A5037463025- CRISPR and Genetic Engineering
- Innovation and Socioeconomic Development
- Pluripotent Stem Cells Research
- RNA regulation and disease
- Advanced biosensing and bioanalysis techniques
- Virus-based gene therapy research
- RNA and protein synthesis mechanisms
- Skin and Cellular Biology Research
- Insect Resistance and Genetics
- Signaling Pathways in Disease
- Cytomegalovirus and herpesvirus research
- Genetics, Aging, and Longevity in Model Organisms
- Chromosomal and Genetic Variations
- Plant Virus Research Studies
- Hair Growth and Disorders
- Retinal Development and Disorders
- Single-cell and spatial transcriptomics
- Insect symbiosis and bacterial influences
- Cancer Genomics and Diagnostics
- Cancer-related gene regulation
- Photoreceptor and optogenetics research
- Renal function and acid-base balance
- Photochromic and Fluorescence Chemistry
- RNA modifications and cancer
- Mosquito-borne diseases and control
Seoul National University Hospital
2023-2024
Myongji University
2024
New Generation University College
2024
Seoul National University
2024
Korea Advanced Institute of Science and Technology
2024
Kangwon National University
2024
Hanyang University
2017-2024
National University College
2024
As a result of its simplicity and high efficiency, the CRISPR-Cas system has been widely used as genome editing tool. Recently, CRISPR base editors, which consist deactivated Cas9 (dCas9) or nickase (nCas9) linked with cytidine guanine deaminase, have developed. Base tools will be very useful for gene correction because they can produce highly specific DNA substitutions without introduction any donor DNA, but dedicated web-based to facilitate use such not yet developed.We present two web...
Abstract Prime editing technology is capable of generating targeted insertions, deletions, and base conversions. However, the process designing prime guide RNAs (pegRNAs), which contain a primer binding site reverse-transcription template at 3′ end, more complex than that for single used with CRISPR nucleases or editors. Furthermore, assessment high-throughput sequencing data after editors (PEs) have been employed should consider unique feature PEs; thus, pre-existing tools cannot directly...
Prime editing is a versatile and precise genome technique that can directly copy desired genetic modifications into target DNA sites without the need for donor DNA. This holds great promise analysis of gene function, disease modeling, correction pathogenic mutations in clinically relevant cells such as human pluripotent stem (hPSCs). Here, we comprehensively tested prime hPSCs by generating doxycycline-inducible platform. successfully induced all types nucleotide substitutions small...
Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin fragility disorder caused by loss-of-function mutations in the COL7A1 gene, which encodes type VII collagen (C7), protein that functions adherence. From 36 Korean RDEB patients, we identified total of 69 pathogenic (40 variants without recurrence), including point (72.5%) and insertion/deletion (27.5%). For fibroblasts from two patients (Pat1 Pat2), applied adenine base editors (ABEs) to correct mutation or bypass premature...
Genome editing using CRISPR-Cas9 nucleases is based on the repair of DNA double-strand break (DSB). In eukaryotic cells, DSBs are rejoined through homology-directed (HDR), non-homologous end joining (NHEJ) or microhomology-mediated (MMEJ) pathways. Among these, it thought that NHEJ pathway dominant and occurs throughout a cell cycle. NHEJ-based DSB known to be error-prone; however, there few studies delve into deeply in endogenous genes. Here, we quantify degree accuracy (termed accuracy)...
Abstract Precise genome editing is crucial for establishing isogenic human disease models and ex vivo stem cell therapy from the patient-derived hPSCs. Unlike Cas9-mediated knock-in, cytosine base editor prime achieve desirable gene correction without inducing DNA double strand breaks. However, hPSCs possess highly active repair pathways are particularly susceptible to p53-dependent death. These unique characteristics impede efficiency of in Here, we demonstrate that dual inhibition...
Abstract Genome sequencing studies have identified numerous cancer mutations across a wide spectrum of tumor types, but determining the phenotypic consequence these remains challenge. Here, we developed high-throughput, multiplexed single-cell technology called TISCC-seq to engineer predesignated in cells using CRISPR base editors, directly delineate their genotype among individual and determine each mutation’s transcriptional phenotype. Long-read target gene’s transcript identifies...
A nonsense mutation is a substitutive in DNA sequence that causes premature termination during translation and produces stalled proteins, resulting dysfunction of gene. Although it usually induces severe genetic disorders, there are no definite methods for inducing read through codons (PTCs). Here, we present targeted tool bypassing PTCs, named CRISPR-pass, uses CRISPR-mediated adenine base editors. which should be applicable to 95.5% clinically significant mutations the ClinVar database,...
We present a novel genome-wide off-target prediction method named Extru-seq and compare it with cell-based (GUIDE-seq), in vitro (Digenome-seq), silico methods using promiscuous guide RNAs large numbers of valid sites. demonstrates high validation rate retention information about the intracellular environment, both beneficial characteristics methods. also shows low miss could easily be performed clinically relevant cell types little optimization, which are major positive features In summary,
Abstract CRISPR-Cas9 nucleases are versatile tools for genetic engineering cells and function by producing targeted double-strand breaks (DSBs) in the DNA sequence. However, unintended production of large deletions (>100 bp) represents a challenge to effective application this genome-editing system. We optimized long-range amplicon sequencing system developed k-mer sequence-alignment algorithm simultaneously detect small alteration events deletions. With workflow, we determined that...
Other SectionsABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS METHODSACKNOWLEDGEMENTSCONFLICTS OF INTERESTFIGURESREFERENCES
CRISPR-Cas9 induces DNA cleavages at desired target sites in a guide RNA-dependent manner; editing occurs through the resulting activity of repair processes including non-homologous end joining (NHEJ), which is dominant mammalian cells. NHEJ frequently causes small insertions and deletions (indels) near cleavage but only rarely nucleotide substitutions. High-throughput sequencing primary means assessing indel substitution frequencies bulk populations cells gene field. However, it difficult...
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 the host genome, but their integration sites generally unpredictable, which may increase uncertainty for use in therapeutics. To determine viral several PCR-based methods developed. However, sensitivities highly dependent on primer sequences, and optimized design is required individual target sites. In order 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 many organs. However, curative treatment ALD has not yet been achieved. To treat ALD, we applied two different gene-editing strategies, base editing and homology-independent targeted integration (HITI), patient-derived fibroblasts. Next, performed vivo HITI-mediated gene using AAV9 vectors delivered via intravenous...
Genome editing technology is a powerful tool for programming microbial cell factories. However, rat APOBEC1-derived cytosine base editor (CBE) that converts C•G to T•A at target genes induced DNA off-targets, regardless of single-guide RNA (sgRNA) sequences. Although the high efficiencies bacterial CBEs have been developed, risk unidentified off-targets impeded genome To address issues, we demonstrate engineering Corynebacterium glutamicum as GC-rich model industrial bacterium by generating...
<title>Abstract</title> CRISPR-Cas9 nucleases are versatile tools for genetic engineering cells and function by producing targeted double-strand breaks (DSBs) in the DNA sequence. However, unintended production of large deletions (> 100 bp) represents a challenge to effective application this genome-editing system. We optimized long-range amplicon sequencing system developed k-mer sequence-alignment algorithm simultaneously detect small alteration events deletions. With workflow, we...
When used to edit genomes, Cas9 nucleases produce targeted double-strand breaks in DNA. Subsequent DNA-repair pathways can induce large genomic deletions (larger than 100 bp), which constrains the applicability of genome editing. Here we show that Cas9-mediated at varying frequencies cancer cell lines, human embryonic stem cells and primary T cells, most are produced by two repair pathways: end resection DNA-polymerase theta-mediated joining. These findings required optimization long-range...
CRISPR-Cas nucleases, base editors (BEs), and prime (PEs) are efficient genome editing tools used widely in diverse research fields, including biology, biotechnology, medicine. While the mechanism is different for each tool, target specificity conferred common by binding of guide RNAs (gRNAs) their complementary sequences. However, gRNAs can bind to off-target sequences with a few mismatches, provoking editing, activities/outcomes vary depending on gRNAs. Therefore, selection as well...