Development of an efficient and precise adenine base editor (ABE) with expanded target range in allotetraploid cotton (Gossypium hirsutum)

Guanying Wang,Zhongping Xu,Xiangqian Zhu,Yuefan Huang,Bo Li,Lin Sun,Keith Lindsey,Luo Chen,Sijia Liang,Qiongqiong Wang,Yanfeng Xin,Huan Si,Lu Yu,Xiao Ding,Fuqiu Wang,Xianlong Zhang,Shuangxia Jin

doi:10.1186/s12915-022-01232-3

Abstract

BackgroundBase 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.ResultsWe 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.ConclusionsOur 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.

Highlights

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)
These eight adenine base editors (ABEs), namely GhABE6.3n, GhABE6.3d, GhABE7.8n, GhABE7.8d, GhABE7.9n, GhABE7.9d, GhABE7.10n, and GhABE7.10d, were all codon-optimized based on cotton genomic preference for high expression level in transgenic cotton
A novel dead LbCpf1 (dCpf1) protein was synthesized after codon optimization and fused with adenine deaminase from the GhABE7.10n vector and was designated as GhABE7.10dCpf1 (Fig. 1, Additional file 1: Appendix S1 and Additional file 1: Fig S1)

Summary

Introduction

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). The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is the most powerful and widely adopted gene editing tool for research in life science [1]. This system causes DNA double-stranded breakage (DSB) in a site-specific manner and leads to insertions and deletions (Indels) at the target sites by an endogenous repair mechanism, including high-fidelity homologous recombination (HR) and errorprone non-homologous end joining (NHEJ) repair pathway [2,3,4,5,6]. Base editing based on CRISPR/Cas system is a promising precise point mutation technology without inducing DSBs at the target genomic locus. It normally uses a Cas variant (nCas or dCas9) and cytosine deaminase or adenine deaminase that was evolved artificially to perform precise single-base editing of target sites without DSBs, enabling the replacement of C by T or A by G [2, 9, 10]

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Conclusion