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Kim, Heon Seok,Lee, Kyungjin,Bae, Sangsu,Park, Jeongbin,Lee, Chong-Kyo,Kim, Meehyein,Kim, Eunji,Kim, Minju,Kim, Seokjoong,Kim, Chonsaeng,Kim, Jin-Soo American Society for Biochemistry and Molecular Bi 2017 The Journal of biological chemistry Vol.292 No.25
<P>Several groups have used genome-wide libraries of lentiviruses encoding small guide RNAs (sgRNAs) for genetic screens. In most cases, sgRNA expression cassettes are integrated into cells by using lentiviruses, and target genes are statistically estimated by the readout of sgRNA sequences after targeted sequencing. We present a new virus-free method for human gene knockout screens using a genome-wide library of CRISPR/Cas9 sgRNAs based on plasmids and target gene identification via whole-genome sequencing (WGS) confirmation of authentic mutations rather than statistical estimation through targeted amplicon sequencing. We used 30,840 pairs of individually synthesized oligonucleotides to construct the genome-scale sgRNA library, collectively targeting 10,280 human genes (<I>i.e.</I> three sgRNAs per gene). These plasmid libraries were co-transfected with a Cas9-expression plasmid into human cells, which were then treated with cytotoxic drugs or viruses. Only cells lacking key factors essential for cytotoxic drug metabolism or viral infection were able to survive. Genomic DNA isolated from cells that survived these challenges was subjected to WGS to directly identify CRISPR/Cas9-mediated causal mutations essential for cell survival. With this approach, we were able to identify known and novel genes essential for viral infection in human cells. We propose that genome-wide sgRNA screens based on plasmids coupled with WGS are powerful tools for forward genetics studies and drug target discovery.</P>
Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells
Kim, Daesik,Bae, Sangsu,Park, Jeongbin,Kim, Eunji,Kim, Seokjoong,Yu, Hye Ryeong,Hwang, Jinha,Kim, Jong-Il,Kim, Jin-Soo Nature Publishing Group, a division of Macmillan P 2015 NATURE METHODS Vol.12 No.3
Although RNA-guided genome editing via the CRISPR-Cas9 system is now widely used in biomedical research, genome-wide target specificities of Cas9 nucleases remain controversial. Here we present Digenome-seq, in vitro Cas9-digested whole-genome sequencing, to profile genome-wide Cas9 off-target effects in human cells. This in vitro digest yields sequence reads with the same 5′ ends at cleavage sites that can be computationally identified. We validated off-target sites at which insertions or deletions were induced with frequencies below 0.1%, near the detection limit of targeted deep sequencing. We also showed that Cas9 nucleases can be highly specific, inducing off-target mutations at merely several, rather than thousands of, sites in the entire genome and that Cas9 off-target effects can be avoided by replacing 'promiscuous' single guide RNAs (sgRNAs) with modified sgRNAs. Digenome-seq is a robust, sensitive, unbiased and cost-effective method for profiling genome-wide off-target effects of programmable nucleases including Cas9.
Application of Genome Editing Nucleases to Crop Breeding
Geung-Joo Lee,Heon-Joong Kim,Seokjoong Kim,Gabbin Wee,Jin-Soo Kim 한국육종학회 2012 한국육종학회 심포지엄 Vol.2012 No.07
Reliable and precise techniques for targeting modification of plant genomes have been explored in plant breeding communities. Initiated in the animal genome first, now the genome editing tool using a nuclease has been reported in some plant species including Arabidopsis, Maize, Tobacco, and other model systems. When the artificial nuclease is introduced into a plant cell and breaks the genomic sites randomly, endogenously operating DNA-repair mechanisms including non-homologous end joining(NHEJ) or homologous recombination(HR) are anticipated, leading to insertion of foreign DNA or deletion of the target locus, which collectively allows changes in plant traits of interest. Traditionally custom designed for induction of double-strand DNA break(DSB) at a predetermined locus was based on zinc-finger nuclease which contains nonspecific cleavage domains with target specificities of DNA binding zinc finger domains(three to four). The binding domains containing more than 20 DNA bases with high affinity to the target gene enable recognition of the locus efficiently. From this project, we focus on a petunia chalcone synthase(CHS) as a model system. The engineered nuclease will target the CHS gene, which is expected to be modified either constitutely or transiently. The derived transformed plants will be genetically or phenotypicly screened, along with molecular confirmation analysis by using various tools. We eventually extend the tools to various crop species and target genes, which makes the brand-new breeding technique more reliable and robust.
Generation of interleukin 2 receptor gamma (IL2Rg) knockout rats with TALEN-mediated gene targeting
Tae-Shin Park,Jung Hwan Hwang,Yong-Hoon Kim,Jung-Ran Noh,Dong-Hee Choi,In-Bok Lee,Yun Jeong Seo,Kyoung-Shim Kim,Doo-Jin Kim,Seokjoong Kim,Jin-Soo Kim,Chul-Ho Lee 한국실험동물학회 2015 한국실험동물학회 학술발표대회 논문집 Vol.2015 No.2
Seokjoong Kim,Naeun Zang,Juho Kim 대한전자공학회 2007 ITC-CSCC :International Technical Conference on Ci Vol.2007 No.7
In this paper, we propose an accurate and efficient statistical leakage power analysis methodology that considers process variance in chip fabrication. The proposed methodology utilizes probability density function and guarantees generality and efficiency by simplifying the algebraic sum of each component in estimation of the total current leakage. The proposed leakage current analysis method uses MOFET's BSIM3 to verify and compare with Monte-Carlo simulation method. The method used in this paper resulted in improvement in accuracy of leakage current estimation and led to a 4.53% improvement.