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.

Genome surgery using Cas9 ribonucleoproteins for the treatment of age-related macular degeneration

Kim, Kyoungmi,Park, Sung Wook,Kim, Jin Hyoung,Lee, Seung Hwan,Kim, Daesik,Koo, Taeyoung,Kim, Kwang-eun,Kim, Jeong Hun,Kim, Jin-Soo Cold Spring Harbor Laboratory 2017 Genome research Vol.27 No.3

<P>RNA-guided genome surgery using CRISPR-Cas9 nucleases has shown promise for the treatment of diverse genetic diseases. Yet, the potential of such nucleases for therapeutic applications in nongenetic diseases is largely unexplored. Here, we focus on age-related macular degeneration (AMD), a leading cause of blindness in adults, which is associated with retinal overexpression of, rather than mutations in, the VEGFA gene. Subretinal injection of preassembled, Vegfa gene-specific Cas9 ribonucleoproteins (RNPs) into the adult mouse eye gave rise to mutagenesis at the target site in the retinal pigment epithelium. Furthermore, Cas9 RNPs effectively reduced the area of laser-induced choroidal neovascularization (CNV) in a mouse model of AMD. Genome-wide profiling of Cas9 off-target effects via Digenome-seq showed that off-target mutations were rarely induced in the human genome. Because Cas9 RNPs can function immediately after in vivo delivery and are rapidly degraded by endogenous proteases, their activities are unlikely to be hampered by antibody-and cell-mediated adaptive immune systems. Our results demonstrate that in vivo genome editing with Cas9 RNPs has the potential for the local treatment for nongenetic degenerative diseases, expanding the scope of RNA-guided genome surgery to a new dimension.</P>

Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins

Kim, Sojung,Kim, Daesik,Cho, Seung Woo,Kim, Jungeun,Kim, Jin-Soo Cold Spring Harbor Laboratory Press 2014 Genome Research Vol.24 No.6

<P>RNA-guided engineered nucleases (RGENs) derived from the prokaryotic adaptive immune system known as CRISPR (clustered, regularly interspaced, short palindromic repeat)/Cas (CRISPR-associated) enable genome editing in human cell lines, animals, and plants, but are limited by off-target effects and unwanted integration of DNA segments derived from plasmids encoding Cas9 and guide RNA at both on-target and off-target sites in the genome. Here, we deliver purified recombinant Cas9 protein and guide RNA into cultured human cells including hard-to-transfect fibroblasts and pluripotent stem cells. RGEN ribonucleoproteins (RNPs) induce site-specific mutations at frequencies of up to 79%, while reducing off-target mutations associated with plasmid transfection at off-target sites that differ by one or two nucleotides from on-target sites. RGEN RNPs cleave chromosomal DNA almost immediately after delivery and are degraded rapidly in cells, reducing off-target effects. Furthermore, RNP delivery is less stressful to human embryonic stem cells, producing at least twofold more colonies than does plasmid transfection.</P>

Genome-wide target specificities of CRISPR RNA-guided programmable deaminases

Kim, Daesik,Lim, Kayeong,Kim, Sang-Tae,Yoon, Sun-heui,Kim, Kyoungmi,Ryu, Seuk-Min,Kim, Jin-Soo Nature Publishing Group, a division of Macmillan P 2017 Nature biotechnology Vol.35 No.5

<P>Cas9-linked deaminases, also called base editors, enable targeted mutation of single nucleotides in eukaryotic genomes. However, their off-target activity is largely unknown. Here we modify digested-genome sequencing (Digenome-seq) to assess the specificity of a programmable deaminase composed of a Cas9 nickase (nCas9) and the deaminase APOBEC1 in the human genome. Genomic DNA is treated with the base editor and a mixture of DNA-modifying enzymes in vitro to produce DNA double-strand breaks (DSBs) at uracil-containing sites. Off-target sites are then computationally identified from whole genome sequencing data. Testing seven different single guide RNAs (sgRNAs), we find that the rAPOBEC1-nCas9 base editor is highly specific, inducing cytosine-to-uracil conversions at only 18 +/- 9 sites in the human genome for each sgRNA. Digenome-seq is sensitive enough to capture off-target sites with a substitution frequency of 0.1%. Notably, off-target sites of the base editors are often different from those of Cas9 alone, calling for independent assessment of their genome-wide specificities.</P>

Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq

Kim, Daesik,Kim, Sojung,Kim, Sunghyun,Park, Jeongbin,Kim, Jin-Soo Cold Spring Harbor Laboratory Press 2016 Genome Research Vol.26 No.3

<P>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.</P>

Genome-wide target specificity of CRISPR RNA-guided adenine base editors

Kim, Daesik,Kim, Da-eun,Lee, Gyeorae,Cho, Sung-Ik,Kim, Jin-Soo Springer Science and Business Media LLC 2019 Nature biotechnology Vol.37 No.4

Highly efficient RNA-guided base editing in mouse embryos

Kim, Kyoungmi,Ryu, Seuk-Min,Kim, Sang-Tae,Baek, Gayoung,Kim, Daesik,Lim, Kayeong,Chung, Eugene,Kim, Sunghyun,Kim, Jin-Soo Nature Publishing Group, a division of Macmillan P 2017 Nature biotechnology Vol.35 No.5

<P>Base editors (BEs) composed of a cytidine deaminase fused to CRISPR-Cas9 convert cytidine to uridine, leading to single-base-pair substitutions in eukaryotic cells. We delivered BE mRNA or ribonucleoproteins targeting the Dmd or Tyr gene via electroporation or microinjection into mouse zygotes. F0 mice showed nonsense mutations with an efficiency of 44-57% and allelic frequencies of up to 100%, demonstrating an efficient method to generate mice with targeted point mutations.</P>

Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells

Kim, Daesik,Kim, Jungeun,Hur, Junho K,Been, Kyung Wook,Yoon, Sun-heui,Kim, Jin-Soo Nature Publishing Group, a division of Macmillan P 2016 Nature biotechnology Vol.34 No.8

<P>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.</P>

이종(異種) 차량간 운행시험 방안 연구

김대식(Daesik Kim),김주원(Juwon Kim),소진섭(JinSub So),김진우(Jinwoo Kim),정형일(Hyeongil Jeong),이성준(Seoungjun Lee),김용한(Yonghan Kim) 한국철도학회 2014 한국철도학회 학술발표대회논문집 Vol.2014 No.5

철도운영의 효율화와 최적의 유지보수를 위해서는 KTK, KTX-산천, 전동차와 같이 고정 편성으로 동일한 차종으로 구성하여 운행하는 것이 가장 효율적이며, 이런 이유로 국내는 물론 세계적으로 동종의 차량으로 열차 운행 다이아를 설정하고 있으며, 화물열차도 최대한 동일 사양으로 묶어 운행을 하고 있다. 본 연구는 국가R&D 사업으로 개발하고 있는 2층 고속열차, 고속화물열차 및 여객/화물 복합열차의 종합편성시험을 위한 안전 및 인터페이스 측면에서 고려되어야 할 사항에 대하여 연구하고 분석하였다. In light of operational efficiency and maintenance effectiveness, it is the most effective way that a train is composed of same cars such as KTX, KTX-Sancheon and Electric Multiple Unit. Therefore, world trend of a train formation is toward the same kind of cars’ and even a freight train often run with the same kind of cars. This study is aimed at analysis of essential elements about safety and interface of a developing high speed train which is composed of duplex passenger cars and passenger/freight complex cars.

시군관리 저수지의 물관리 정보화를 위한 시스템 분석

김대식 ( Daesik Kim ),김진택 ( Jintaek Kim ),김정대 ( Jeongdae Kim ),구희동,임혜지 한국농공학회 2015 한국농공학회 학술대회초록집 Vol.2015 No.-

한국농어촌공사가 사용하고 있는 물관리 관련 정보시스템은 RIMS, RAWRIS 등 다양하게 운영되고 있으며, 한국농어촌공사 외부 시스템들도 수자원공사의 WAMIS 등과 같이 다양한 부처에서 각각의 시스템들로 운영되고 있다. 시군관리 소규모 저수지는 전체적으로 수자원의 비율은 12% 정도에 이르지만, 14,200개에 이르는 전국적인 분포와 대부분 소유역의 주요 위치에 입지하고 있으므로 장기적으로 농업용 수자원의 관리와 수자원의 추가 확보 측면에서 매우 중요하다. 물관리 측면에서 한국농어촌공사와 시군관리 저수지를 통합한 정보화가 절실하다. 본 연구에서는 첫째, 시군관리 저수지를 포함한 물관리통합정보시스템을 구성하기 위하여 체계적인 분석을 실시하였으며, 둘째, 시군관리 저수지의 정보화를 위한 개선방안을 제시하였다. 첫째, 물관리통합정보시스템의 구성 체계를 설계하고 개념과 해결과제를 6단계의 모듈로 체계화하여 제시하였다. 물관리통합정보시스템 6단계 모듈은 ① 지능형통합정보시스템 구성요소설계, ② 기존 시스템 활용 내용 도출, ③ 신규개발 요소 설계, ④ 지능형 시군저수지 정보시스템 설계, ⑤ 시군 위험저수지 지정 및 정보화 방안, ⑥ 기존시스템 연결 자료/정보공유 체계 및 제도 마련 등이다. 둘째, 시군관리 저수지의 정보화를 위한 개선방안도 저수지 현장에서 중앙 정보시스템에 이르는 6단계의 오브젝트로 구분하여 현재실태를 현장조사를 통하여 분석하고 개선사항을 체계적으로 제시하였다. 시군관리 저수지 정보화 6단계 오브젝트는 ① 저수지(유역, 시설, 수위내용적곡선, 몽리구역), ② 저수지 현장관리조직, ③ 지자체 행정조직, ④ 중앙조직, ⑤ 정보전달네트워크, ⑥ 통합정보시스템 등이다. 시군관리저수지는 시설관리와 물관리측면에서 매우 중요하지만 예산문제로 지자체에서 최소한의 예산으로 관리하고 있는데, 충분한 예산확보를 통하여 시설관리 및 이치수 측면의 물관리를 통합한 정보시스템과 전문기관에서 관리할 수 있는 조직이 마련되어야 한다.