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Abstract Cytosine base editors (CBEs) are promising tools for precise genome editing in plants. It is important to investigate potential off-target effects of an efficient CBE at the genome and transcriptome levels in a major crop. Based on comparison of five cytidine deaminases and two different promoters for expressing sgRNAs, we tested a highly efficient A3A/Y130F-BE3 system for efficient C-to-T base editing in tomato (Solanum lycopersicum). We then conducted whole-genome sequencing (WGS) of four base-edited tomato plants, three GFP-expressing control plants, and two wild-type (WT) plants. The sequencing depths ranged from 25X to 49X with read mapping rates above 97%. No sgRNA-dependent off-target mutations were detected. Our data show an average of ∼1000 single nucleotide variations (SNVs) and ∼100 insertions and deletions (indels) per GFP control plant. Base-edited plants had on average elevated levels of SNVs (∼1250) and indels (∼300) per plant. On average, about 200 more C-to-T (G-to-A) mutations were found in a base-edited plant than a GFP control plant, suggesting some level of sgRNA-independent off-target effects, though the difference is not statistically significant. We also conducted RNA sequencing (RNA-seq) of the same four base-edited plants and three GFP control plants. An average of ∼200 RNA SNVs was discovered per plant for either base-edited or GFP control plants. Furthermore, no specific enrichment of C-to-U mutations can be found in the base-edited plants. Hence, we cannot find any evidence for bona fide off-target mutations by A3A/Y130F-BE3 at the transcriptome level. 

Abstract CRISPR-Cas12a is a promising genome editing system for targeting AT-rich genomic regions. Comprehensive genome engineering requires simultaneous targeting of multiple genes at defined locations. Here, to expand the targeting scope of Cas12a, we screen nine Cas12a orthologs that have not been demonstrated in plants, and identify six, ErCas12a, Lb5Cas12a, BsCas12a, Mb2Cas12a, TsCas12a and MbCas12a, that possess high editing activity in rice. Among them, Mb2Cas12a stands out with high editing efficiency and tolerance to low temperature. An engineered Mb2Cas12a-RVRR variant enables editing with more relaxed PAM requirements in rice, yielding two times higher genome coverage than the wild type SpCas9. To enable large-scale genome engineering, we compare 12 multiplexed Cas12a systems and identify a potent system that exhibits nearly 100% biallelic editing efficiency with the ability to target as many as 16 sites in rice. This is the highest level of multiplex edits in plants to date using Cas12a. Two compact single transcript unit CRISPR-Cas12a interference systems are also developed for multi-gene repression in rice and Arabidopsis . This study greatly expands the targeting scope of Cas12a for crop genome engineering. 

Cytosine base editors (CBEs) are great additions to the expanding genome editing toolbox. To improve C‐to‐T base editing in plants, we first compared seven cytidine deaminases in the BE3‐like configuration in rice. We found A3A/Y130F‐CBE_V01 resulted in the highest C‐to‐T base editing efficiency in both rice andArabidopsis. Furthermore, we demonstrated this A3A/Y130F cytidine deaminase could be used to improve iSpyMacCas9‐mediated C‐to‐T base editing at A‐rich PAMs. To showcase its applications, we first applied A3A/Y130F‐CBE_V01 for multiplexed editing to generate microRNA‐resistant mRNA transcripts as well as pre‐mature stop codons in multiple seed trait genes. In addition, we harnessed A3A/Y130F‐CBE_V01 for efficient artificial evolution of novelALSandEPSPSalleles which conferred herbicide resistance in rice. To further improve C‐to‐T base editing, multiple CBE_V02, CBE_V03 and CBE_V04 systems were developed and tested in rice protoplasts. The CBE_V04 systems were found to have improved editing activity and purity with focal recruitment of more uracil DNA glycosylase inhibitors (UGIs) by the engineered single guide RNA 2.0 scaffold. Finally, we used whole‐genome sequencing (WGS) to compare six CBE_V01 systems and four CBE_V04 systems for genome‐wide off‐target effects in rice. Different levels of cytidine deaminase‐dependent and sgRNA‐independent off‐target effects were indeed revealed by WGS among edited lines by these CBE systems. We also investigated genome‐wide sgRNA‐dependent off‐target effects by different CBEs in rice. This comprehensive study compared 21 different CBE systems, and benchmarked PmCDA1‐CBE_V04 and A3A/Y130F‐CBE_V04 as next‐generation plant CBEs with high editing efficiency, purity, and specificity.