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Summary CRISPR‐Cas‐based cytosine base editors (CBEs) are prominent tools that perform site‐specific and precise C‐to‐T conversions catalysed by cytidine deaminases. However, their use is often constrained by stringent editing preferences for genomic contexts, off‐target effects and restricted editing windows. To expand the repertoire of CBEs, we systematically screened 66 novel cytidine deaminases sourced from various organisms, predominantly from the animal kingdom and benchmarked them in rice protoplasts using the nCas9‐BE3 configuration. After selecting candidates in rice protoplasts and further validation in transgenic rice lines, we unveiled a few cytidine deaminases exhibiting high editing efficiencies and wide editing windows. CBEs based on these cytidine deaminases also displayed minimal frequencies of indels and C‐to‐R (R = A/G) conversions, suggesting high purity in C‐to‐T base editing. Furthermore, we highlight the highly efficient cytidine deaminase OoA3GX2 derived from Orca (killer whale) for its comparable activity across GC/CC/TC/AC sites, thus broadening the targeting scope of CBEs for robust multiplexed base editing. Finally, the whole‐genome sequencing analyses revealed very few sgRNA‐dependent and ‐independent off‐target effects in independent T₀lines. This study expands the cytosine base‐editing toolkit with many cytidine deaminases sourced from mammals, providing better‐performing CBEs that can be further leveraged for sophisticated genome engineering strategies in rice and likely in other plant species. 

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (Cas) technologies facilitate routine genome engineering of one or a few genes at a time. However, large-scale CRISPR screens with guide RNA libraries remain challenging in plants. Here, we have developed a comprehensive all-in-one CRISPR toolbox for Cas9-based genome editing, cytosine base editing, adenine base editing (ABE), Cas12a-based genome editing and ABE, and CRISPR-Act3.0-based gene activation in both monocot and dicot plants. We evaluated all-in-one T-DNA expression vectors in rice (Oryza sativa, monocot) and tomato (Solanum lycopersicum, dicot) protoplasts, demonstrating their broad and reliable applicability. To showcase the applications of these vectors in CRISPR screens, we constructed guide RNA (gRNA) pools for testing in rice protoplasts, establishing a high-throughput approach to select high-activity gRNAs. Additionally, we demonstrated the efficacy of sgRNA library screening for targeted mutagenesis of ACETOLACTATE SYNTHASE in rice, recovering novel candidate alleles for herbicide resistance. Furthermore, we carried out a CRISPR activation screen in Arabidopsis thaliana, rapidly identifying potent gRNAs for FLOWERING LOCUS T activation that confer an early-flowering phenotype. This toolbox contains 61 versatile all-in-one vectors encompassing nearly all commonly used CRISPR technologies. It will facilitate large-scale genetic screens for loss-of-function or gain-of-function studies, presenting numerous promising applications in plants. 

Abstract Cytosine base editors (CBEs) and adenine base editors (ABEs) enable precise C-to-T and A-to-G edits. Recently, ABE8e, derived from TadA-8e, enhances A-to-G edits in mammalian cells and plants. Interestingly, TadA-8e can also be evolved to confer C-to-T editing. This study compares engineered CBEs derived from TadA-8e in rice and tomato cells, identifying TadCBEa, TadCBEd, and TadCBEd_V106W as efficient CBEs with high purity and a narrow editing window. A dual base editor, TadDE, promotes simultaneous C-to-T and A-to-G editing. Multiplexed base editing with TadCBEa and TadDE is demonstrated in transgenic rice, with no off-target effects detected by whole genome and transcriptome sequencing, indicating high specificity. Finally, two crop engineering applications using TadDE are shown: introducing herbicide resistance alleles inOsALSand creating synonymous mutations inOsSPL14to resistOsMIR156-mediated degradation. Together, this study presents TadA-8e derived CBEs and a dual base editor as valuable additions to the plant editing toolbox. 

Abstract Adenine base editors (ABEs) are valuable, precise genome editing tools in plants. In recent years, the highly promising ADENINE BASE EDITOR8e (ABE8e) was reported for efficient A-to-G editing. However, compared to monocots, comprehensive off-target analyses for ABE8e are lacking in dicots. To determine the occurrence of off-target effects in tomato (Solanum lycopersicum), we assessed ABE8e and a high-fidelity version, ABE8e-HF, at 2 independent target sites in protoplasts, as well as stable T0 lines. Since ABE8e demonstrated higher on-target efficiency than ABE8e-HF in tomato protoplasts, we focused on ABE8e for off-target analyses in T0 lines. We conducted whole-genome sequencing (WGS) of wild-type (WT) tomato plants, green fluorescent protein (GFP)–expressing T0 lines, ABE8e-no-gRNA control T0 lines, and edited T0 lines. No guide RNA (gRNA)–dependent off-target edits were detected. Our data showed an average of approximately 1,200 to 1,500 single-nucleotide variations (SNVs) in either GFP control plants or base-edited plants. Also, no specific enrichment of A-to-G mutations were found in base-edited plants. We also conducted RNA sequencing (RNA-seq) of the same 6 base-edited and 3 GFP control T0 plants. On average, approximately 150 RNA–level SNVs were discovered per plant for either base-edited or GFP controls. Furthermore, we did not find enrichment of a TA motif on mutated adenine in the genomes and transcriptomes in base-edited tomato plants, as opposed to the recent discovery in rice (Oryza sativa). Hence, we could not find evidence for genome- and transcriptome-wide off-target effects by ABE8e in tomato. 

Abstract BackgroundCas12a (formerly known as Cpf1), the class II type V CRISPR nuclease, has been widely used for genome editing in mammalian cells and plants due to its distinct characteristics from Cas9. Despite being one of the most robust Cas12a nucleases, LbCas12a in general is less efficient than SpCas9 for genome editing in human cells, animals, and plants. ResultsTo improve the editing efficiency of LbCas12a, we conduct saturation mutagenesis inE. coliand identify 1977 positive point mutations of LbCas12a. We selectively assess the editing efficiency of 56 LbCas12a variants in human cells, identifying an optimal LbCas12a variant (RVQ: G146R/R182V/E795Q) with the most robust editing activity. We further test LbCas12a-RV, LbCas12a-RRV, and LbCas12a-RVQ in plants and find LbCas12a-RV has robust editing activity in rice and tomato protoplasts. Interestingly, LbCas12a-RRV, resulting from the stacking of RV and D156R, displays improved editing efficiency in stably transformed rice and poplar plants, leading to up to 100% editing efficiency inT₀plants of both plant species. Moreover, this high-efficiency editing occurs even at the non-canonical TTV PAM sites. ConclusionsOur results demonstrate that LbCas12a-RVQ is a powerful tool for genome editing in human cells while LbCas12a-RRV confers robust genome editing in plants. Our study reveals the tremendous potential of these LbCas12a variants for advancing precision genome editing applications across a wide range of organisms.