Base Editors for Citrus Gene Editing.

Xiaoen Huang,Nian Wang,Yuanchun Wang

doi:10.3389/fgeed.2022.852867

Xiaoen Huang, Nian Wang + Show 1 more

Open Access

https://doi.org/10.3389/fgeed.2022.852867

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Abstract

Base editors, such as adenine base editors (ABE) and cytosine base editors (CBE), provide alternatives for precise genome editing without generating double-strand breaks (DSBs), thus avoiding the risk of genome instability and unpredictable outcomes caused by DNA repair. Precise gene editing mediated by base editors in citrus has not been reported. Here, we have successfully adapted the ABE to edit the TATA box in the promoter region of the canker susceptibility gene LOB1 from TATA to CACA in grapefruit (Citrus paradise) and sweet orange (Citrus sinensis). TATA-edited plants are resistant to the canker pathogen Xanthomonas citri subsp. citri (Xcc). In addition, CBE was successfully used to edit the acetolactate synthase (ALS) gene in citrus. ALS-edited plants were resistant to the herbicide chlorsulfuron. Two ALS-edited plants did not show green fluorescence although the starting construct for transformation contains a GFP expression cassette. The Cas9 gene was undetectable in the herbicide-resistant citrus plants. This indicates that the ALS edited plants are transgene-free, representing the first transgene-free gene-edited citrus using the CRISPR technology. In summary, we have successfully adapted the base editors for precise citrus gene editing. The CBE base editor has been used to generate transgene-free citrus via transient expression.

Highlights

Unlike classical clustered regularly interspaced short palindromic repeats (CRISPR) systems that use Cas proteins, such as Cas9 and Cas12, nickase Cas9 derived base editors do not create double-strand breaks (DSBs)
The effector binding elements (EBE) region of the LATERAL ORGAN BOUNDARIES 1 (LOB1) promoter in citrus is responsible for binding by the transcriptional activator-like (TAL) effector PthA4 of Xanthomonas citri subsp. citri (Xcc) (Hu et al, 2014) to activate its expression
The TATA box of the LOB1 promoter overlaps with the EBE region (Figures 1B,D)

Summary

Introduction

Unlike classical CRISPR systems that use Cas proteins, such as Cas and Cas, nickase Cas (nCas9) derived base editors do not create double-strand breaks (DSBs). It is well known that base editors can introduce specific amino acid changes in a protein, can be used for site-specific mutagenesis. They can be deployed to disrupt gene functions by altering splicing sites (splice donor, splice acceptor, and branch point). CBEs can introduce premature stop codons to knock out genes Both ABEs and CBEs can modify cis-regulatory elements to fine-tune gene functions. They can be utilized to mutate start codon ATG to interrupt protein translation (Kluesner et al, 2021; Molla et al, 2021)

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