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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)
Sources
Abstract
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)
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References33
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#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
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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
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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
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Increased efficiency of base editing in methylated DNA using human APOBEC3A as a deaminase.
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The precision of base editing is enhanced with an engineered version of the APOBEC3A deaminase.
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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
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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
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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
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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...
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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...
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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...
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#1Dawei Wang (SJTU: Shanghai Jiao Tong University)H-Index: 1
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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...
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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...
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