Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos

Published on Feb 28, 2019in Science41.063
· DOI :10.1126/science.aav9973
Erwei Zuo7
Estimated H-index: 7
(CAS: Chinese Academy of Sciences),
Yidi Sun8
Estimated H-index: 8
(CAS-MPG Partner Institute for Computational Biology)
+ 7 AuthorsHui Yang19
Estimated H-index: 19
(CAS: Chinese Academy of Sciences)
Genome editing holds promise for correcting pathogenic mutations. However, it is difficult to determine off-target effects of editing due to single nucleotide polymorphism in individuals. Here, we developed a method named GOTI (Genome-wide Off-target analysis by Two-cell embryo Injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. Comparison of the whole genome sequences of progeny cells of edited vs. non-edited blastomeres at E14.5 showed that off-target single nucleotide variants (SNVs) were rare in embryos edited by CRISPR-Cas9 or adenine base editor, with a frequency close to the spontaneous mutation rate. In contrast, cytosine base editing induced SNVs with over 20-fold higher frequencies, requiring a solution to address its fidelity.
  • References (33)
  • Citations (66)
📖 Papers frequently viewed together
5 Authors (Alexis C. Komor, ..., David R. Liu)
866 Citations
457 Citations
11 Authors (Shuai Jin, ..., Caixia Gao)
55 Citations
78% of Scinapse members use related papers. After signing in, all features are FREE.
#1Shuai Jin (CAS: Chinese Academy of Sciences)H-Index: 4
#2Yuan Zong (CAS: Chinese Academy of Sciences)H-Index: 10
Last. Caixia Gao (CAS: Chinese Academy of Sciences)H-Index: 34
view all 11 authors...
Cytosine and adenine base editors (CBEs and ABEs) are promising new tools for achieving the precise genetic changes required for disease treatment and trait improvement. However, genome-wide and unbiased analyses of their off-target effects in vivo are still lacking. Our whole genome sequencing (WGS) analysis of rice plants treated with BE3, high-fidelity BE3 (HF1-BE3), or ABE revealed that BE3 and HF1-BE3, but not ABE, induce substantial genome-wide off-target mutations, which are mostly the C→...
55 CitationsSource
#1Cicera R. Lazzarotto (St. Jude Children's Research Hospital)H-Index: 3
#2Nhu T. Nguyen (Harvard University)H-Index: 8
Last. Shengdar Tsai (St. Jude Children's Research Hospital)H-Index: 3
view all 8 authors...
Circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq) is a sensitive and unbiased method for defining the genome-wide activity (on-target and off-target) of CRISPR–Cas9 nucleases by selective sequencing of nuclease-cleaved genomic DNA (gDNA). Here, we describe a detailed experimental and analytical protocol for CIRCLE-seq. The principle of our method is to generate a library of circularized gDNA with minimized numbers of free ends. Highly purified gDNA circles are...
10 CitationsSource
#1Holly A. Rees (Broad Institute)H-Index: 12
#2David R. Liu (Broad Institute)H-Index: 81
RNA-guided programmable nucleases from CRISPR systems generate precise breaks in DNA or RNA at specified positions. In cells, this activity can lead to changes in DNA sequence or RNA transcript abundance. Base editing is a newer genome-editing approach that uses components from CRISPR systems together with other enzymes to directly install point mutations into cellular DNA or RNA without making double-stranded DNA breaks. DNA base editors comprise a catalytically disabled nuclease fused to a nuc...
75 CitationsSource
#1Gavin J. Knott (University of California, Berkeley)H-Index: 11
#2Jennifer A. DoudnaH-Index: 101
The diversity, modularity, and efficacy of CRISPR-Cas systems are driving a biotechnological revolution. RNA-guided Cas enzymes have been adopted as tools to manipulate the genomes of cultured cells, animals, and plants, accelerating the pace of fundamental research and enabling clinical and agricultural breakthroughs. We describe the basic mechanisms that set the CRISPR-Cas toolkit apart from other programmable gene-editing technologies, highlighting the diverse and naturally evolved systems no...
125 CitationsSource
#1Xiao WangH-Index: 2
#2Jianan LiH-Index: 8
Last. Li YangH-Index: 38
view all 10 authors...
Increased efficiency of base editing in methylated DNA using human APOBEC3A as a deaminase.
31 CitationsSource
#1Jason M. GehrkeH-Index: 3
Last. J. Keith JoungH-Index: 68
view all 8 authors...
The precision of base editing is enhanced with an engineered version of the APOBEC3A deaminase.
59 CitationsSource
#1Keith R. Anderson (Genentech)H-Index: 5
#2Maximilian Haeussler (UCSC: University of California, Santa Cruz)H-Index: 26
Last. Søren Warming (Genentech)H-Index: 28
view all 22 authors...
Despite widespread use of CRISPR, comprehensive data on the frequency and impact of Cas9-mediated off-targets in modified rodents are limited. Here we present deep-sequencing data from 81 genome-editing projects on mouse and rat genomes at 1,423 predicted off-target sites, 32 of which were confirmed, and show that high-fidelity Cas9 versions reduced off-target mutation rates in vivo. Using whole-genome sequencing data from ten mouse embryos, treated with a single guide RNA (sgRNA), and from thei...
43 CitationsSource
#1Erwei Zuo (CAS: Chinese Academy of Sciences)H-Index: 7
#2Xiaona Huo (CAS: Chinese Academy of Sciences)H-Index: 2
Last. Hui Yang (CAS: Chinese Academy of Sciences)H-Index: 19
view all 18 authors...
The CRISPR/Cas9 system has become an efficient gene editing method for generating cells carrying precise gene mutations, including the rearrangement and deletion of chromosomal segments. However, whether an entire chromosome could be eliminated by this technology is still unknown. Here we demonstrate the use of the CRISPR/Cas9 system to eliminate targeted chromosomes. Using either multiple cleavages induced by a single-guide RNA (sgRNA) that targets multiple chromosome-specific sites or a cockta...
34 CitationsSource
#1Nicole M. GaudelliH-Index: 7
#2Alexis C. KomorH-Index: 13
Last. David R. LiuH-Index: 81
view all 7 authors...
A new DNA ‘base editor’ can change targeted A•T base pairs to G•C, allowing disease-associated mutations to be corrected and disease-suppressing mutations to be introduced into cells.
457 CitationsSource
#1Erwei Zuo (CAS: Chinese Academy of Sciences)H-Index: 7
#2Yijun Cai (CAS: Chinese Academy of Sciences)H-Index: 9
Last. Hui Yang (CAS: Chinese Academy of Sciences)H-Index: 19
view all 29 authors...
One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs
47 CitationsSource
Cited By66
#1Yawei Zhao (SHNU: Shanghai Normal University)H-Index: 1
#2Jinzhong Tian (CAS: Chinese Academy of Sciences)H-Index: 3
Last. Yinhua Lu (SHNU: Shanghai Normal University)H-Index: 5
view all 11 authors...
CRISPR/Cas-mediated genome editing has greatly facilitated the study of gene function in Streptomyces. However, it could not be efficiently employed in streptomycetes with low homologous recombination (HR) ability. Here, a deaminase-assisted base editor dCas9-CDA-ULstr was developed in Streptomyces, which comprises the nuclease-deficient Cas9 (dCas9), the cytidine deaminase from Petromyzon marinus (PmCDA1), the uracil DNA glycosylase inhibitor (UGI) and the protein degradation tag (LVA tag). Usi...
1 CitationsSource
#1Gue-Ho Hwang (Hanyang University)H-Index: 3
#2Jihyeon Yu (Hanyang University)H-Index: 7
Last. Sangsu Bae (Hanyang University)H-Index: 19
view all 8 authors...
Abstract CRISPR-Cas9 induces DNA cleavages at desired target sites in a guide RNA-dependent manner; DNA editing occurs through the resulting activity of DNA repair processes including non-homologous end joining (NHEJ), which is dominant in mammalian cells. NHEJ repair frequently causes small insertions and deletions (indels) near DNA cleavage sites but only rarely causes nucleotide substitutions. High-throughput sequencing is the primary means of assessing indel and substitution frequencies in b...
#1Andrew V. Anzalone (Broad Institute)H-Index: 2
#2Luke W. Koblan (Broad Institute)H-Index: 6
Last. David R. Liu (Broad Institute)H-Index: 81
view all 3 authors...
The development of new CRISPR–Cas genome editing tools continues to drive major advances in the life sciences. Four classes of CRISPR–Cas-derived genome editing agents—nucleases, base editors, transposases/recombinases and prime editors—are currently available for modifying genomes in experimental systems. Some of these agents have also moved rapidly into the clinic. Each tool comes with its own capabilities and limitations, and major efforts have broadened their editing capabilities, expanded t...
#1Yuxi Chen (SYSU: Sun Yat-sen University)H-Index: 7
#2Shengyao Zhi (SYSU: Sun Yat-sen University)H-Index: 2
Last. Yizhi Liu (SYSU: Sun Yat-sen University)H-Index: 29
view all 12 authors...
#1Chao Li (CAS: Chinese Academy of Sciences)H-Index: 4
#2Yuan Zong (CAS: Chinese Academy of Sciences)H-Index: 10
Last. Caixia Gao (CAS: Chinese Academy of Sciences)H-Index: 34
view all 9 authors...
We describe here a CRISPR simultaneous and wide-editing induced by a single system (SWISS), in which RNA aptamers engineered in crRNA scaffold recruit their cognate binding proteins fused with cytidine deaminase and adenosine deaminase to Cas9 nickase target sites to generate multiplexed base editing. By using paired sgRNAs, SWISS can produce insertions/deletions in addition to base editing. Rice mutants are generated using the SWISS system with efficiencies of cytosine conversion of 25.5%, aden...
#1Dawei Wang (SJTU: Shanghai Jiao Tong University)H-Index: 1
#2Kang Wang (Shanghai University of Political Science and Law)H-Index: 1
Last. Yujia Cai (SJTU: Shanghai Jiao Tong University)H-Index: 8
view all 3 authors...
After setbacks related to serious adverse events 20 years ago, gene therapy is now coming back to the central stage worldwide. In the past few years, gene therapy has shown astonishing efficacy against genetic diseases and cancers. In history, China carried out the world’s second gene therapy clinical trial in 1991 for hemophilia B and approved the world’s first gene therapy product—Gendicine—in 2003. In recent years, numerous efforts have been made on gene editing. Here, we reviewed the past of...
2 CitationsSource
#1Thomas L. Maurissen (Kyoto University)H-Index: 1
#2Knut Woltjen (Kyoto University)H-Index: 24
Precise gene editing aims at generating single-nucleotide modifications to correct or model human disease. However, precision editing with nucleases such as CRIPSR-Cas9 has seen limited success due to poor efficiency and limited practicality. Here, we establish a fluorescent DNA repair assay in human induced pluripotent stem (iPS) cells to visualize and quantify the frequency of DNA repair outcomes during monoallelic and biallelic targeting. We found that modulating both DNA repair and cell cycl...
#1Napisa Pattharaprachayakul (SKKU: Sungkyunkwan University)H-Index: 1
#2Mieun Lee (SKKU: Sungkyunkwan University)
Last. Han Min Woo (SKKU: Sungkyunkwan University)H-Index: 23
view all 4 authors...
Abstract Cyanobacteria are photosynthetic microorganisms that are capable of converting CO2 to value-added chemicals. Engineering of cyanobacteria with synthetic biology tools, including the CRISPR-Cas system, has allowed an opportunity for biological CO2 utilization. Here, we described natural CRISPR-Cas systems for understanding cyanobacterial genomics and synthetic CRISPR-Cas systems for metabolic engineering applications. The natural CRISPR-Cas systems in cyanobacteria have been identified a...
#1Yu-Chan Yang (NTU: National Taiwan University)H-Index: 1
#2Yu-Hsiang Chen (NTU: National Taiwan University)H-Index: 1
Last. Pei-JerChenH-Index: 87
view all 12 authors...
Abstract Current antiviral therapy fails to cure chronic hepatitis B virus (HBV) infection because of persistent covalently closed circular (ccc) DNA. CRISPR/Cas9-mediated specific cleavage of cccDNA is a potentially curative strategy for chronic hepatitis B (CHB). However, the CRISPR/Cas system inevitably targets integrated HBV DNA and induces double-strand breaks (DSBs) of host genome, bearing the risk of genomic rearrangement and damage. Herein, we examined the utility of recently developed C...
1 CitationsSource
#1Xiujuan Lv (WMU: Wenzhou Medical College)H-Index: 2
#2Kairui Qiu (WMU: Wenzhou Medical College)H-Index: 1
Last. Jinyu Wu (WMU: Wenzhou Medical College)H-Index: 1
view all 13 authors...
Abstract Base editing is a form of genome editing that can directly convert a single base (C or A) to another base (T or G), which is of great potential in biomedical applications. The broad application of base editing is limited by its low activity and specificity which still needs to be resolved. To address this, a simple and quick method for the determination of its activity/specificity is highly desired. Here, we developed a novel system, which could be harnessed for quick detection of editi...
1 CitationsSource