Showing papers by "Jin-Soo Kim published in 2021"

ISSCR Guidelines for Stem Cell Research and Clinical Translation: The 2021 update.

Abstract: The International Society for Stem Cell Research has updated its Guidelines for Stem Cell Research and Clinical Translation in order to address advances in stem cell science and other relevant fields, together with the associated ethical, social, and policy issues that have arisen since the last update in 2016. While growing to encompass the evolving science, clinical applications of stem cells, and the increasingly complex implications of stem cell research for society, the basic principles underlying the Guidelines remain unchanged, and they will continue to serve as the standard for the field and as a resource for scientists, regulators, funders, physicians, and members of the public, including patients. A summary of the key updates and issues is presented here.

Mitochondrial DNA editing in mice with DddA-TALE fusion deaminases

Abstract: DddA-derived cytosine base editors (DdCBEs), composed of the split interbacterial toxin DddAtox, transcription activator-like effector (TALE), and uracil glycosylase inhibitor (UGI), enable targeted C-to-T base conversions in mitochondrial DNA (mtDNA). Here, we demonstrate highly efficient mtDNA editing in mouse embryos using custom-designed DdCBEs. We target the mitochondrial gene, MT-ND5 (ND5), which encodes a subunit of NADH dehydrogenase that catalyzes NADH dehydration and electron transfer to ubiquinone, to obtain several mtDNA mutations, including m.G12918A associated with human mitochondrial diseases and m.C12336T that incorporates a premature stop codon, creating mitochondrial disease models in mice and demonstrating a potential for the treatment of mitochondrial disorders. Split DddA-derived base editors fused to TALEs enable mitochondrial DNA editing. Here the authors demonstrate their use in mouse embryos with germline transmission.

Decarbonizing the food and beverages industry: A critical and systematic review of developments, sociotechnical systems and policy options

Abstract: From farm to fork, food and beverage consumption can have significant negative impacts on energy consumption, water consumption, climate change, and other environmental subsystems. This paper presents a comprehensive, critical and systematic review of more than 350,000 sources of evidence, and a short list of 701 studies, on the topic of greenhouse gas emissions from the food and beverage industry. Utilizing a sociotechnical lens that examines food supply and agriculture, manufacturing, retail and distribution, and consumption and use, the review identifies the most carbon-intensive processes in the industry, as well as the corresponding energy and carbon “footprints”. It discusses multiple current and emerging options and practices for decarbonization, including 78 potentially transformative technologies. It examines the benefits to sector decarbonization—including energy and carbon savings, cost savings, and other co-benefits related to sustainability or health—as well as barriers across financial and economic, institutional and managerial, and behavioral and consumer dimensions. It lastly discusses how financing, business models, and policy can be harnessed to help overcome these barriers, and identifies a set of research gaps.

Chloroplast and mitochondrial DNA editing in plants

Abstract: Plant organelles including mitochondria and chloroplasts contain their own genomes, which encode many genes essential for respiration and photosynthesis, respectively. Gene editing in plant organelles, an unmet need for plant genetics and biotechnology, has been hampered by the lack of appropriate tools for targeting DNA in these organelles. In this study, we developed a Golden Gate cloning system1, composed of 16 expression plasmids (8 for the delivery of the resulting protein to mitochondria and the other 8 for delivery to chloroplasts) and 424 transcription activator-like effector subarray plasmids, to assemble DddA-derived cytosine base editor (DdCBE)2 plasmids and used the resulting DdCBEs to efficiently promote point mutagenesis in mitochondria and chloroplasts. Our DdCBEs induced base editing in lettuce or rapeseed calli at frequencies of up to 25% (mitochondria) and 38% (chloroplasts). We also showed DNA-free base editing in chloroplasts by delivering DdCBE mRNA to lettuce protoplasts to avoid off-target mutations caused by DdCBE-encoding plasmids. Furthermore, we generated lettuce calli and plantlets with edit frequencies of up to 99%, which were resistant to streptomycin or spectinomycin, by introducing a point mutation in the chloroplast 16S rRNA gene.

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

Abstract: 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.

Adenine base editor engineering reduces editing of bystander cytosines.

Abstract: Adenine base editors (ABEs) catalyze specific A-to-G conversions at genomic sites of interest. However, ABEs also induce cytosine deamination at the target site. To reduce the cytosine editing activity, we engineered a commonly used adenosine deaminase, TadA7.10, and found that ABE7.10 with a D108Q mutation in TadA7.10 exhibited tenfold reduced cytosine deamination activity. The D108Q mutation also reduces cytosine deamination activity in two recently developed high-activity versions of ABE, ABE8e and ABE8s, and is compatible with V106W, a mutation that reduces off-target RNA editing. ABE7.10 containing a P48R mutation displayed increased cytosine deamination activity and a substantially reduced adenine editing rate, yielding a TC-specific base editing tool for TC-to-TT or TC-to-TG conversions that broadens the utility of base editors.

Small-molecule inhibitors of histone deacetylase improve CRISPR-based adenine base editing.

Abstract: CRISPR-based base editors (BEs) are widely used to induce nucleotide substitutions in living cells and organisms without causing the damaging DNA double-strand breaks and DNA donor templates Cytosine BEs that induce C:G to T:A conversion and adenine BEs that induce A:T to G:C conversion have been developed Various attempts have been made to increase the efficiency of both BEs; however, their activities need to be improved for further applications Here, we describe a fluorescent reporter-based drug screening platform to identify novel chemicals with the goal of improving adenine base editing efficiency The reporter system revealed that histone deacetylase inhibitors, particularly romidepsin, enhanced base editing efficiencies by up to 49-fold by increasing the expression levels of proteins and target accessibility The results support the use of romidepsin as a viable option to improve base editing efficiency in biomedical research and therapeutic genome engineering

Adenine Base Editor Ribonucleoproteins Delivered by Lentivirus-Like Particles Show High On-Target Base Editing and Undetectable RNA Off-Target Activities.

Abstract: Adenine base editors (ABEs) can correct gene mutations without creating double-strand breaks. However, in recent reports, these editors showed guide-independent RNA off-target activities. This work describes our development of a delivery method to minimize ABEs' RNA off-target activity. After discovering a RNA off-target hot spot for sensitive detection of RNA off-target activities, we found that delivering ribonucleoproteins (RNPs) by electroporation generated undetectable non-specific RNA editing, but on-target base editing activity was also relatively low. We then explored a lentivirus capsid-based delivery strategy to deliver ABE. We used aptamer/aptamer-binding protein (ABP) interactions to package ABE RNPs into lentiviral capsids. Capsid RNPs were delivered to human cells for highly efficient guided base editing. Importantly, RNA off-target activities from the capsid RNPs were undetectable. Our new lentiviral capsid-based ABE RNP delivery method with minimal RNA off-target activities makes ABE one step closer to possible therapeutic applications.

The efficacy of CRISPR-mediated cytosine base editing with the RPS5a promoter in Arabidopsis thaliana.

Abstract: CRISPR/Cas9-mediated genome editing is an important and versatile technology in modern biological research. Recent advancements include base-editing CRISPR tools that enable targeted nucleotide substitutions using a fusion protein comprising a nickase variant of Cas9 and a base deaminase. Improvements in base editing efficiencies and inheritable of edited loci need to be made to make CRISPR a viable system in plants. Here, we report efficiency of cytosine base editors (CBEs) in Arabidopsis thaliana by applying the strong endogenous RPS5a promoter to drive the expression of nickase Cas9 and either rAPOBEC1 from rat (BE3) or the PmCDA1 activation-induced cytidine deaminase from sea lamprey (AIDv2). Compared with the strong heterologous CaMV35S promoter of viral origin, the RPS5a promoter improved CBE efficiency by 32% points with the number of T1 plants showing over 50% conversion ratio when the LFY gene was targeted. CBE induced nonsense mutations in LFY via C-to-T conversion, which resulted in loss-of-function lfy phenotypes; defects in LFY function were associated with the targeted base substitutions. Our data suggest that optimal promoter choice for CBE expression may affect base-editing efficiencies in plants. The results provide a strategy to optimize low-efficiency base editors and demonstrate their applicability for functional assays and trait development in crop research.

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

Abstract: 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.

Production of MSTN-mutated cattle without exogenous gene integration using CRISPR-Cas9.

Abstract: Many genome-edited animals have been produced using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology to edit specific genes. However, there are few guidelines for the application of this technique to cattle. The goal of this study was to produce trait-improved cattle using the genome-editing technology CRISPR-Cas9. Myostatin (MSTN) was selected as a target locus, and synthetic mRNA of sgRNA and Cas9 were microinjected into fertilized bovine embryos in vitro. As a result, 17 healthy calves were born, and three of them showed MSTN mutation rates of 10.5%, 45.4%, and 99.9%, respectively. Importantly, the offspring with the 99.9% MSTN mutation rate had a biallelic mutation (-12 bps) and a double-muscling phenotype. In conclusion, we demonstrate that the genome-editing technology CRISPR-Cas9 can produce genetically modified calves with improved traits.

Identifying genome-wide off-target sites of CRISPR RNA-guided nucleases and deaminases with Digenome-seq.

Abstract: Digested genome sequencing (Digenome-seq) is a highly sensitive, easy-to-carry-out, cell-free method for experimentally identifying genome-wide off-target sites of programmable nucleases and deaminases (also known as base editors). Genomic DNA is digested in vitro using clustered regularly interspaced short palindromic repeats ribonucleoproteins (RNPs; plus DNA-modifying enzymes to cleave both strands of DNA at sites containing deaminated base products, in the case of base editors) and subjected to whole-genome sequencing (WGS) with a typical sequencing depth of 30×. A web-based program is available to map in vitro cleavage sites corresponding to on- and off-target sites. Chromatin DNA, in parallel with histone-free genomic DNA, can also be used to account for the effects of chromatin structure on off-target nuclease activity. Digenome-seq is more sensitive and comprehensive than cell-based methods for identifying off-target sites. Unlike other cell-free methods, Digenome-seq does not involve enrichment of DNA ends through PCR amplification. The entire process other than WGS, which takes ~1–2 weeks, including purification and preparation of RNPs, digestion of genomic DNA and bioinformatic analysis after WGS, takes about several weeks. This protocol describes a cell-free method for experimentally identifying genome-wide off-target sites of CRISPR nucleases and deaminases through in vitro digestion of genomic DNA or chromatin followed by whole-genome sequencing.

Off-the-Shelf, Immune-Compatible Human Embryonic Stem Cells Generated Via CRISPR-Mediated Genome Editing

Abstract: Human embryonic stem cells (hESCs) hold promise in regenerative medicine but allogeneic immune rejections caused by highly polymorphic human leukocyte antigens (HLAs) remain a barrier to their clinical applications. Here, we used a CRISPR/Cas9-mediated HLA-editing strategy to generate a variety of HLA homozygous-like hESC lines from pre-established hESC lines. We edited four pre-established HLA-heterozygous hESC lines and created a mini library of 14 HLA-edited hESC lines in which single HLA-A and HLA-B alleles and both HLA-DR alleles are disrupted. The HLA-edited hESC derivatives elicited both low T cell- and low NK cell-mediated immune responses. Our library would cover about 40% of the Asian-Pacific population. We estimate that HLA-editing of only 19 pre-established hESC lines would give rise to 46 different hESC lines to cover 90% of the Asian-Pacific population. This study offers an opportunity to generate an off-the-shelf HLA-compatible hESC bank, available for immune-compatible cell transplantation, without embryo destruction. Graphical Abstract.

Novel Positive Working Photosensitive Polyimide-Carbon Black (PSPI-CB) Composites Containing Triphenylene Vinyl Ether (TP-VE) Monomers

Abstract: The first positive working chemically amplified photosensitive polyimide-carbon black (PSPI-CB) composite with 2,2′-bis(4-(2′-(vinyloxy)ethoxy)phenyl)propane (BPA-DEVE) as a crosslinking agent was prepared. In addition, the triphenylene vinyl ether (TP-VE) monomers 2,3-di-[(2-vinyloxy)ethoxy]triphenylene (TP-DEVE) and 2,3,6,7,10,11-hexa-[(2-vinyloxy)ethoxy]triphenylene (TP-HEVE) were used as novel CB dispersing aids as well as crosslinking agents. The photolithographic performance of TP-DEVE and TP-HEVE as crosslinking agents in the positive working PSPI and PSPI-CB composites was similar to that of BPA-DEVE. The CB dispersion ability of the crosslinking agents used in the present study was evaluated on the basis of the shading ability (optical density) and surface roughness of the PSPI-CB films. Given the same amount of crosslinking agent in the formulation of the PSPI-CB composite, the optical density and surface roughness of the corresponding PSPI-CB composite film were 1.4 and 116.0 nm for PSPI-CB-1 with BPA-DEVE, 1.7 and 78.2 nm for PSPI-CB-2 with TP-DEVE, and 1.9 and 58.4 nm for PSPI-CB-3 with TP-HEVE, respectively. The results confirm that the CB dispersion ability of these crosslinking agents decreases in the order TP-HEVE > TP-DEVE > BPA-DEVE.

Base Editing in Progeria.

Abstract: Extending Life Span in a Mouse Model of Progeria The Hutchinson–Gilford progeria syndrome is an autosomal dominant disease caused by a single point mutation in LMNA, which encodes the lamin A and C...

Tailored Polymer Gate Dielectric Engineering to Optimize Flexible Organic Field-Effect Transistors and Complementary Integrated Circuits

Abstract: The increasing demand for solution-processed and flexible organic electronics has promoted the fabrication of integrated logic circuits using organic field-effect transistors (OFETs) instead of fundamental unit devices. This has been made possible through the rapid development of materials and processes in the past few decades. It is important for the p- and n-type OFETs using different organic semiconductors (OSCs) to have complementarily matched electrical characteristics, which significantly improve the performance of organic logic circuits. In this study, an efficient strategy to optimize the performance of flexible organic electronics, such as OFETs and complementary inverters, is proposed using a combination of polymer insulators tailored to each OSC type. Photopatternable soluble copolyimides (ScoPIs), which exhibit excellent insulating properties and chemical resistance, are synthesized and applied as gate dielectric layers in the OFETs. The material and electrical properties are systematically investigated by varying the molecular ratio of ScoPIs to determine the optimal conditions for each OFET type. As a result, complementary inverters report 1.67 times higher integration density compared to the conventional ones while maintaining gain, switching threshold, and static noise margin of 23.7 V/V, 22.1 V, and 12.1 V, respectively, at a supply voltage of 40 V. The flexible complementary inverters are successfully demonstrated by fully exploiting the advantages of ScoPIs.