Directed evolution rice genes with randomly multiplexed sgRNAs assembly of base editors

Abstract

SummaryCRISPR‐based directed evolution is an effective breeding biotechnology to improve agronomic traits in plants. However, its gene diversification is still limited using individual single guide RNA. We described here a multiplexed orthogonal base editor (MoBE), and a randomly multiplexed sgRNAs assembly strategy to maximize gene diversification. MoBE could induce efficiently orthogonal ABE (<36.6%), CBE (<36.0%), and A&CBE (<37.6%) on different targets, while the sgRNA assembling strategy randomized base editing events on various targets. With respective 130 and 84 targets from each strand of the 34th exon of rice acetyl‐coenzyme A carboxylase (OsACC), we observed the target‐scaffold combination types up to 27 294 in randomly dual and randomly triple sgRNA libraries. We further performed directed evolution of OsACC using MoBE and randomly dual sgRNA libraries in rice, and obtained single or linked mutations of stronger herbicide resistance. These strategies are useful for in situ directed evolution of functional genes and may accelerate trait improvement in rice.

High performance TadA-8e derived cytosine and dual base editors with undetectable off-target effects in plants

“…The STEME systems mostly induce C-to-T base editing than simultaneous C-to-T and A-to-G base editing, which could be partly attributed to the use of a low efficiency adenine deaminase 35 . A recent study adopted a similar strategy of the SWISS system and took one more step further to install two distinct RNA aptamers on the sgRNA scaffold for the recruitment of cytidine deaminase and adenine deaminase simultaneously to the same target site 37 . This multiplexed orthogonal base editor (MoBE) system greatly outperformed the STEME system by inducing simultaneous C-to-T and A-to-G mutations at the same target sites efficiently.…”

“…This suggests that this novel dual base editor is not only simple and efficient but also highly specific. Construction of the MoBE dual base editing system is complicated as it requires the expression of four protein effectors (e.g., two different deaminases and two different RNA binding proteins) to achieve the desired dual base editing 37 . By contrast, the TadDE that we demonstrated here is very simple, only requiring a small engineered TadA-8e variant that is fused to the N-terminus of nCas9.…”

“…The cytosine base editor consists of nCas9 and cytidine deaminase, while the adenine base editor comprises nCas9 and adenosine deaminase 34 . Fusion of nCas9, cytidine deaminase, and adenosine deaminase allows simultaneous C-to-T and A-to-G editing [35][36][37] . However, these dual-base editors have larger molecular weight, lower editing efficiency, and significant differences in editing windows due to the characteristics of deaminases.…”

“…Dual base editors are powerful tools for targeted saturation mutagenesis and directed evolution in crops 34 . Currently, there is limited exploration of dual base editors in plants, with notable systems including STEME 35 , SIWSS 36 , and MoBE 37 . These systems require the use of both deaminases simultaneously, resulting in larger molecular weights of the base editors which will hinder ribonucleoprotein (RNP) delivery for transgene-free genome editing in plants 38 .…”

A Critical Review of Conventional and Modern Approaches to Develop Herbicide‐Resistance in Rice

“…The utilization of CRISPR‐based gene‐directed evolution represents a powerful breeding technique for improving the agronomic traits of crops. In a recent study by Zhang et al (2023), a multiple orthogonal base editor (MoBE) was employed alongside a randomized assembly strategy for sgRNA to maximize gene editing diversity. Specifically, they designed 130 and 84 targeted editing sites with each strand of the 34th exon of rice OsACCase .…”