skip to main content


Title: Improved plant cytosine base editors with high editing activity, purity, and specificity
Summary

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.

 
more » « less
Award ID(s):
1758745 2029889
PAR ID:
10388215
Author(s) / Creator(s):
 ;  ;  ;  ;  ;  ;  ;  ;  ;  ;  ;  ;  ;  ;  ;  ;  ;  ;  ;  more » ;   « less
Publisher / Repository:
Wiley-Blackwell
Date Published:
Journal Name:
Plant Biotechnology Journal
Volume:
19
Issue:
10
ISSN:
1467-7644
Page Range / eLocation ID:
p. 2052-2068
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. 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. 
    more » « less
  2. Cytosine base editors (CBEs) enable efficient cytidine-to-thymidine (C-to-T) substitutions at targeted loci without double-stranded breaks. However, current CBEs edit all Cs within their activity windows, generating undesired bystander mutations. In the most challenging circumstance, when a bystander C is adjacent to the targeted C , existing base editors fail to discriminate them and edit both Cs. To improve the precision of CBE, we identified and engineered the human APOBEC3G (A3G) deaminase; when fused to the Cas9 nickase, the resulting A3G-BEs exhibit selective editing of the second C in the 5′-C C -3′ motif in human cells. Our A3G-BEs could install a single disease-associated C-to-T substitution with high precision. The percentage of perfectly modified alleles is more than 6000-fold for disease correction and more than 600-fold for disease modeling compared with BE4max. On the basis of the two-cell embryo injection method and RNA sequencing analysis, our A3G-BEs showed minimum genome- and transcriptome-wide off-target effects, achieving high targeting fidelity. 
    more » « less
  3. 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.

     
    more » « less
  4. Abstract

    Phytopathogenic bacteria play important roles in plant productivity, and developments in gene editing have potential for enhancing the genetic tools for the identification of critical genes in the pathogenesis process. CRISPR-based genome editing variants have been developed for a wide range of applications in eukaryotes and prokaryotes. However, the unique mechanisms of different hosts restrict the wide adaptation for specific applications. Here, CRISPR-dCas9 (dead Cas9) and nCas9 (Cas9 nickase) deaminase vectors were developed for a broad range of phytopathogenic bacteria. A gene for a dCas9 or nCas9, cytosine deaminase CDA1, and glycosylase inhibitor fusion protein (cytosine base editor, or CBE) was applied to base editing under the control of different promoters. Results showed that the RecA promoter led to nearly 100% modification of the target region. When residing on the broad host range plasmid pHM1, CBERecApis efficient in creating base edits in strains ofXanthomonas,Pseudomonas,ErwiniaandAgrobacterium. CBE based on nCas9 extended the editing window and produced a significantly higher editing rate inPseudomonas. Strains with nonsynonymous mutations in test genes displayed expected phenotypes. By multiplexing guide RNA genes, the vectors can modify up to four genes in a single round of editing. Whole-genome sequencing of base-edited isolates ofXanthomonas oryzaepv.oryzaerevealed guide RNA-independent off-target mutations. Further modifications of the CBE, using a CDA1 variant (CBERecAp-A) reduced off-target effects, providing an improved editing tool for a broad group of phytopathogenic bacteria.

     
    more » « less
  5. Summary

    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.

     
    more » « less