PAM-relaxed Cas9 nucleases, cytosine base editors and adenine base editors are promising tools for precise genome editing in plants. However, their genome-wide off-target effects are largely unexplored. Here, we conduct whole-genome sequencing (WGS) analyses of transgenic plants edited by xCas9, Cas9-NGv1, Cas9-NG, SpRY, nCas9-NG-PmCDA1, nSpRY-PmCDA1 and nSpRY-ABE8e in rice. Our results reveal that Cas9 nuclease and base editors, when coupled with the same guide RNA (gRNA), prefer distinct gRNA-dependent off-target sites. De novo generated gRNAs by SpRY editors lead to additional, but insubstantial, off-target mutations. Strikingly, ABE8e results in ~500 genome-wide A-to-G off-target mutations at TA motif sites per transgenic plant. ABE8e's preference for the TA motif is also observed at the target sites. Finally, we investigate the timeline and mechanism of somaclonal variation due to tissue culture, which chiefly contributes to the background mutations. This study provides a comprehensive understanding on the scale and mechanisms of off-target and background mutations occurring during PAM-relaxed genome editing in plants.

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Summary Cytosine base editors (CBEs) can install a predefined stop codon at the target site, representing a more predictable and neater method for creating genetic knockouts without altering the genome size. Due to the enhanced predictability of the editing outcomes, it is also more efficient to obtain homozygous mutants in the first generation. With the recent advancement of CBEs on improved editing activity, purify and specificity in plants and animals, base editing has become a more appealing technology for generating knockouts. However, there is a lack of design tools that can aid the adoption of CBEs for achieving such a purpose, especially in plants. Here, we developed a user‐friendly design tool named CRISPR‐BETS (base editing to stop), which helps with guide RNA (gRNA) design for introducing stop codons in the protein‐coding genes of interest. We demonstrated in rice and tomato that CRISPR‐BETS is easy‐to‐use, and its generated gRNAs are highly specific and efficient for generating stop codons and obtaining homozygous knockout lines. While we tailored the tool for the plant research community, CRISPR‐BETS can also serve non‐plant species.

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DNA methylation is a conserved epigenetic modification which is vital for regulating gene expression and maintaining genome stability in both mammals and plants. Homozygous mutation of rice methyltransferase 1 (met1) gene can cause host death in rice, making it difficult to obtain plant material needed for hypomethylation research. To circumvent this challenge, the methylation inhibitor, 5-Aza-2′-deoxycytidine (AzaD), is used as a cytosine nucleoside analogue to reduce genome wide hypomethylation and is widely used in hypomethylation research. However, how AzaD affects plant methylation profiles at the genome scale is largely unknown. Here, we treated rice seedlings with AzaD and compared the AzaD treatment with osmet1-2 mutants, illustrating that there are similar CG hypomethylation and distribution throughout the whole genome. Along with global methylation loss class I transposable elements (TEs) which are farther from genes compared with class II TEs, were more significantly activated, and the RNA-directed DNA Methylation (RdDM) pathway was activated in specific genomic regions to compensate for severe CG loss. Overall, our results suggest that AzaD is an effective DNA methylation inhibitor that can influence genome wide methylation and cause a series of epigenetic variations.

DNA methylation is a conserved epigenetic modification which is vital for regulating gene expression and maintaining genome stability in both mammals and plants. Homozygous mutation of rice methyltransferase 1 (met1) gene can cause host death in rice, making it difficult to obtain plant material needed for hypomethylation research. To circumvent this challenge, the methylation inhibitor, 5-Aza-2′-deoxycytidine (AzaD), is used as a cytosine nucleoside analogue to reduce genome wide hypomethylation and is widely used in hypomethylation research. However, how AzaD affects plant methylation profiles at the genome scale is largely unknown. Here, we treated rice seedlings with AzaD and compared the AzaD treatment with osmet1-2 mutants, illustrating that there are similar CG hypomethylation and distribution throughout the whole genome. Along with global methylation loss class I transposable elements (TEs) which are farther from genes compared with class II TEs, were more significantly activated, and the RNA-directed DNA Methylation (RdDM) pathway was activated in specific genomic regions to compensate for severe CG loss. Overall, our results suggest that AzaD is an effective DNA methylation inhibitor that can influence genome wide methylation and cause a series of epigenetic variations.