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Genome Editing using CRISPR-Cas9 and -Cpf1
Hyongbum (Henry) Kim 한국동물생명공학회(구 한국동물번식학회) 2017 Reproductive & Developmental Biology(Supplement) Vol.41 No.2
Programmable nucleases, which include zinc-finger nucleases, transcriptional activatorlike effector nucleases, and RNA-guided engineered nucleases derived from the prokaryotic CRISPR/Cas system, enable targeted genetic modifications in cultured cells, animals, and plants, tools of great value in research, medicine, and biotechnology. Cpf1 is a recently reported effector endonuclease protein of the class 2 CRISPR-Cas system. Cpf1 has several differences from Cas9: cleavage with 5’ overhangs, shorter guide RNA, and a longer distance between the seed sequence and cleavage site, which could provide potential advantages for some cases of genome editing such as nonhomologous end joining-based gene insertion and efficient genome editing using homology- directed repair. However, limited information is available about Cpf1 activity profiles in mammalian cells, precluding its wide use for genome editing. Here, we performed en masse evaluation of guide RNA and Cpf1 activity using synthetic target sequences and deep sequencing. Using this approach, we determined target sequence- dependent activity profiles and protospacer adjacent motif sequences of Cpf1. We found that sequence features of high activity AsCpf1 guide RNAs are distinct from those of SpCas9. Evaluation of activity at mismatched target sequences showed that Cpf1 target sequences can be divided into seed, trunk, and promiscuous regions depending on mismatch tolerability. The Cpf1 characterization profile will greatly facilitate Cpf1-based genome editing.
Bone Marrow Mononuclear Cells Have Neurovascular Tropism and Improve Diabetic Neuropathy
Kim, Hyongbum,Park, Jong-seon,Choi, Yong Jin,Kim, Mee-Ohk,Huh, Yang Hoon,Kim, Sung-Whan,Han, Ji Woong,Lee, JiYoon,Kim, Sinae,Houge, Mackenzie A.,Ii, Masaaki,Yoon, Young-sup Wiley (John WileySons) 2009 Stem Cells Vol.27 No.7
<P>Bone marrow-derived mononuclear cells (BMNCs) have been shown to effectively treat ischemic cardiovascular diseases. Because diabetic neuropathy (DN) is causally associated with impaired angiogenesis and deficiency of angiogenic and neurotrophic factors in the nerves, we investigated whether DN can be ameliorated by local injection of BMNCs. Severe peripheral neuropathy, characterized by a significant decrease in the motor and sensory nerve conduction velocities (NCVs), developed 12 weeks after the induction of diabetes with streptozotocin in rats. The injection of BMNCs restored motor and sensory NCVs to normal levels and significantly improved vascular density and blood flow in diabetic nerves over 4 weeks. Fluorescent microscopic observation revealed that DiI-labeled BMNCs preferentially engrafted in sciatic nerves. Whole-mount fluorescent imaging and confocal microscopic evaluation demonstrated that many of the BMNCs localized following the course of the vasa nervorum in close proximity to blood vessels without incorporation into vasa nervorum as endothelial cells at a detectable level. Real-time reverse transcription-polymerase chain reaction analysis showed that the levels of angiogenic and neurotrophic factors were significantly increased in the nerves by BMNC injection. Local transplantation of BMNCs improved experimental DN by augmenting angiogenesis and increasing angiogenic and neurotrophic factors in peripheral nerves. These findings suggest that BMNC transplantation may represent a novel therapeutic option for treating DN.</P>
A guide to genome engineering with programmable nucleases
Kim, Hyongbum,Kim, Jin-Soo Nature Publishing Group, a division of Macmillan P 2014 Nature reviews. Genetics Vol.15 No.5
Programmable nucleases — including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided engineered nucleases (RGENs) derived from the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)–Cas (CRISPR-associated) system — enable targeted genetic modifications in cultured cells, as well as in whole animals and plants. The value of these enzymes in research, medicine and biotechnology arises from their ability to induce site-specific DNA cleavage in the genome, the repair (through endogenous mechanisms) of which allows high-precision genome editing. However, these nucleases differ in several respects, including their composition, targetable sites, specificities and mutation signatures, among other characteristics. Knowledge of nuclease-specific features, as well as of their pros and cons, is essential for researchers to choose the most appropriate tool for a range of applications.
Recent developments and clinical studies utilizing engineered zinc finger nuclease technology.
Jo, Young-Il,Kim, Hyongbum,Ramakrishna, Suresh Birkhäuser ; Springer 2015 Cellular and molecular life sciences Vol.72 No.20
<P>Efficient methods for creating targeted genetic modifications have long been sought for the investigation of gene function and the development of therapeutic modalities for various diseases, including genetic disorders. Although such modifications are possible using homologous recombination, the efficiency is extremely low. Zinc finger nucleases (ZFNs) are custom-designed artificial nucleases that make double-strand breaks at specific sequences, enabling efficient targeted genetic modifications such as corrections, additions, gene knockouts and structural variations. ZFNs are composed of two domains: (i) a DNA-binding domain comprised of zinc finger modules and (ii) the FokI nuclease domain that cleaves the DNA strand. Over 17?years after ZFNs were initially developed, a number of improvements have been made. Here, we will review the developments and future perspectives of ZFN technology. For example, ZFN activity and specificity have been significantly enhanced by modifying the DNA-binding domain and FokI cleavage domain. Advances in culture methods, such as the application of a cold shock and the use of small molecules that affect ZFN stability, have also increased ZFN activity. Furthermore, ZFN-induced mutant cells can be enriched using episomal surrogate reporters. Additionally, we discuss several ongoing clinical studies that are based on ZFN-mediated genome editing in humans. These breakthroughs have substantially facilitated the use of ZFNs in research, medicine and biotechnology.</P>
Seo, Jung Hwa,Kim, Hyongbum,Park, Eun Sook,Lee, Jong Eun,Kim, Dong Wook,Kim, Hyun Ok,Im, Sang Hee,Yu, Ji Hea,Kim, Ji Yeon,Lee, Min-Young,Kim, Chul Hoon,Cho, Sung-Rae Pergamon Press ; Elsevier Science Ltd ; Elsevier S 2013 Cell transplantation Vol.22 No.9
<P>We investigated the effects of environmental enrichment (EE) on the function of transplanted adipose stem cells (ASCs) and the combined effect of EE and ASC transplantation on neurobehavioral function in an animal model of chronic hypoxic-ischemic (HI) brain injury. HI brain damage was induced in 7-day-old mice by unilateral carotid artery ligation and exposure to hypoxia (8% O2 for 90 min). At 6 weeks of age, the mice were randomly injected with either ASCs or PBS into the striatum and were randomly assigned to either EE or standard cages (SC), comprising ASC-EE (n=18), ASC-SC (n=19), PBS-EE (n=12), PBS-SC (n=17), and untreated controls (n=23). Rotarod, forelimb-use asymmetry, and grip strength tests were performed to evaluate neurobehavioral function. The fate of transplanted cells and the levels of endogenous neurogenesis, astrocyte activation, and paracrine factors were also measured. As a result, EE and ASC transplantation synergistically improved rotarod latency, forelimb-use asymmetry, and grip strength compared to those of the other groups. The number of engrafted ASCs and βIII-tubulin(+) neurons derived from the transplanted ASCs was significantly higher in mice in EE than those in SC. EE and ASC transplantation also synergistically increased BrdU(+)βIII-tubulin(+) neurons, GFAP(+) astrocytic density, and fibroblast growth factor 2 (FGF2) level but not the level of CS-56(+) glial scarring in the striatum. In conclusion, EE and ASC transplantation synergistically improved neurobehavioral functions. The underlying mechanisms of this synergism included enhanced repair processes such as higher engraftment of the transplanted ASCs, increased endogenous neurogenesis and astrocytic activation coupled with upregulation of FGF2.</P>
Jeong, Jin-Ok,Kim, Mee-Ohk,Kim, Hyongbum,Lee, Min-Young,Kim, Sung-Whan,Ii, Masaaki,Lee, Jung-uek,Lee, Jiyoon,Choi, Yong Jin,Cho, Hyun-Jai,Lee, Namho,Silver, Marcy,Wecker, Andrea,Kim, Dong-Wook,Yoon, Y Ovid Technologies Wolters Kluwer -American Heart A 2009 CIRCULATION - Vol.119 No.5
<P>BACKGROUND: Endothelial progenitor cells (EPCs) are known to promote neovascularization in ischemic diseases. Recent evidence suggested that diabetic neuropathy is causally related to impaired angiogenesis and deficient growth factors. Accordingly, we investigated whether diabetic neuropathy could be reversed by local transplantation of EPCs. METHODS AND RESULTS: We found that motor and sensory nerve conduction velocities, blood flow, and capillary density were reduced in sciatic nerves of streptozotocin-induced diabetic mice but recovered to normal levels after hind-limb injection of bone marrow-derived EPCs. Injected EPCs were preferentially and durably engrafted in the sciatic nerves. A portion of engrafted EPCs were uniquely localized in close proximity to vasa nervorum, and a smaller portion of these EPCs were colocalized with endothelial cells. Multiple angiogenic and neurotrophic factors were significantly increased in the EPC-injected nerves. These dual angiogenic and neurotrophic effects of EPCs were confirmed by higher proliferation of Schwann cells and endothelial cells cultured in EPC-conditioned media. CONCLUSIONS: We demonstrate for the first time that bone marrow-derived EPCs could reverse various manifestations of diabetic neuropathy. These therapeutic effects were mediated by direct augmentation of neovascularization in peripheral nerves through long-term and preferential engraftment of EPCs in nerves and particularly vasa nervorum and their paracrine effects. These findings suggest that EPC transplantation could represent an innovative therapeutic option for treating diabetic neuropathy.</P>