Development of an efficient and precise adenine base editor (ABE) with expanded target range in allotetraploid cotton (Gossypium hirsutum).
Development of an efficient and precise adenine base editor (ABE) with expanded target range in allotetraploid cotton (Gossypium hirsutum).
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DOI：
10.1186/s12915-022-01232-3
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发表时间：
2022-02-15
期刊：
影响因子：
5.4
通讯作者：
Jin S
中科院分区：
文献类型：
--
作者：
Wang G;Xu Z;Wang F;Huang Y;Xin Y;Liang S;Li B;Si H;Sun L;Wang Q;Ding X;Zhu X;Chen L;Yu L;Lindsey K;Zhang X;Jin S
碱基编辑器（BEs）在拟南芥、水稻、小麦、玉米、大豆和棉花等多种植物物种中显示出不同的应用，它们被用来介导精确的碱基对转换，而不会产生不良的双链断裂（DSB）。支持植物性状的单核苷酸多态性（snp）的研究仍然具有挑战性，特别是在多倍体物种中，这些snp存在于多个拷贝中，并且需要同时修改所有等位基因以进行功能分析。同种异体四倍体棉花在a和D亚基因组中存在大量同源基因对，且具有相当大的snp，因此需要开发腺嘌呤碱基编辑器（ABEs），在如此复杂的基因组中进行高效、精确的a -to- g单碱基编辑，而不需要DSB。我们基于不同的工程腺苷脱氨酶（TadA）蛋白与Cas9变异体（dCas9, nCas9）融合建立了多种ABE载体，使异源四倍体棉花基因组靶位点上的A到G编辑效率高达64%。综合分析显示，GhABE7.10n的编辑效率最高，主要编辑位点位于A5位点（将PAM计算为21-23位点）。利用全基因组和全转录组测序对GhABE7.10n和GhABE7.10d编辑的棉花植株进行DNA和RNA脱靶分析，未发现DNA脱靶突变，而RNA脱靶突变非常低。首次开发了识别富t PAM的新碱基编辑器GhABE7.10dCpf1 （7.10TadA + dCpf1）。靶向a -to- g取代导致棉花磷脂酰乙醇胺结合蛋白（GhPEBP）中单个氨基酸发生变化，导致紧凑的棉花植株结构，这是现代棉花生产机械化收获的理想模式。我们的数据说明了腺嘌呤碱基编辑在具有复杂基因组的植物物种中的稳健性，为棉花功能基因组学和精确分子育种提供了高效和精确的工具包。在线版本包含补充材料，可在10.1186/s12915-022-01232-3获得。
Base editors (BEs) display diverse applications in a variety of plant species such as Arabidopsis, rice, wheat, maize, soybean, and cotton, where they have been used to mediate precise base pair conversions without the collateral generation of undesirable double-stranded breaks (DSB). Studies of single-nucleotide polymorphisms (SNPs) underpinning plant traits are still challenging, particularly in polyploidy species where such SNPs are present in multiple copies, and simultaneous modification of all alleles would be required for functional analysis. Allotetraploid cotton has a number of homoeologous gene pairs located in the A and D sub-genomes with considerable SNPs, and it is desirable to develop adenine base editors (ABEs) for efficient and precise A-to-G single-base editing without DSB in such complex genome. We established various ABE vectors based on different engineered adenosine deaminase (TadA) proteins fused to Cas9 variants (dCas9, nCas9), enabling efficient A to G editing up to 64% efficiency on-target sites of the allotetraploid cotton genome. Comprehensive analysis showed that GhABE7.10n exhibited the highest editing efficiency, with the main editing sites specifically located at the position A5 (counting the PAM as positions 21–23). Furthermore, DNA and RNA off-target analysis of cotton plants edited with GhABE7.10n and GhABE7.10d by whole genome and whole-transcriptome sequencing revealed no DNA off-target mutations, while very low-level RNA off-target mutations were detected. A new base editor, namely GhABE7.10dCpf1 (7.10TadA + dCpf1), that recognizes a T-rich PAM, was developed for the first time. Targeted A-to-G substitutions generated a single amino acid change in the cotton phosphatidyl ethanolamine-binding protein (GhPEBP), leading to a compact cotton plant architecture, an ideotype for mechanized harvesting of modern cotton production. Our data illustrate the robustness of adenine base editing in plant species with complex genomes, which provides efficient and precise toolkit for cotton functional genomics and precise molecular breeding. The online version contains supplementary material available at 10.1186/s12915-022-01232-3.
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影响因子：
13.8
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Li B;Liang S;Alariqi M;Wang F;Wang G;Wang Q;Xu Z;Yu L;Naeem Zafar M;Sun L;Si H;Yuan D;Guo W;Wang Y;Lindsey K;Zhang X;Jin S
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