Showing papers by "Jin-Soo Kim published in 2016"
TL;DR: It is found that Cpf1 could tolerate single or double mismatches in the 3′ Pam-distal region, but not in the 5′ PAM-proximal region, and off-target effects were completely abrogated by using preassembled, recombinant CpF1 ribonucleoproteins.
Abstract: Programmable clustered regularly interspaced short palindromic repeats (CRISPR) Cpf1 endonucleases are single-RNA-guided (crRNA) enzymes that recognize thymidine-rich protospacer-adjacent motif (PAM) sequences and produce cohesive double-stranded breaks (DSBs). Genome editing with CRISPR-Cpf1 endonucleases could provide an alternative to CRISPR-Cas9 endonucleases, but the determinants of targeting specificity are not well understood. Using mismatched crRNAs we found that Cpf1 could tolerate single or double mismatches in the 3' PAM-distal region, but not in the 5' PAM-proximal region. Genome-wide analysis of cleavage sites in vitro for eight Cpf1 nucleases using Digenome-seq revealed that there were 6 (LbCpf1) and 12 (AsCpf1) cleavage sites per crRNA in the human genome, fewer than are present for Cas9 nucleases (>90). Most Cpf1 off-target cleavage sites did not produce mutations in cells. We found mismatches in either the 3' PAM-distal region or in the PAM sequence of 12 off-target sites that were validated in vivo. Off-target effects were completely abrogated by using preassembled, recombinant Cpf1 ribonucleoproteins.
588 citations
TL;DR: It is demonstrated that direct delivery of purified CRISPR/Cas9 RNPs to the protoplast system enables targeted gene editing and paves the way to the generation of DNA-free genome edited grapevine and apple plants.
Abstract: The combined availability of whole genome sequences and genome editing tools is set to revolutionize the field of fruit biotechnology by enabling the introduction of targeted genetic changes with unprecedented control and accuracy, both to explore emergent phenotypes and to introduce new functionalities. Although plasmid-mediated delivery of genome editing components to plant cells is very efficient, it also presents some drawbacks, such as possible random integration of plasmid sequences in the host genome. Additionally, it may well be intercepted by current process-based GMO regulations, complicating the path to commercialization of improved varieties. Here, we explore direct delivery of purified CRISPR/Cas9 ribonucleoproteins (RNPs) to the protoplast of grape cultivar Chardonnay and apple cultivar such as Golden delicious fruit crop plants for efficient targeted mutagenesis. We targeted MLO-7, a susceptible gene in order to increase resistance to powdery mildew in grape cultivar and DIPM-1, DIPM-2, and DIPM-4 in the apple to increase resistance to fire blight disease. Furthermore, efficient protoplast transformation, the molar ratio of Cas9 and sgRNAs were optimized for each grape and apple cultivar. The targeted mutagenesis insertion and deletion rate was analyzed using targeted deep sequencing. Our results demonstrate that direct delivery of CRISPR/Cas9 RNPs to the protoplast system enables targeted gene editing and paves the way to the generation of DNA-free genome edited grapevine and apple plants.
465 citations
TL;DR: Findings of the NHEJ-mediated knock-in events will allow applications of the CRISPR/Cas9 system in microalgae, including “safe harboring” techniques shown in other organisms.
Abstract: Genome editing is crucial for genetic engineering of organisms for improved traits, particularly in microalgae due to the urgent necessity for the next generation biofuel production. The most advanced CRISPR/Cas9 system is simple, efficient and accurate in some organisms; however, it has proven extremely difficult in microalgae including the model alga Chlamydomonas. We solved this problem by delivering Cas9 ribonucleoproteins (RNPs) comprising the Cas9 protein and sgRNAs to avoid cytotoxicity and off-targeting associated with vector-driven expression of Cas9. We obtained CRISPR/Cas9-induced mutations at three loci including MAA7, CpSRP43 and ChlM, and targeted mutagenic efficiency was improved up to 100 fold compared to the first report of transgenic Cas9-induced mutagenesis. Interestingly, we found that unrelated vectors used for the selection purpose were predominantly integrated at the Cas9 cut site, indicative of NHEJ-mediated knock-in events. As expected with Cas9 RNPs, no off-targeting was found in one of the mutagenic screens. In conclusion, we improved the knockout efficiency by using Cas9 RNPs, which opens great opportunities not only for biological research but also industrial applications in Chlamydomonas and other microalgae. Findings of the NHEJ-mediated knock-in events will allow applications of the CRISPR/Cas9 system in microalgae, including “safe harboring” techniques shown in other organisms.
300 citations
TL;DR: A one-step transformation of Chlamydomonas reinhardtii is reported by the DNA-free CRISPR-Cas9 method rather than plasmids that encode Cas9 and guide RNAs, resulting in a strain constitutively producing zeaxanthin and showing improved photosynthetic productivity.
Abstract: Microalgae are versatile organisms capable of converting CO2, H2O, and sunlight into fuel and chemicals for domestic and industrial consumption. Thus, genetic modifications of microalgae for enhancing photosynthetic productivity, and biomass and bio-products generation are crucial for both academic and industrial applications. However, targeted mutagenesis in microalgae with CRISPR-Cas9 is limited. Here we report, a one-step transformation of Chlamydomonas reinhardtii by the DNA-free CRISPR-Cas9 method rather than plasmids that encode Cas9 and guide RNAs. Outcome was the sequential CpFTSY and ZEP two-gene knockout and the generation of a strain constitutively producing zeaxanthin and showing improved photosynthetic productivity.
222 citations
TL;DR: It is found that many false-positive, bulge-type off-target sites were cleaved by sgRNAs transcribed from an oligonucleotide duplex but not by those transcribes from a plasmid template.
Abstract: We present multiplex Digenome-seq to profile genome-wide specificities of up to 11 CRISPR-Cas9 nucleases simultaneously, saving time and reducing cost. Cell-free human genomic DNA was digested using multiple sgRNAs combined with the Cas9 protein and then subjected to whole-genome sequencing. In vitro cleavage patterns, characteristic of on- and off-target sites, were computationally identified across the genome using a new DNA cleavage scoring system. We found that many false-positive, bulge-type off-target sites were cleaved by sgRNAs transcribed from an oligonucleotide duplex but not by those transcribed from a plasmid template. Multiplex Digenome-seq captured many bona fide off-target sites, missed by other genome-wide methods, at which indels were induced at frequencies <0.1%. After analyzing 964 sites cleaved in vitro by these sgRNAs and measuring indel frequencies at hundreds of off-target sites in cells, we propose a guideline for the choice of target sites for minimizing CRISPR-Cas9 off-target effects in the human genome.
180 citations
TL;DR: It is concluded that both tracrRNA and crRNA regulate the conformations and kinetics of the Cas9 complex, which are crucial in the DNA cleavage activity of the CRISPR-Cas9 system.
Abstract: The type II CRISPR-associated protein Cas9 recognizes and cleaves target DNA with the help of two guide RNAs (gRNAs; tracrRNA and crRNA). However, the detailed mechanisms and kinetics of these gRNAs in the Cas9 nuclease activity are unclear. Here, we investigate the structural roles of gRNAs in the CRISPR-Cas9 system by single-molecule spectroscopy and reveal a new conformation of inactive Cas9 that is thermodynamically more preferable than active apo-Cas9. We find that tracrRNA prevents Cas9 from changing into the inactive form and leads to the Cas9:gRNA complex. For the Cas9:gRNA complex, we identify sub-conformations of the RNA-DNA heteroduplex during R-loop expansion. Our single-molecule study indicates that the kinetics of the sub-conformations is controlled by the complementarity between crRNA and target DNA. We conclude that both tracrRNA and crRNA regulate the conformations and kinetics of the Cas9 complex, which are crucial in the DNA cleavage activity of the CRISPR-Cas9 system.
87 citations
TL;DR: This work highlights the key advances that set the foundation for the rapid and widespread implementation of CRISPR–Cas9 genome editing approaches that has revolutionized the field.
Abstract: Genome editing harnesses programmable nucleases to cut and paste genetic information in a targeted manner in living cells and organisms. Here, I review the development of programmable nucleases, including zinc finger nucleases (ZFNs), TAL (transcription-activator-like) effector nucleases (TALENs) and CRISPR (cluster of regularly interspaced palindromic repeats)-Cas9 (CRISPR-associated protein 9) RNA-guided endonucleases (RGENs). I specifically highlight the key advances that set the foundation for the rapid and widespread implementation of CRISPR-Cas9 genome editing approaches that has revolutionized the field.
78 citations
TL;DR: It is concerned that the approved mushroom may still contain tiny fragments of foreign DNA in its genome, and if foreign DNA is present after CRISPR-Cas9 editing, regulatory approval would be required under current GMO legislation.
Abstract: VOLUME 34 NUMBER 10 OCTOBER 2016 NATURE BIOTECHNOLOGY To the Editor: The US Department of Agriculture (USDA) recently announced that regulation of a CRISPR-Cas9 gene-edited mushroom, in which a polyphenol oxidase (PPO) gene had been mutated to avoid (or delay) browning, fell outside of genetically modified organism (GMO) legislation1. However, we remain concerned that the approved mushroom may still contain tiny fragments of foreign DNA in its genome. If foreign DNA is present after CRISPR-Cas9 editing, regulatory approval would be required under current GMO legislation. In a letter (available in ref. 1 and at https://www.aphis.usda.gov/aphis/home/) submitted to the USDA, Yinong Yang, a researcher at Pennsylvania State University, outlined how the gene-edited mushroom was produced. Mushroom protoplasts (fungal cells with no cell wall) were transfected with plasmids encoding Cas9 and a guide RNA (gRNA) specific for a PPO gene. Transfected protoplasts were regenerated to produce mushrooms that contained small deletions (1–14 base pairs (bp)) in the PPO gene. The researchers analyzed the genome of edited fungi using PCR and Southern blot analysis and found no evidence of foreign DNA in the genome. In response, the USDA decided that the gene-edited mushroom fell outside of GMO regulations and that the US government had no authority to regulate this product. Melbourne, Victoria, Australia. 19Division of Surgical Oncology, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. 20Molecular Genomics, Research Division, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. 21Bioinformatics and Cancer Genomics, Research Division, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. 22The Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. 23Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. 24The Department of Surgery, the University of Melbourne, Parkville, Victoria, Australia. 25The Jack Brockhoff Familial Cancer Centre, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. 26Hereditary Cancer Program, BC Cancer Agency, Vancouver, Canada. 27Molecular Biomarkers and Translational Genomics Laboratory, Research Department, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. 28Department of Nursing, the Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. 29Department of Pathology, the Royal Women’s Hospital, Parkville, Victoria, Australia. 30Department of Pathology, University of Dammam, Dammam, Saudi Arabia. 31Department of Forensic Medicine, School of Public Health and Preventive Medicine, Monash University, Victoria, Australia. 32The Garvan Institute, Sydney, New South Wales, Australia. Correspondence should be addressed to: d.bowtell@petermac.org
64 citations
TL;DR: A simple and high-throughput binary vector system to clone a 19-20 bp of sgRNA, which binds to the reverse complement of a target locus, in a large T-DNA binary vector containing an SpCas9 expressing cassette that will be useful to generate targeted large-scale knock-out lines of model as well as non-model plant.
Abstract: CRISPR-Cas9 system is now widely used to edit a target genome in animals and plants. Cas9 protein derived from Streptococcus pyogenes (SpCas9) cleaves double-stranded DNA targeted by a chimeric single-guide RNA (sgRNA). For plant genome editing, Agrobacterium-mediated T-DNA transformation has been broadly used to express Cas9 proteins and sgRNAs under the control of CaMV 35S and U6/U3 promoter, respectively. We here developed a simple and high-throughput binary vector system to clone a 19-20 bp of sgRNA, which binds to the reverse complement of a target locus, in a large T-DNA binary vector containing an SpCas9 expressing cassette. Two-step cloning procedures: (1) annealing two target-specific oligonucleotides with overhangs specific to the AarI restriction enzyme site of the binary vector; and (2) ligating the annealed oligonucleotides into the two AarI sites of the vector, facilitate the high-throughput production of the positive clones. In addition, Cas9-coding sequence and U6/U3 promoter can be easily exchanged via the Gateway(TM) system and unique EcoRI/XhoI sites on the vector, respectively. We examined the mutation ratio and patterns when we transformed these constructs into Arabidopsis thaliana and a wild tobacco, Nicotiana attenuata. Our vector system will be useful to generate targeted large-scale knock-out lines of model as well as non-model plant.
62 citations
TL;DR: This review compares available methods for detecting, measuring, and analyzing off-target mutations and focuses on CRISPR/Cas9 regarding various methods, tweaks, and software tools available to nullify off- target effects.
55 citations
TL;DR: An online tool, Cas-Database, a genome-wide gRNA library design tool for Cas9 nucleases from Streptococcus pyogenes (SpCas9) with an easy-to-use web interface, which provides a powerful way to select multiple optimal target sequences from thousands of genes at once for the creation of a genomes-wide library.
Abstract: Motivation: CRISPR-derived RNA guided endonucleases (RGENs) have been widely used for both gene knockout and knock-in at the level of single or multiple genes. RGENs are now available for forward genetic screens at genome scale, but single guide RNA (sgRNA) selection at this scale is difficult.
Results: We develop an online tool, Cas-Database, a genome-wide gRNA library design tool for Cas9 nucleases from Streptococcus pyogenes (SpCas9). With an easy-to-use web interface, Cas-Database allows users to select optimal target sequences simply by changing the filtering conditions. Furthermore, it provides a powerful way to select multiple optimal target sequences from thousands of genes at once for the creation of a genome-wide library. Cas-Database also provides a web application programming interface (web API) for advanced bioinformatics users.
Availability and implementation: Free access at http://www.rgenome.net/cas-database/.
Contact: rk.ca.gnaynah@eabusgnas or rk.ca.uns@10miksj
Supplementary information: Supplementary data are available at Bioinformatics online.
TL;DR: This study provides a comprehensive overview of the epidemiology of hand injuries in the pediatric population and finds that with increasing age, closed injuries tended to increase more sharply than open injuries, extensor tendon rupture more than flexor injuries, and the level of injury moved proximally.
Abstract: BACKGROUND The purpose of this study was to identify comprehensive hand injury patterns in different pediatric age groups and to assess their risk factors. METHODS This retrospective study was conducted among patients younger than 16-year-old who presented to the emergency room of a general hospital located in Gyeonggi-do, Republic of Korea, and were treated for an injury of the finger or hand from January 2010 to December 2014. The authors analyzed the medical records of 344 patients. Age was categorized according to five groups. RESULTS A total of 391 injury sites of 344 patients were evaluated for this study. Overall and in each group, male patients were in the majority. With regard to dominant or non-dominant hand involvement, there were no significant differences. Door-related injuries were the most common cause in the age groups of 0 to 3, 4 to 6, and 7 to 9 years. Sport/recreational activities or physical conflict injuries were the most common cause in those aged 10 to 12 and 13 to 15. Amputation and crushing injury was the most common type in those aged 0 to 3 and 4 to 6 years. However, in those aged 10 to 12 and 13 to 15, deep laceration and closed fracture was the most common type. With increasing age, closed injuries tended to increase more sharply than open injuries, extensor tendon rupture more than flexor injuries, and the level of injury moved proximally. CONCLUSIONS This study provides a comprehensive overview of the epidemiology of hand injuries in the pediatric population.
TL;DR: It is found that cellular RNH1 affords marked protection from the lethal ribonucleolytic activity of RNase 1 but not from oxidants, which concludes that R NH1 protects cytosolic RNA from invading ribon nucleases.
Abstract: Ribonuclease inhibitor (RNH1) is a cytosolic protein that binds with femtomolar affinity to human ribonuclease 1 (RNase 1) and homologous secretory ribonucleases. RNH1 contains 32 cysteine residues and has been implicated as an antioxidant. Here, we use CRISPR-Cas9 to knock out RNH1 in HeLa cells. We find that cellular RNH1 affords marked protection from the lethal ribonucleolytic activity of RNase 1 but not from oxidants. We conclude that RNH1 protects cytosolic RNA from invading ribonucleases.
TL;DR: Dorsal veins in the lateral nail fold can be found easily because of the constant anatomical location and the new incision provides not only sufficient operative field for anastomosis but also additional opportunity of successful venous anastOMosis in the selected cases.
Abstract: BACKGROUND Successful venous anastomosis is one of the most important factors in fingertip replantation. Volar veins in the fingertip course proximally in a random pattern, which makes it difficult to find out the exact locations. Although dorsal veins in the lateral nail fold have constant location and adequate diameter for anastomosis, they have been known as hard to dissect from the immobile subcutaneous tissue. The authors present a new lateral nail fold incision technique for venous anastomosis in the fingertip amputations. METHODS From February 2010 to October 2010, 9 replantations using the new incision and venous anastomosis technique were performed in 9 patients. The levels of amputations were from the nail base to half of the nail bed. After repairing the proper digital arteries, a skin incision was made along the junction between the lateral nail fold and nail bed. Careful dissection was performed to isolate the veins in the lateral nail fold. After evaluation of the suitability of the vessel, venous anastomosis was performed. RESULTS Seven male and 2 female patients were enrolled in this study. Appropriate dorsal veins for anastomosis could be found in 8 of 9 patients. All the replanted stumps survived without venous congestion and following additional procedures. A sizable volar or dorsal vein could not be found in 1 patient. The salvage technique was required in this patient. CONCLUSIONS Dorsal veins in the lateral nail fold can be found easily because of the constant anatomical location. The new incision on the lateral nail fold provides not only sufficient operative field for anastomosis but also additional opportunity of successful venous anastomosis in the selected cases. The authors, therefore, propose this technique as an effective method for an alternative venous anastomosis in the zone I replantation.
TL;DR: The partial second toe pulp flap was associated with low rates of wound complications and favorable long-term outcomes, and donor-site morbidities appear acceptable in this patient population.
Abstract: Background In this study, we characterize the morbidity at the donor-site of partial second toe pulp free flaps in terms of wound management as well as long-term outcomes. Methods A single-institutional retrospective review was performed for patients who had undergone partial second toe pulp free flap transfer to the fingertip. Patient charts were reviewed for infection, skin necrosis, wound dehiscence, and hematoma for the donor site. Additionally, a questionnaire survey was given to patients who had a follow-up of longer than 1 year to characterize long-term postoperative pain and appearance. Results The review identified a total of 246 cases. Early wound complications were significant for wound dehiscence (n=8) and hematoma (n=5) for a wound complication rate of 5.3%. The questionnaire was distributed to 109 patients, and 54 patients completed the survey. Out of these 54 patients, 15 patients continued to have donor-site pain (28%) at a mean follow-up period of 32.4 months. However, the pain intensity was relatively low in the range between 2 to 5, on a 0-10 scale. None of these patients felt this donor-site pain interfered significantly with daily activity, nor did any patient require pain medications of any type. Donor-site appearance was satisfactory to most patients. Conclusions The partial second toe pulp flap was associated with low rates of wound complications and favorable long-term outcomes. Given the functional and aesthetic gain in the recipient finger, donor-site morbidities appear acceptable in this patient population. This study can be helpful in counseling patients regarding donor-site morbidity during the informed consent process.
TL;DR: Using a fascial free flap for reconstruction of the hand, the reconstructed tissue was approximately 1.5× as thick as the contour of the normal side, which led to positive responses regarding aesthetic satisfaction.
Abstract: BACKGROUND Fascial free flaps have been widely used for reconstruction of the hand because they are thin. However, studies reporting objective data regarding the advantages of this approach are lacking. Thus, we report our experience with such flaps. METHODS Forty-five cases of fascial free flaps between November 2006 and March 2014 were reviewed. Nine cases involving reconstructed dorsal or lateral defects were included. Four anterolateral thigh fascial free flaps and 5 lateral arm fascial free flaps were examined. Maximal flap contour was assessed by measuring reconstructed tissue thickness at the central area from the surface of the skin to below the bone in a vertical manner using ultrasonography and X-ray data. Contralateral regions were examined in the same manner and a comparative analysis was performed. A questionnaire survey regarding aesthetic satisfaction was also administered. RESULTS All reconstructed parts had a thicker contour than the contralateral side. The average relative percentage of reconstructed tissue thickness was found to be 152% using ultrasonography and 143% using X-ray imaging. According to the aesthetic satisfaction survey, the average rate of satisfaction for patients was 62%, and satisfaction with the flap contour was 72%. CONCLUSIONS Using a fascial free flap, the reconstructed tissue was approximately 1.5× as thick as the contour of the normal side, which led to positive responses regarding aesthetic satisfaction.
TL;DR: The foremost goal of managing a mutilated hand is provision of adequate skin coverage and the most suitable method is free tissue transfer.
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Weizmann Institute of Science1, University of Washington2, University of Cambridge3, Massachusetts Institute of Technology4, Stanford University5, Harvard University6, University of Trento7, University of California, Berkeley8, University of California, San Diego9, Max Planck Society10, University of Pennsylvania11, Amgen12, University of Massachusetts Medical School13, Seoul National University14, University of Chicago15, Columbia University16, University of California, Los Angeles17, University of Birmingham18, Royal Institute of Technology19, Dartmouth College20, John Innes Centre21, Genentech22, RIKEN Brain Science Institute23, Leiden University Medical Center24, University of California, San Francisco25, Georgia Institute of Technology26, ETH Zurich27, Baylor College of Medicine28, University of Illinois at Urbana–Champaign29, Technical University of Denmark30, Imperial College London31, Icahn School of Medicine at Mount Sinai32, Peking University33, Wellcome Trust Sanger Institute34, International Institute of Tropical Agriculture35
TL;DR: Nature Biotechnology asks a selection of researchers about the most exciting frontier in their field and the most needed technologies for advancing knowledge and applications.
Abstract: Nature Biotechnology asks a selection of researchers about the most exciting frontier in their field and the most needed technologies for advancing knowledge and applications.
TL;DR: “ 구조의 복 원이라고 봐야 할 것이다.”
Abstract: 산업시설의 자동화가 보편화되면서 수부외상의 빈도는 많 이 감소하고 있고 고령화가 진행되면서 환자의 양상도 변화 하고 있다. 국내 수부외과학회에서는 수부외과 세부전문의 제도를 시행하여 치료의 질적 향상을 도모하면서 수부외상 에 대한 전문성을 강조하고 있다. 또한 의료의 접근성이 향 상되고 미용적인 관심이 증가하면서 예전에는 병원문을 두 드리지 않았을 환자들도 포함하여 여전히 많은 사람들이 수 부문제로 병원을 찾고 있다. 수부재건은 외형적 개선도 중요 하지만 수부 본연의 기능회복만큼 중요한 것은 없을 것이다. 따라서 수부재건의 일차적인 목표는 단순히 결손된 부분을 만들어 주는 것이 아니라 실제적으로 기능을 하는 구조의 복 원이라고 봐야 할 것이다. 여기에 더불어 미용적으로도 개선 이 된다면 이상적인 치료라 할 수 있으며 이에 관한 다양한 재건방법에 대해 알아보고자 한다.