Yongsub Kim

Bio: Yongsub Kim is an academic researcher from University of Ulsan. The author has contributed to research in topics: CRISPR & Cas9. The author has an hindex of 14, co-authored 29 publications receiving 2176 citations. Previous affiliations of Yongsub Kim include Seoul National University & Asan Medical Center.

Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases

Abstract: RNA-guided endonucleases (RGENs), derived from the prokaryotic adaptive immune system known as CRISPR/Cas, enable targeted genome engineering in cells and organisms. RGENs are ribonucleoproteins that consist of guide RNA and Cas9, a protein component originated from Streptococcus pyogenes. These enzymes cleave chromosomal DNA, whose sequence is complementary, to guide RNA in a targeted manner, producing site-specific DNA double-strand breaks (DSBs), the repair of which gives rise to targeted genome modifications. Despite broad interest in RGEN-mediated genome editing, these nucleases are limited by off-target mutations and unwanted chromosomal translocations associated with off-target DNA cleavages. Here, we show that off-target effects of RGENs can be reduced below the detection limits of deep sequencing by choosing unique target sequences in the genome and modifying both guide RNA and Cas9. We found that both the composition and structure of guide RNA can affect RGEN activities in cells to reduce off-target effects. RGENs efficiently discriminated on-target sites from off-target sites that differ by two bases. Furthermore, exome sequencing analysis showed that no off-target mutations were induced by two RGENs in four clonal populations of mutant cells. In addition, paired Cas9 nickases, composed of D10A Cas9 and guide RNA, which generate two single-strand breaks (SSBs) or nicks on different DNA strands, were highly specific in human cells, avoiding off-target mutations without sacrificing genome-editing efficiency. Interestingly, paired nickases induced chromosomal deletions in a targeted manner without causing unwanted translocations. Our results highlight the importance of choosing unique target sequences and optimizing guide RNA and Cas9 to avoid or reduce RGEN-induced off-target mutations.

A library of TAL effector nucleases spanning the human genome

Abstract: A collection of TALENs targeted to 18,740 human protein-coding genes will facilitate genetic engineering of human cells.

Generation of knockout mice by Cpf1-mediated gene targeting

Abstract: VOLUME 34 NUMBER 8 AUGUST 2016 NATURE BIOTECHNOLOGY target sites by four mismatches. We also electroporated an AsCpf1 RNP targeting Tyr, the gene encoding tyrosinase, into mouse embryos and observed mutations in 4 out of 12 blastocysts (33%) (Supplementary Fig. 6). Taken together, these results show that Cas9 and Cpf1 RNPs can be delivered efficiently by electroporation into animal embryos, resulting in high mutation frequencies. We next transplanted mouse embryos after Cpf1 microinjection or electroporation into surrogate mothers and obtained mice with targeted mutations in Foxn1 (Fig. 1d and Supplementary Fig. 7a) or Tyr (Supplementary Table 2 and Supplementary Fig. 8a). Three out of seven mice carried mutations at the Cpf1 cleavage site in the Foxn1 gene. One Tyr mutant mouse showed a partial coat color change, consistent with its mosaic genotype (Supplementary Fig. 8b). To investigate whether Cpf1 had offtarget effects, we performed whole genome sequencing using genomic DNA isolated from one Foxn1 mutant mouse and its wildtype sibling (Supplementary Note). The sequence analysis showed that no off-target mutations were introduced at homologous sites with up to 7-nucleotide mismatches. Notably, the mutant allele in a female Foxn1 mutant mouse was transmitted to embryos (Supplementary Fig. 7b,c). In summary, our results show that electroporation of AsCpf1 RNPs resulted in efficient and specific genome editing in mouse embryos. RNPs7 are as effective as mRNA8 or plasmids9, but are degraded rapidly by endogenous proteases and RNases in cells, and have been previously shown to reduce off-target effects9 and mosaicism10. Unlike microinjection, electroporation is easy to carry out, fast, and scalable. Up to 50 embryos can be electroporated simultaneously. We propose that electroporation of Cpf1 RNPs is a potentially useful new method for genome editing in animals.

TALENs and ZFNs are associated with different mutation signatures

Abstract: 1. Bibikova, M., Beumer, K., Trautman, J.K. & Carroll, D. Science 300, 764 (2003). 2. Urnov, F.D. et al. Nature 435, 646–651 (2005). 3. Kim, H.J., Lee, H.J., Kim, H., Cho, S.W. & Kim, J.S. Genome Res. 19, 1279–1288 (2009). 4. Kim, S., Lee, M.J., Kim, H., Kang, M. & Kim, J.S. Nat. Methods 8, 7 (2011). 5. Miller, J.C. et al. Nat. Biotechnol. 29, 143–148 (2011). 6. Maresca, M., Lin, V.G., Guo, N. & Yang, Y. Genome Res. published online, doi:10.1101/gr.145441.112 (14 November 2012). ZFN vs. TALEN (literature) a

Chemical screening identifies ATM as a target for alleviating senescence

Abstract: Ataxia telangiectasia mutated (ATM) directly interacts with and phosphorylates the V-ATPase V1 subunit ATP6V1G1, thereby decreasing V1-V0 assembly in the V-ATPase. Attenuation of ATM activity results in lysosomal pH acidification, recovery of autophagy and alleviation of senescence. Senescence, defined as irreversible cell-cycle arrest, is the main driving force of aging and age-related diseases. Here, we performed high-throughput screening to identify compounds that alleviate senescence and identified the ataxia telangiectasia mutated (ATM) inhibitor KU-60019 as an effective agent. To elucidate the mechanism underlying ATM's role in senescence, we performed a yeast two-hybrid screen and found that ATM interacted with the vacuolar ATPase V1 subunits ATP6V1E1 and ATP6V1G1. Specifically, ATM decreased E-G dimerization through direct phosphorylation of ATP6V1G1. Attenuation of ATM activity restored the dimerization, thus consequently facilitating assembly of the V1 and V0 domains with concomitant reacidification of the lysosome. In turn, this reacidification induced the functional recovery of the lysosome/autophagy system and was coupled with mitochondrial functional recovery and metabolic reprogramming. Together, our data reveal a new mechanism through which senescence is controlled by the lysosomal–mitochondrial axis, whose function is modulated by the fine-tuning of ATM activity.

The new frontier of genome engineering with CRISPR-Cas9

Abstract: The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics.

CRISPR-Cas systems for editing, regulating and targeting genomes

Abstract: Targeted genome editing using engineered nucleases has rapidly gone from being a niche technology to a mainstream method used by many biological researchers. This widespread adoption has been largely fueled by the emergence of the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology, an important new approach for generating RNA-guided nucleases, such as Cas9, with customizable specificities. Genome editing mediated by these nucleases has been used to rapidly, easily and efficiently modify endogenous genes in a wide variety of biomedically important cell types and in organisms that have traditionally been challenging to manipulate genetically. Furthermore, a modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells. Although the genome-wide specificities of CRISPR-Cas9 systems remain to be fully defined, the power of these systems to perform targeted, highly efficient alterations of genome sequence and gene expression will undoubtedly transform biological research and spur the development of novel molecular therapeutics for human disease.

Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor

Abstract: PLoS BIOLOGY Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor Jean-Philippe Coppe 1 , Christopher K. Patil 1[ , Francis Rodier 1,2[ , Yu Sun 3 , Denise P. Mun oz 1,2 , Joshua Goldstein 1¤ , Peter S. Nelson 3 , Pierre-Yves Desprez 1,4 , Judith Campisi 1,2* 1 Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America, 2 Buck Institute for Age Research, Novato, California, United States of America, 3 Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 4 California Pacific Medical Center Research Institute, San Francisco, California, United States of America Cellular senescence suppresses cancer by arresting cell proliferation, essentially permanently, in response to oncogenic stimuli, including genotoxic stress. We modified the use of antibody arrays to provide a quantitative assessment of factors secreted by senescent cells. We show that human cells induced to senesce by genotoxic stress secrete myriad factors associated with inflammation and malignancy. This senescence-associated secretory phenotype (SASP) developed slowly over several days and only after DNA damage of sufficient magnitude to induce senescence. Remarkably similar SASPs developed in normal fibroblasts, normal epithelial cells, and epithelial tumor cells after genotoxic stress in culture, and in epithelial tumor cells in vivo after treatment of prostate cancer patients with DNA- damaging chemotherapy. In cultured premalignant epithelial cells, SASPs induced an epithelial–mesenchyme transition and invasiveness, hallmarks of malignancy, by a paracrine mechanism that depended largely on the SASP factors interleukin (IL)-6 and IL-8. Strikingly, two manipulations markedly amplified, and accelerated development of, the SASPs: oncogenic RAS expression, which causes genotoxic stress and senescence in normal cells, and functional loss of the p53 tumor suppressor protein. Both loss of p53 and gain of oncogenic RAS also exacerbated the promalignant paracrine activities of the SASPs. Our findings define a central feature of genotoxic stress-induced senescence. Moreover, they suggest a cell-nonautonomous mechanism by which p53 can restrain, and oncogenic RAS can promote, the development of age-related cancer by altering the tissue microenvironment. Citation: Coppe JP, Patil CK, Rodier F, Sun Y, Mun oz DP, et al. (2008) Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biol 6(12): e301. doi:10.1371/journal.pbio.0060301 Introduction Cancer is a multistep disease in which cells acquire increasingly malignant phenotypes. These phenotypes are acquired in part by somatic mutations, which derange normal controls over cell proliferation (growth), survival, invasion, and other processes important for malignant tumorigenesis [1]. In addition, there is increasing evidence that the tissue microenvironment is an important determinant of whether and how malignancies develop [2,3]. Normal tissue environ- ments tend to suppress malignant phenotypes, whereas abnormal tissue environments such at those caused by inflammation can promote cancer progression. Cancer development is restrained by a variety of tumor suppressor genes. Some of these genes permanently arrest the growth of cells at risk for neoplastic transformation, a process termed cellular senescence [4–6]. Two tumor suppressor pathways, controlled by the p53 and p16INK4a/pRB proteins, regulate senescence responses. Both pathways integrate multiple aspects of cellular physiology and direct cell fate towards survival, death, proliferation, or growth arrest, depending on the context [7,8]. Several lines of evidence indicate that cellular senescence is a potent tumor-suppressive mechanism [4,9,10]. Many poten- tially oncogenic stimuli (e.g., dysfunctional telomeres, DNA PLoS Biology | www.plosbiology.org damage, and certain oncogenes) induce senescence [6,11]. Moreover, mutations that dampen the p53 or p16INK4a/pRB pathways confer resistance to senescence and greatly increase cancer risk [12,13]. Most cancers harbor mutations in one or both of these pathways [14,15]. Lastly, in mice and humans, a senescence response to strong mitogenic signals, such as those delivered by certain oncogenes, prevents premalignant lesions from progressing to malignant cancers [16–19]. Academic Editor: Julian Downward, Cancer Research UK, United Kingdom Received June 27, 2008; Accepted October 22, 2008; Published December 2, 2008 Copyright: O 2008 Coppe et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abbreviations: CM, conditioned medium; DDR, DNA damage response; ELISA, enzyme-linked immunosorbent assay; EMT, epithelial–mesenchymal transition; GSE, genetic suppressor element; IL, interleukin; MIT, mitoxantrone; PRE, presenescent; PrEC, normal human prostate epithelial cell; REP, replicative exhaustion; SASP, senescence-associated secretory phenotype; SEN, senescent; shRNA, short hairpin RNA; XRA, X-irradiation * To whom correspondence should be addressed. E-mail: jcampisi@lbl.gov [ These authors contributed equally to this work. ¤ Current address: Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America December 2008 | Volume 6 | Issue 12 | e301