Nature Protocols
Abstract
Base editors can achieve targeted genomic base conversion. However, the off-target issue is one of the major concerns in their application. Whole-genome sequencing (WGS) at the individual level can provide direct information on genome-wide specificity, but it is difficult to distinguish true off-target single-nucleotide variants (SNVs) induced by base editors from background variation. Here we describe an unbiased WGS method for evaluating the specificity of base editors in rice. In this protocol, we describe the experimental design and provide details of vector construction, rice transformation and tissue culture, as well as a comprehensive WGS data analysis pipeline for overcoming two related core problems in various plant species: high background mutation rates and the heterogeneity of examined populations. Using this protocol, researchers can straightforwardly and accurately assess the genome-wide specificity of base editors and other genome editing tools in 12–15 weeks.
| 论文编号: | DOI:10.1038/s41596-020-00423-y |
| 论文题目: | An Unbiased Method for Evaluating the Genome-Wide Specificity of Base Editors in Rice |
| 英文论文题目: | An Unbiased Method for Evaluating the Genome-Wide Specificity of Base Editors in Rice |
| 第一作者: | Shuai Jin, Qiang Gao & Caixia Gao |
| 英文第一作者: | Shuai Jin, Qiang Gao & Caixia Gao |
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| 发表年度: | 2020-12-22 |
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| 摘要: | Base editors can achieve targeted genomic base conversion. However, the off-target issue is one of the major concerns in their application. Whole-genome sequencing (WGS) at the individual level can provide direct information on genome-wide specificity, but it is difficult to distinguish true off-target single-nucleotide variants (SNVs) induced by base editors from background variation. Here we describe an unbiased WGS method for evaluating the specificity of base editors in rice. In this protocol, we describe the experimental design and provide details of vector construction, rice transformation and tissue culture, as well as a comprehensive WGS data analysis pipeline for overcoming two related core problems in various plant species: high background mutation rates and the heterogeneity of examined populations. Using this protocol, researchers can straightforwardly and accurately assess the genome-wide specificity of base editors and other genome editing tools in 12–15 weeks. |
| 英文摘要: | Base editors can achieve targeted genomic base conversion. However, the off-target issue is one of the major concerns in their application. Whole-genome sequencing (WGS) at the individual level can provide direct information on genome-wide specificity, but it is difficult to distinguish true off-target single-nucleotide variants (SNVs) induced by base editors from background variation. Here we describe an unbiased WGS method for evaluating the specificity of base editors in rice. In this protocol, we describe the experimental design and provide details of vector construction, rice transformation and tissue culture, as well as a comprehensive WGS data analysis pipeline for overcoming two related core problems in various plant species: high background mutation rates and the heterogeneity of examined populations. Using this protocol, researchers can straightforwardly and accurately assess the genome-wide specificity of base editors and other genome editing tools in 12–15 weeks. |
| 刊物名称: | Nature Protocols |
| 英文刊物名称: | Nature Protocols |
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| 其它备注: | Shuai Jin, Qiang Gao & Caixia Gao. An Unbiased Method for Evaluating the Genome-Wide Specificity of Base Editors in Rice. Nature Protocols. DOI:10.1038/s41596-020-00423-y |
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