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Dual base editors (DBEs) enable simultaneous A-to-G and C-to-T conversions, expanding mutation types. However, low editing efficiency and narrow targeting range limit the widespread use of DBEs in plants. The single-strand DNA binding domain of RAD51 DBD can be fused to base editors to improve their editing efficiency. However, it remains unclear how the DBD affects dual base editing performance in plants. In this study, we generated a series of novel plant DBE-SpGn tools consisting of nine constructs using the high-activity cytidine deaminase evoFERNY, adenosine deaminase TadA8e and DBD in various fusion modes with the PAM-flexible Streptococcus pyogenes Cas9 (SpCas9) nickase variant SpGn (with NG-PAM). By analysing their editing performance on 48 targets in rice, we found that DBE-SpGn constructs containing a single DBD and deaminases located at the N-terminus of SpGn exhibited the highest editing efficiencies. Meanwhile, constructs with deaminases located at the C-terminus and/or multiple DBDs failed to function normally and exhibited inhibited editing activity. We identified three particularly high-efficiency dual base editors (C-A-SpGn, C-A-D-SpGn and A-C-D-SpGn), named PhieDBEs (Plant high-efficiency dual base editors), capable of producing efficient dual base conversions within a narrow editing window (M5 ~ M9, M = A/C). The editing efficiency of C-A-D-SpGn was as high as 95.2% at certain target sites, with frequencies of simultaneous C-to-T and A-to-G conversions as high as 81.0%. In summary, PhieDBEs (especially C-A-D-SpGn) can produce diverse mutants and may prove useful in a wide variety of applications, including plant functional genomics, precise mutagenesis, directed evolution and crop genetic improvement, among others.

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CRISPR-mediated base editors have been widely used to correct defective alleles and create novel alleles by artificial evolution for the rapid genetic improvement of crops. The editing capabilities of base editors strictly rely on the performance of various nucleotide modification enzymes. Compared with the well-developed adenine base editors (ABEs), cytosine base editors (CBEs) and dual base editors suffer from unstable editing efficiency and patterns at different genomic loci in rice, significantly limiting their application. Here, we comprehensively examined the base editing activities of multiple evolved TadA8e variants in rice. We found that both TadA-CDd and TadA-E27R/N46L achieved more robust C-to-T editing than previously reported hyperactive hAID∗Δ, and TadA-CDd outperformed TadA-E27R/N46L. A C-to-G base editor (CGBE) engineered with TadA-CDd and OsUNG performed highly efficient C-to-G editing in rice compared with that of TadA-N46P. In addition, a dual base editor constructed with a single protein, TadDE, enabled simultaneous, highly efficient C-to-T and A-to-G editing in rice. Collectively, our results demonstrate that TadA8e derivatives improve both CBEs and dual base editors in rice, providing a powerful way to induce diverse nucleotide substitutions for plant genome editing.

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CRISPR-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.

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Genome-editing (GE) techniques like base editing are ideal for introducing novel gain-of-function mutations and in situ protein evolution. Features of base editors (BEs) such as higher efficacy, relaxed protospacer adjacent motif (PAM), and a broader editing window enables diversification of user-defined targeted locus. Cytosine (CBE) or adenine (ABE) BEs alone can only alter C-to-T or A-to-G in target sites. In contrast, dual BEs (ACBEs) can concurrently generate C-to-T and A-to-G modifications. Although BE tools have recently been applied in microbes, there is no report of ACBE for microbial GE. In this study, we engineered four improved ACBEs (iACBEs) tethering highly active CBE and ABE variants that can introduce synchronized C-to-T and A-to-G mutations in targeted loci. iACBE4 generated by evoCDA1-ABE9e fusion demonstrated a broader editing window (positions -6 to 15) and is also compatible with the multiplex editing approach in Escherichia coli. We further show that the iACBE4-NG containing PAM-relaxed nCas9-NG expands the targeting scope beyond NGG (N-A/G/C/T) PAM. As a proof-of-concept, iACBE was effectively utilized to identify previously unknown mutations in the rpoB gene, conferring gain-of-function, i.e., rifampicin resistance. The iACBE tool would expand the CRISPR-GE toolkit for microbial genome engineering and synthetic biology. IMPORTANCE Dual base editors are DSB-free CRISPR tools applied in eukaryotes but not yet in bacteria. We developed an improved ACBE toolset for bacteria, combining highly processive deaminases. We believe that the bacterial optimized iACBE toolset is a significant advancement in CRISPR-based E. coli genome editing and adaptable to other microbes.

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VP64 is the smallest transactivation domain that can be packaged together with the sgRNA into a single adeno-associated virus (AAV) vector. However, VP64-based CRISPRa often exerts modest activation to the target gene when only one sgRNA is used. Herein, we used PAM-flexible dual base editor-mediated mutagenesis and self-activation strategies to derive VP64 variants with gain-of-function mutations. First, we generated an HEK293FT transgenic clone to stably expressing pTK-CRISPRa-GFP. The sgRNA of CRISPRa was designed to target the TK promoter, thereby allowing self-activation of CRISPRa-GFP. Base editors were then used to randomly mutagenesis VP64 in this transgenic cell. VP64 with enhanced potency would translate into increment of GFP fluorescence intensity, thereby allowing positive selection of the desired VP64 mutants. This strategy has enabled us to identify several VP64 variants that are more potent than the wild-type VP64. ΔCRISPRa derived from these VP64 variants also efficiently activated the endogenous promoter of anti-aging and longevity genes (KLOTHO, SIRT6, and NFE2L2) in human cells. Since the overall size of these ΔCRISPRa transgenes is not increased, it remains feasible for all-in-one AAV applications. The strategies described here can facilitate high-throughput screening of the desired protein variants and adapted to evolve any other effector domains.

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An enhanced CDA-like (eCDAL) was established from Japanese lamprey CDA1-like 4 to achieve a high editing frequency in a broad region as a C-terminal cytosine base editors (CT-CBE). Then, a novel plant dual-base editor version 1(pDuBE1) was developed by integrating TadA-8e into eCDAL. The editing efficiency of pDuBE1 could reach to 87.6%, with frequencies of concurrent A-to-G and C-to-T conversions as high as 49.7% in stably transformed plant cells. Our results showed that pDuBE1 could mediate robust dual editing in plant genome, providing a powerful manipulation tool for precise crop breeding and screening platforms for in planta direct evolution.

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