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1. Genome- and transcriptome-wide off-target analyses of an improved cytosine base editor

   https://doi.org/10.1093/plphys/kiab264  

   Randall, Linnell Bentley ; Sretenovic, Simon ; Wu, Yuechao ; Yin, Desuo ; Zhang, Tao ; Van Eck, Joyce ; Qi, Yiping ( June 2021 , Plant Physiology)

   null (Ed.)

   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. 

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2. Expanding plant genome editing scope and profiles with CRISPR‐FrCas9 systems targeting palindromic TA sites

   https://doi.org/10.1111/pbi.14363  

   He, Yao ; Han, Yangshuo ; Ma, Yanqin ; Liu, Shishi ; Fan, Tingting ; Liang, Yanling ; Tang, Xu ; Zheng, Xuelian ; Wu, Yuechao ; Zhang, Tao ; et al ( May 2024 , Plant Biotechnology Journal)

   CRISPR‐Cas9 is widely used for genome editing, but its PAM sequence requirements limit its efficiency. In this study, we exploreFaecalibaculum rodentiumCas9 (FrCas9) for plant genome editing, especially in rice. FrCas9 recognizes a concise 5′‐NNTA‐3′ PAM, targeting more abundant palindromic TA sites in plant genomes than the 5′‐NGG‐3′ PAM sites of the most popular SpCas9. FrCas9 shows cleavage activities at all tested 5′‐NNTA‐3′ PAM sites with editing outcomes sharing the same characteristics of a typical CRISPR‐Cas9 system. FrCas9 induces high‐efficiency targeted mutagenesis in stable rice lines, readily generating biallelic mutants with expected phenotypes. We augment FrCas9's ability to generate larger deletions through fusion with the exonuclease, TREX2. TREX2‐FrCas9 generates much larger deletions than FrCas9 without compromise in editing efficiency. We demonstrate TREX2‐FrCas9 as an efficient tool for genetic knockout of a microRNA gene. Furthermore, FrCas9‐derived cytosine base editors (CBEs) and adenine base editors (ABE) are developed to produce targeted C‐to‐T and A‐to‐G base edits in rice plants. Whole‐genome sequencing‐based off‐target analysis suggests that FrCas9 is a highly specific nuclease. Expression of TREX2‐FrCas9 in plants, however, causes detectable guide RNA‐independent off‐target mutations, mostly as single nucleotide variants (SNVs). Together, we have established an efficient CRISPR‐FrCas9 system for targeted mutagenesis, large deletions, C‐to‐T base editing, and A‐to‐G base editing in plants. The simple palindromic TA motif in the PAM makes the CRISPR‐FrCas9 system a promising tool for genome editing in plants with an expanded targeting scope.

    

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

   https://doi.org/10.1038/s41467-024-49473-w  

   Fan, Tingting ; Cheng, Yanhao ; Wu, Yuechao ; Liu, Shishi ; Tang, Xu ; He, Yao ; Liao, Shanyue ; Zheng, Xuelian ; Zhang, Tao ; Qi, Yiping ; et al ( June 2024 , Nature Communications)

   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.

    

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4. Improved plant cytosine base editors with high editing activity, purity, and specificity

   https://doi.org/10.1111/pbi.13635  

   Ren, Qiurong ; Sretenovic, Simon ; Liu, Guanqing ; Zhong, Zhaohui ; Wang, Jiaheng ; Huang, Lan ; Tang, Xu ; Guo, Yachong ; Liu, Li ; Wu, Yuechao ; et al ( June 2021 , Plant Biotechnology Journal)

   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.

    

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5. Multiplex knockout of trichome-regulating MYB duplicates in hybrid poplar using a single gRNA

   https://doi.org/10.1093/plphys/kiac128  

   Bewg, William P. ; Harding, Scott A. ; Engle, Nancy L. ; Vaidya, Brajesh N. ; Zhou, Ran ; Reeves, Jacob ; Horn, Thomas W. ; Joshee, Nirmal ; Jenkins, Jerry W. ; Shu, Shengqiang ; et al ( March 2022 , Plant Physiology)

   As the focus for CRISPR/Cas-edited plants moves from proof-of-concept to real-world applications, precise gene manipulation will increasingly require concurrent multiplex editing for polygenic traits. A common approach for editing across multiple sites is to design one guide RNA (gRNA) per target; however, this complicates construct assembly and increases the possibility of off-target mutations. In this study, we utilized one gRNA to target MYB186, a known positive trichome regulator, as well as its paralogs MYB138 and MYB38 at a consensus site for mutagenesis in hybrid poplar (Populus tremula × P. alba INRA 717-1B4). Unexpected duplications of MYB186 and MYB138 resulted in eight alleles for the three targeted genes in the hybrid poplar. Deep sequencing and polymerase chain reaction analyses confirmed editing across all eight targets in nearly all of the resultant glabrous mutants, ranging from small indels to large genomic dropouts, with no off-target activity detected at four potential sites. This highlights the effectiveness of a single gRNA targeting conserved exonic regions for multiplex editing. Additionally, cuticular wax and whole-leaf analyses showed a complete absence of triterpenes in the trichomeless mutants, hinting at a previously undescribed role for the nonglandular trichomes of poplar.

    

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