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Programmable Base Editing of the Sheep Genome Revealed No Genome-Wide Off-Target Mutations

Published on Mar 15, 2019in Frontiers in Genetics3.517
· DOI :10.3389/fgene.2019.00215
Shiwei Zhou6
Estimated H-index: 6
(NWAFU: Northwest A&F University),
Bei Cai6
Estimated H-index: 6
(NWAFU: Northwest A&F University)
+ 16 AuthorsXiaolong Wang10
Estimated H-index: 10
(NWAFU: Northwest A&F University)
Abstract
Since its emergence, CRISPR/Cas9-mediated base editors (BEs) with cytosine deaminase activity have been used to precisely and efficiently introduce single-base mutations in genomes, including those of human cells, mice, and crop species. Most production traits in livestock are induced by point mutations, and genome editing using BEs without homology-directed repair of double-strand breaks can directly alter single nucleotides. The p.96R>C variant of Suppressor cytokine signaling 2 (SOCS2) has profound effects on body weight, body size, and milk production in sheep. In the present study, we successfully obtained lambs with defined point mutations resulting in a p.96R>C substitution in SOCS2 by the co-injection of Base Editor 3 (BE3) mRNA and a single guide RNA (sgRNA) into sheep zygotes. The observed efficiency of the single nucleotide exchange in newborn animals was as high as 25%. Observations of body size and body weight in the edited group showed that gene modification contributes to enhanced growth traits in sheep. Moreover, targeted deep sequencing and unbiased family trio-based whole genome sequencing revealed undetectable off-target mutations in the edited animals. This study demonstrates the potential for the application of BE-mediated point mutations in large animals for the improvement of production traits in livestock species.
  • References (44)
  • Citations (3)
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References44
Newest
#1Shiwei Zhou (NWAFU: Northwest A&F University)H-Index: 6
#2Honghao Yu (NWAFU: Northwest A&F University)H-Index: 1
Last. Xiaolong Wang (NWAFU: Northwest A&F University)H-Index: 10
view all 16 authors...
Since its emergence, the clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated (Cas) 9 system has been increasingly used to generate animals for economically important traits. However, most CRISPR/Cas9 applications have been focused on non-homologous end joining, which results in base deletions and insertions, leading to a functional knockout of the targeted gene. The Booroola fecundity gene (FecBB) mutation (p.Q249R) in bone morphogenetic protein receptor type 1B (...
3 CitationsSource
#1Chao Li (NWAFU: Northwest A&F University)H-Index: 5
#2Shiwei Zhou (NWAFU: Northwest A&F University)H-Index: 6
Last. Xiaolong Wang (NWAFU: Northwest A&F University)H-Index: 10
view all 14 authors...
Unintended off-target mutations induced by CRISPR/Cas9 nucleases may result in unwanted consequences, which will impede the efficient applicability of this technology for genetic improvement. We have recently edited the goat genome through CRISPR/Cas9 by targeting MSTN and FGF5, which increased muscle fiber diameter and hair fiber length, respectively. Using family trio-based sequencing that allow better discrimination of variant origins, we herein generated offspring from edited goats, and sequ...
9 CitationsSource
#1Michaela Willi (NIH: National Institutes of Health)H-Index: 8
#2Harold E. Smith (NIH: National Institutes of Health)H-Index: 12
Last. Lothar Hennighausen (NIH: National Institutes of Health)H-Index: 89
view all 5 authors...
9 CitationsSource
#1Jason M. GehrkeH-Index: 3
Last. J. Keith JoungH-Index: 62
view all 8 authors...
The precision of base editing is enhanced with an engineered version of the APOBEC3A deaminase.
59 CitationsSource
#1Hiroki Sasaguri (Tokyo Medical and Dental University)H-Index: 6
#2Kenichi NagataH-Index: 9
Last. Takaomi C. SaidoH-Index: 77
view all 10 authors...
Base Editor (BE) and Target-AID (activation-induced cytidine deaminase) are engineered genome-editing proteins composed of Cas9 and cytidine deaminases. These base-editing tools convert C:G base pairs to T:A at target sites. Here, we inject either BE or Target-AID mRNA together with identical single-guide RNAs (sgRNAs) into mouse zygotes, and compare the base-editing efficiencies of the two distinct tools in vivo. BE consistently show higher base-editing efficiency (10.0–62.8%) compared to that ...
9 CitationsSource
#1Vivek IyerH-Index: 24
#2Katharina BoroviakH-Index: 5
Last. David J. AdamsH-Index: 60
view all 7 authors...
CRISPR-Cas9 technologies have transformed genome-editing of experimental organisms and have immense therapeutic potential. Despite significant advances in our understanding of the CRISPR-Cas9 system, concerns remain over the potential for off-target effects. Recent studies have addressed these concerns using whole-genome sequencing (WGS) of gene-edited embryos or animals to search for de novo mutations (DNMs), which may represent candidate changes introduced by poor editing fidelity. Critically,...
26 CitationsSource
#1Guanwei LiH-Index: 3
#2Shiwei ZhouH-Index: 6
Last. Xiaolong WangH-Index: 10
view all 23 authors...
The ability to alter single bases without DNA double strand breaks provides a potential solution for multiplex editing of livestock genomes for quantitative traits. Here, we report using a single base editing system, Base Editor 3 (BE3), to induce nonsense codons (C-to-T transitions) at four target sites in caprine FGF5. All five progenies produced from microinjected single-cell embryos had alleles with a targeted nonsense mutation and yielded expected phenotypes. The effectiveness of BE3 to mak...
1 CitationsSource
#1Luke W. KoblanH-Index: 3
#2Jordan L. DomanH-Index: 3
Last. David R. LiuH-Index: 73
view all 9 authors...
67 CitationsSource
#1Xiaolong WangH-Index: 10
#2Jing LiuH-Index: 4
Last. Yulin ChenH-Index: 15
view all 13 authors...
Background The simplicity of the CRISPR/Cas9 system has enabled its widespread applications in generating animal models, functional genomic screening and in treating genetic and infectious diseases. However, unintended mutations produced by off-target CRISPR/Cas9 nuclease activity may lead to negative consequences. Especially, a very recent study found that gene editing can introduce hundreds of unintended mutations into the genome, and have attracted wide attention. Results To address the off-t...
8 CitationsSource
#1Yiyuan Niu (NWAFU: Northwest A&F University)H-Index: 7
#2Xiaoe Zhao (NWAFU: Northwest A&F University)H-Index: 6
Last. Yulin Chen (NWAFU: Northwest A&F University)H-Index: 15
view all 15 authors...
7 CitationsSource
Cited By3
Newest
#1Peter Girgis Tawfek Kalds (NWAFU: Northwest A&F University)H-Index: 1
#2Yawei Gao (NWAFU: Northwest A&F University)
Last. Yulin Chen (NWAFU: Northwest A&F University)H-Index: 15
view all 7 authors...
Abstract Genetic modification is a rapidly developing field in which numerous significant breakthroughs have been achieved. Over the last few decades, genetic modification has evolved from insertional transgenesis to gene targeting and editing and, more recently, to base and prime editing using CRISPR-derived systems. Currently, CRISPR-based genome editing systems are showing great potential for generating gene-edited offspring with defined genetic characteristics. Domestic small ruminants (shee...
Source
#1Hye-Kyung Lee (NIH: National Institutes of Health)H-Index: 13
#2Harold E. Smith (NIH: National Institutes of Health)H-Index: 12
Last. Lothar Hennighausen (NIH: National Institutes of Health)H-Index: 89
view all 5 authors...
Deaminase base editing has emerged as a tool to install or correct point mutations in the genomes of living cells in a wide range of organisms. However, the genome-wide off-target effects introduced by base editors in the mammalian genome have been examined in only one study. Here, we have investigated the fidelity of cytosine base editor 4 (BE4) and adenine base editors (ABE) in mouse embryos using unbiased whole-genome sequencing of a family-based trio cohort. The same sgRNA was used for BE4 a...
3 CitationsSource
#1Guanwei Li (NWAFU: Northwest A&F University)H-Index: 3
#2Shiwei Zhou (NWAFU: Northwest A&F University)H-Index: 6
Last. Xiaolong Wang (NWAFU: Northwest A&F University)H-Index: 10
view all 23 authors...
The ability to alter single bases without homology directed repair (HDR) of double-strand breaks provides a potential solution for editing livestock genomes for economic traits, which are often multigenic. Progress toward multiplex editing in large animals has been hampered by the costly inefficiencies of HDR via microinjection of in vitro manipulated embryos. Here, we designed sgRNAs to induce nonsense codons (C-to-T transitions) at four target sites in caprine FGF5, which is a crucial regulato...
Source
#1Peter Girgis Tawfek Kalds (NWAFU: Northwest A&F University)H-Index: 1
#2Shiwei Zhou (NWAFU: Northwest A&F University)H-Index: 6
Last. Yulin Chen (NWAFU: Northwest A&F University)H-Index: 15
view all 9 authors...
Sheep and goats are valuable livestock species that have been raised for their production of meat, milk, fiber, and other by-products. Due to their suitable size, short gestation period, and abundant secretion of milk, sheep and goats have become important model animals in agricultural, pharmaceutical, and biomedical research. Genome engineering has been widely applied to sheep and goat research. Pronuclear injection and somatic cell nuclear transfer represent the two primary procedures for the ...
Source
#1Hye-Kyung Lee (NIH: National Institutes of Health)H-Index: 13
#2Harold E. Smith (NIH: National Institutes of Health)H-Index: 12
Last. Lothar Hennighausen (NIH: National Institutes of Health)H-Index: 89
view all 5 authors...
Deaminase base editing has emerged as a tool to install or correct point mutations in the genomes of living cells in a wide range of organisms and its ultimate success therapeutically depends on its accuracy. Here we have investigated the fidelity of cytosine base editor 4 (BE4) and adenine base editor (ABE) in mouse embryos using unbiased whole genome sequencing of a family-based trio cohort. We demonstrate that BE4-edited mice carry an excess of single-nucleotide variants and deletions compare...
Source