DNA editing, the generation of bystander mutations, and promiscuous deamination effects in both DNA and RNA, which arise from the main DNA base editor constituents, a Cas nuclease variant and a deaminase. In this review, we summarize information about the DNA base editors that have been developed to date, introduce their associated potential challenges, and describe current efforts to minimize or mitigate those issues of DNA base editors.
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http://dx.doi.org/10.1016/j.ymthe.2020.07.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7474268PMC
September 2020

Generation and Transcriptome Profiling of Slr1-d7 and Slr1-d8 Mutant Lines with a New Semi-Dominant Dwarf Allele of Using the CRISPR/Cas9 System in Rice.

Int J Mol Sci 2020 Jul 31;21(15). Epub 2020 Jul 31.

Division of Horticultural Biotechnology, Hankyong National University, Anseong 17579, Korea.

The rice gene encodes the DELLA protein, and a loss-of-function mutation is dwarfed by inhibiting plant growth. We generate slr1-d mutants with a semi-dominant dwarf phenotype to target mutations of the DELLA/TVHYNP domain using CRISPR/Cas9 genome editing in rice. Sixteen genetic edited lines out of 31 transgenic plants were generated. Deep sequencing results showed that the mutants had six different mutation types at the target site of the TVHYNP domain of the gene. The homo-edited plants selected individuals without DNA (T-DNA) transcribed by segregation in the T1 generation. The slr1-d7 and slr1-d8 plants caused a gibberellin (GA)-insensitive dwarf phenotype with shrunken leaves and shortened internodes. A genome-wide gene expression analysis by RNA-seq indicated that the expression levels of two GA-related genes, (Gibberellin oxidase) and , were increased in the edited mutant plants, suggesting that acts as a convert of GA signaling. These mutant plants are required by altering GA responses, at least partially by a defect in the phytohormone signaling system process and prevented cell elongation. The new mutants, namely, the slr1-d7 and slr1-d8 lines, are valuable semi-dominant dwarf alleles with potential application value for molecule breeding using the CRISPR/Cas9 system in rice.
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http://dx.doi.org/10.3390/ijms21155492DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432230PMC
July 2020

CRISPR-sub: Analysis of DNA substitution mutations caused by CRISPR-Cas9 in human cells.

Comput Struct Biotechnol J 2020 25;18:1686-1694. Epub 2020 Jun 25.

Department of Chemistry, Hanyang University, Seoul 04763, South Korea.

CRISPR-Cas9 induces DNA cleavages at desired target sites in a guide RNA-dependent manner; DNA editing occurs through the resulting activity of DNA repair processes including non-homologous end joining (NHEJ), which is dominant in mammalian cells. NHEJ repair frequently causes small insertions and deletions (indels) near DNA cleavage sites but only rarely causes nucleotide substitutions. High-throughput sequencing is the primary means of assessing indel and substitution frequencies in bulk populations of cells in the gene editing field. However, it is difficult to detect bona fide substitutions, which are embedded among experimentally-induced substitution errors, in high-throughput sequencing data. Here, we developed a novel analysis method, named CRISPR-Sub, to statistically detect Cas9-mediated substitutions in high-throughput sequencing data by comparing Mock- and CRISPR-treated samples. We first pinpointed 'hotspot positions' in target sequences at which substitution mutations were quantitatively observed much more often (p > 0.001) in CRISPR- versus Mock-treated samples. We refer to the substitution mutations in defined hotspot positions as 'apparent substitutions' and ultimately calculated 'apparent substitution frequencies' for each target. By examining 51 endogenous target sites in HeLa cells, we found that the average apparent substitution frequency was 0.8% in all queries, that apparent substitutions frequently occur near CRISPR-Cas9 cleavage sites, and that nucleotide conversion showed no meaningful nucleotide preference patterns. Furthermore, we generated NHEJ-inhibited cell lines ( ) by knockout of the gene encoding ligase IV and found that the apparent substitution frequencies were significantly decreased in cells, strongly suggesting that DNA substitutions are generated by the NHEJ pathway.
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http://dx.doi.org/10.1016/j.csbj.2020.06.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7338987PMC
June 2020

Current trends in gene recovery mediated by the CRISPR-Cas system.

Exp Mol Med 2020 07 10;52(7):1016-1027. Epub 2020 Jul 10.

Department of Chemistry and Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul, 04763, South Korea.

The CRISPR-Cas system has undoubtedly revolutionized the genome editing field, enabling targeted gene disruption, regulation, and recovery in a guide RNA-specific manner. In this review, we focus on currently available gene recovery strategies that use CRISPR nucleases, particularly for the treatment of genetic disorders. Through the action of DNA repair mechanisms, CRISPR-mediated DNA cleavage at a genomic target can shift the reading frame to correct abnormal frameshifts, whereas DNA cleavage at two sites, which can induce large deletions or inversions, can correct structural abnormalities in DNA. Homology-mediated or homology-independent gene recovery strategies that require donor DNAs have been developed and widely applied to precisely correct mutated sequences in genes of interest. In contrast to the DNA cleavage-mediated gene correction methods listed above, base-editing tools enable base conversion in the absence of donor DNAs. In addition, CRISPR-associated transposases have been harnessed to generate a targeted knockin, and prime editors have been developed to edit tens of nucleotides in cells. Here, we introduce currently developed gene recovery strategies and discuss the pros and cons of each.
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http://dx.doi.org/10.1038/s12276-020-0466-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8080666PMC
July 2020

Purification of an Intact Human Protein Overexpressed from Its Endogenous Locus Direct Genome Engineering.

ACS Synth Biol 2020 07 2;9(7):1591-1598. Epub 2020 Jul 2.

Department of Chemistry, Hanyang University, Seoul 04763, South Korea.

The overproduction and purification of human proteins is a requisite of both basic and medical research. Although many recombinant human proteins have been purified, current protein production methods have several limitations; recombinant proteins are frequently truncated, fail to fold properly, and/or lack appropriate post-translational modifications. In addition, such methods require subcloning of the target gene into relevant plasmids, which can be difficult for long proteins with repeated domains. Here we devised a novel method for target protein production by introduction of a strong promoter for overexpression and an epitope tag for purification in front of the endogenous human gene, in a sense performing molecular cloning directly in the human genome, which does not require cloning of the target gene. As a proof of concept, we successfully purified intact human Reelin protein, which is lengthy (3460 amino acids) and contains repeating domains, and confirmed that it was biologically functional.
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http://dx.doi.org/10.1021/acssynbio.0c00090DOI Listing
July 2020

Anti-Atherogenic Effect of Stem Cell Nanovesicles Targeting Disturbed Flow Sites.

Small 2020 04 2;16(16):e2000012. Epub 2020 Apr 2.

Department of Medical Engineering, Yonsei University College of Medicine, Seoul, 03722, Republic of Korea.

Atherosclerosis development leads to irreversible cascades, highlighting the unmet need for improved methods of early diagnosis and prevention. Disturbed flow formation is one of the earliest atherogenic events, resulting in increased endothelial permeability and subsequent monocyte recruitment. Here, a mesenchymal stem cell (MSC)-derived nanovesicle (NV) that can target disturbed flow sites with the peptide GSPREYTSYMPH (PREY) (PMSC-NVs) is presented which is selected through phage display screening of a hundred million peptides. The PMSC-NVs are effectively produced from human MSCs (hMSCs) using plasmid DNA designed to functionalize the cell membrane with PREY. The potent anti-inflammatory and pro-endothelial recovery effects are confirmed, similar to those of hMSCs, employing mouse and porcine partial carotid artery ligation models as well as a microfluidic disturbed flow model with human carotid artery-derived endothelial cells. This nanoscale platform is expected to contribute to the development of new theragnostic strategies for preventing the progression of atherosclerosis.
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http://dx.doi.org/10.1002/smll.202000012DOI Listing
April 2020

Generation of albino via SLC45a2 gene targeting by CRISPR/Cas9 in the marine medaka Oryzias melastigma.

Mar Pollut Bull 2020 May 12;154:111038. Epub 2020 Mar 12.

Department of Biological Science, College of Science, Sungkyunkwan University, Suwon 16419, South Korea. Electronic address:

To produce albinism in the marine medaka Oryzias melastigma, we disrupted the solute carrier family 45 (SLC45a2) gene by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 with a single guide RNA (sgRNA). Selected sgRNAs were able to target a SLC45a2 gene as confirmed by genotyping and heteroduplex mobility assay (HMA). Of the survived embryos after injection, 54.2% and 60.0% embryos exhibited albinism phenotype by sgRNA1 and sgRNA2, respectively. Deep sequencing at the on-target sites showed different insertion and deletion (indel) mutation profiles near the DNA cleavage sites, indicating high efficacy of producing SLC45a2 knock-out mutants by this method. Moreover, HMA at the potential off-target sites revealed that off-target activity would be induced at a low rate, or not induced at all. This albino marine medaka will be a good model for marine molecular ecotoxicology in establishment of diverse in vivo endpoints, and the application of this efficient gene targeting method in the marine medaka would be useful tool for mechanistic approaches.
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http://dx.doi.org/10.1016/j.marpolbul.2020.111038DOI Listing
May 2020

CRISPR-mediated gene correction links the ATP7A M1311V mutations with amyotrophic lateral sclerosis pathogenesis in one individual.

Commun Biol 2020 01 20;3(1):33. Epub 2020 Jan 20.

Department of Neurosurgery, Spine & Spinal Cord Institute, College of Medicine, Yonsei University, Seoul, 03722, South Korea.

Amyotrophic lateral sclerosis (ALS) is a severe disease causing motor neuron death, but a complete cure has not been developed and related genes have not been defined in more than 80% of cases. Here we compared whole genome sequencing results from a male ALS patient and his healthy parents to identify relevant variants, and chose one variant in the X-linked ATP7A gene, M1311V, as a strong disease-linked candidate after profound examination. Although this variant is not rare in the Ashkenazi Jewish population according to results in the genome aggregation database (gnomAD), CRISPR-mediated gene correction of this mutation in patient-derived and re-differentiated motor neurons drastically rescued neuronal activities and functions. These results suggest that the ATP7A M1311V mutation has a potential responsibility for ALS in this patient and might be a potential therapeutic target, revealed here by a personalized medicine strategy.
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http://dx.doi.org/10.1038/s42003-020-0755-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6970999PMC
January 2020

i-Silence, Please! An Alternative for Gene Disruption via Adenine Base Editors.

Mol Ther 2020 02 17;28(2):348-349. Epub 2020 Jan 17.

Department of Chemistry, Hanyang University and Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul 04763, South Korea. Electronic address:

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http://dx.doi.org/10.1016/j.ymthe.2020.01.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7001083PMC
February 2020

Adenine base editors catalyze cytosine conversions in human cells.

Nat Biotechnol 2019 10 23;37(10):1145-1148. Epub 2019 Sep 23.

Department of Chemistry, Hanyang University, Seoul, South Korea.

Adenine base editors comprise an adenosine deaminase, evolved in vitro, and a Cas9 nickase. Here, we show that in addition to converting adenine to guanine, adenine base editors also convert cytosine to guanine or thymine in a narrow editing window (positions 5-7) and in a confined TC*N sequence context. Adenine base editor-induced cytosine substitutions occur independently of adenosine conversions with an efficiency of up to 11.2% and reduce the number of suitable targeting sites for high-specificity base editing.
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http://dx.doi.org/10.1038/s41587-019-0254-4DOI Listing
October 2019

CRISPR-Pass: Gene Rescue of Nonsense Mutations Using Adenine Base Editors.

Mol Ther 2019 08 24;27(8):1364-1371. Epub 2019 May 24.

Department of Chemistry, Hanyang University, Seoul 04763, South Korea; Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul 04763, South Korea. Electronic address:

A nonsense mutation is a substitutive mutation in a DNA sequence that causes a premature termination during translation and produces stalled proteins, resulting in dysfunction of a gene. Although it usually induces severe genetic disorders, there are no definite methods for inducing read through of premature termination codons (PTCs). Here, we present a targeted tool for bypassing PTCs, named CRISPR-pass, that uses CRISPR-mediated adenine base editors. CRISPR-pass, which should be applicable to 95.5% of clinically significant nonsense mutations in the ClinVar database, rescues protein synthesis in patient-derived fibroblasts, suggesting potential clinical utility.
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http://dx.doi.org/10.1016/j.ymthe.2019.05.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6698196PMC
August 2019

CRISPR-Cas9 Screening of Kaposi's Sarcoma-Associated Herpesvirus-Transformed Cells Identifies XPO1 as a Vulnerable Target of Cancer Cells.

mBio 2019 05 14;10(3). Epub 2019 May 14.

Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA

The abnormal proliferation of cancer cells is driven by deregulated oncogenes or tumor suppressors, among which the cancer-vulnerable genes are attractive therapeutic targets. Targeting mislocalization of oncogenes and tumor suppressors resulting from aberrant nuclear export is effective for inhibiting growth transformation of cancer cells. We performed a clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) screening in a unique model of matched primary and oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV)-transformed cells and identified genes that were growth promoting and growth suppressive for both types of cells, among which exportin XPO1 was demonstrated to be critical for the survival of transformed cells. Using XPO1 inhibitor KPT-8602 and by small interfering RNA (siRNA) knockdown, we confirmed the essential role of XPO1 in cell proliferation and growth transformation of KSHV-transformed cells and in cell lines of other cancers, including gastric cancer and liver cancer. XPO1 inhibition induced cell cycle arrest through p53 activation, but the mechanisms of p53 activation differed among the different types of cancer cells. p53 activation depended on the formation of promyelocytic leukemia (PML) nuclear bodies in gastric cancer and liver cancer cells. Mechanistically, XPO1 inhibition induced relocalization of autophagy adaptor protein p62 (SQSTM1), recruiting p53 for activation in PML nuclear bodies. Taken the data together, we have identified novel growth-promoting and growth-suppressive genes of primary and cancer cells and have demonstrated that XPO1 is a vulnerable target of cancer cells. XPO1 inhibition induces cell arrest through a novel PML- and p62-dependent mechanism of p53 activation in some types of cancer cells. Using a model of oncogenic virus KSHV-driven cellular transformation of primary cells, we have performed a genome-wide CRISPR-Cas9 screening to identify vulnerable genes of cancer cells. This screening is unique in that this virus-induced oncogenesis model does not depend on any cellular genetic alterations and has matched primary and KSHV-transformed cells, which are not available for similar screenings in other types of cancer. We have identified genes that are both growth promoting and growth suppressive in primary and transformed cells, some of which could represent novel proto-oncogenes and tumor suppressors. In particular, we have demonstrated that the exportin XPO1 is a critical factor for the survival of transformed cells. Using a XPO1 inhibitor (KPT-8602) and siRNA-mediated knockdown, we have confirmed the essential role of XPO1 in cell proliferation and in growth transformation of KSHV-transformed cells, as well as of gastric and liver cancer cells. XPO1 inhibition induces cell cycle arrest by activating p53, but the mechanisms of p53 activation differed among different types of cancer cells. p53 activation is dependent on the formation of PML nuclear bodies in gastric and liver cancer cells. Mechanistically, XPO1 inhibition induces relocalization of autophagy adaptor protein p62 (SQSTM1), recruiting p53 for activation in PML nuclear bodies. These results illustrate that XPO1 is a vulnerable target of cancer cells and reveal a novel mechanism for blocking cancer cell proliferation by XPO1 inhibition as well as a novel PML- and p62-mediated mechanism of p53 activation in some types of cancer cells.
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http://dx.doi.org/10.1128/mBio.00866-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6520457PMC
May 2019

Machine learning finds Cas9-edited genotypes.

Nat Biomed Eng 2018 12;2(12):892-893

Department of Chemistry, Seoul National University and Center for Genome Engineering, Institute for Basic Science, Seoul, South Korea.

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http://dx.doi.org/10.1038/s41551-018-0327-6DOI Listing
December 2018

Construction of non-canonical PAM-targeting adenosine base editors by restriction enzyme-free DNA cloning using CRISPR-Cas9.

Sci Rep 2019 03 20;9(1):4939. Epub 2019 Mar 20.

Department of Chemistry, Hanyang University, Seoul, 04763, South Korea.

Molecular cloning is an essential technique in molecular biology and biochemistry, but it is frequently laborious when adequate restriction enzyme recognition sites are absent. Cas9 endonucleases can induce site-specific DNA double-strand breaks at sites homologous to their guide RNAs, rendering an alternative to restriction enzymes. Here, by combining DNA cleavage via a Cas9 endonuclease and DNA ligation via Gibson assembly, we demonstrate a precise and practical DNA cloning method for replacing part of a backbone plasmid. We first replaced a resistance marker gene as a proof of concept and next generated DNA plasmids that encode engineered Cas9 variants (VQR, VRER and SpCas9-NG), which target non-canonical NGA, NGCG and NG protospacer-adjacent motif (PAM) sequences, fused with adenosine deaminases for adenine base editing (named VQR-ABE, VRER-ABE and NG-ABE, respectively). Ultimately, we confirmed that the re-constructed plasmids can successfully convert adenosine to guanine at endogenous target sites containing the non-canonical NGA, NGCG and NG PAMs, expanding the targetable range of the adenine base editing.
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http://dx.doi.org/10.1038/s41598-019-41356-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6426851PMC
March 2019

Web-based design and analysis tools for CRISPR base editing.

BMC Bioinformatics 2018 Dec 27;19(1):542. Epub 2018 Dec 27.

Department of Chemistry, Hanyang University, Seoul, South Korea.

Background: As a result of its simplicity and high efficiency, the CRISPR-Cas system has been widely used as a genome editing tool. Recently, CRISPR base editors, which consist of deactivated Cas9 (dCas9) or Cas9 nickase (nCas9) linked with a cytidine or a guanine deaminase, have been developed. Base editing tools will be very useful for gene correction because they can produce highly specific DNA substitutions without the introduction of any donor DNA, but dedicated web-based tools to facilitate the use of such tools have not yet been developed.

Results: We present two web tools for base editors, named BE-Designer and BE-Analyzer. BE-Designer provides all possible base editor target sequences in a given input DNA sequence with useful information including potential off-target sites. BE-Analyzer, a tool for assessing base editing outcomes from next generation sequencing (NGS) data, provides information about mutations in a table and interactive graphs. Furthermore, because the tool runs client-side, large amounts of targeted deep sequencing data (< 1 GB) do not need to be uploaded to a server, substantially reducing running time and increasing data security. BE-Designer and BE-Analyzer can be freely accessed at http://www.rgenome.net/be-designer/ and http://www.rgenome.net/be-analyzer /, respectively.

Conclusion: We develop two useful web tools to design target sequence (BE-Designer) and to analyze NGS data from experimental results (BE-Analyzer) for CRISPR base editors.
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http://dx.doi.org/10.1186/s12859-018-2585-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307267PMC
December 2018

Direct observation of DNA target searching and cleavage by CRISPR-Cas12a.

Nat Commun 2018 07 17;9(1):2777. Epub 2018 Jul 17.

Department of Chemistry, Hanyang University, Seoul, 04763, South Korea.

Cas12a (also called Cpf1) is a representative type V-A CRISPR effector RNA-guided DNA endonuclease, which provides an alternative to type II CRISPR-Cas9 for genome editing. Previous studies have revealed that Cas12a has unique features distinct from Cas9, but the detailed mechanisms of target searching and DNA cleavage by Cas12a are still unclear. Here, we directly observe this entire process by using single-molecule fluorescence assays to study Cas12a from Acidaminococcus sp. (AsCas12a). We determine that AsCas12a ribonucleoproteins search for their on-target site by a one-dimensional diffusion along elongated DNA molecules and induce cleavage in the two DNA strands in a well-defined order, beginning with the non-target strand. Furthermore, the protospacer-adjacent motif (PAM) for AsCas12a makes only a limited contribution of DNA unwinding during R-loop formation and shows a negligible role in the process of DNA cleavage, in contrast to the Cas9 PAM.
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http://dx.doi.org/10.1038/s41467-018-05245-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6050341PMC
July 2018

Author Correction: ID3 regulates the MDC1-mediated DNA damage response in order to maintain genome stability.

Nat Commun 2018 06 6;9(1):2284. Epub 2018 Jun 6.

Laboratory of Genomic Instability and Cancer Therapeutics, Cancer Mutation Research Center, Chosun University School of Medicine, Gwangju, 501-759, Republic of Korea.

This Article contains errors in Fig. 3, Fig. 4 and Fig. 7, for which we apologize. In Fig. 3, panel 'b', the 0.5 hour time point after Ku55933 treatment images were inadvertently replaced with duplicates of the 3 hour time point after Ku55933 treatment images in Fig. 3b. Additionally, in panel 'b', the 0.5 hour time point after Nu7026 treatment images were inadvertently replaced with duplicates of the 180 min time point after siMDC1 treatment images in Fig. 3d. In Fig. 4, panel 'g', RNF168 foci in U2OS cell images were inadvertently replaced with duplicates of RNF168 foci in HeLa cell images in Fig. 4f. In Fig. 7, panel 'b', the DAPI images 0.5 hours after IR under siID3 treatment were inadvertently replaced with DAPI images of a different field of view from the same experiment. Additionally, in panel 'i', the shID3 mock-treated GFP-ID3 cells image was inadvertently replace with duplications of the shID3 mock-treated GFP-ID3 cells image in Fig. 7g.
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http://dx.doi.org/10.1038/s41467-018-04599-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5989224PMC
June 2018

Response to "Unexpected mutations after CRISPR-Cas9 editing in vivo".

Nat Methods 2018 04 30;15(4):239-240. Epub 2018 Mar 30.

Center for Genome Engineering, Institute for Basic Science, Seoul, South Korea.

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http://dx.doi.org/10.1038/nmeth.4554DOI Listing
April 2018