Base Editors for Citrus Gene Editing

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.

Genome Editing of Veterinary Relevant Mycoplasmas Using a CRISPR-Cas Base Editor System

“…In the era of synthetic biology, with the fast expansion of the toolbox for modifying the genomes of living organisms, base-editing tools have attracted interest because of specific features: independence of endogenous cellular DNA repair pathways, lower cytotoxic effects than with other Cas9-based tools, high efficacy, which means that there is no need for selection markers, possibility of multitargeting, and the lack of a scar at the edited locus. These properties make BE promising tools for clinical usage in human therapeutics ( 60 ) and for multiple applications in plants ( 51 , 61 ) and microorganisms of medical or biotechnological relevance ( 62 – 64 ). Because of their high efficacy, BE are particularly attractive for bacteria that are difficult to transform, as, ultimately, one single transformant can be enough to obtain a mutant.…”

Efficient transgene-free genome editing in plants in the T0 generation based on a co-editing strategy

“…We first investigated if we could obtain transgene-free, gene-edited tomato in the T0 generation by base-editing SlALS1 (Solyc03g044330) alone. Previous studies suggested such a possibility, but the putative transgene-free plants were not confirmed by whole genome sequencing 10,15,28 . Here, we constructed the CBE-Cas12a-GFP-SlALS1 construct to edit the SlALS1 gene using CBE to target the proline residue at position 186 (Pro186) (Fig.…”

“…it is integrated into the plant genome because of their asexual reproduction nature through apomixis 9,10 .…”

“…Herbicides, such as chlorsulfuron, kills plants by acting as the inhibitors of acetolactate synthase. Mutation in the ALS genes using CBE confers resistance to herbicides such as chlorsulfuron in diverse plant species 10,15,[18][19][20][21][22][23][24][25] , thus providing a useful selection marker. The gene(s) of interest can be edited via Cas12a/crRNA, whereas GFP enables screening of putative transgene-free (GFP-negative) transformants.…”

Efficient generation of transgene-free canker-resistantCitrus sinensisusing Cas12a/crRNA ribonucleoprotein

“…Transgenic expression of antimicrobial peptides [13][14][15] , toxin 16 , resistance genes 17,18 , and immune-related genes [19][20][21][22][23][24][25] enabled increased canker resistance. Recently, CRISPR/Cas-mediated genome editing of the promoter or coding region of LOB1 has conferred citrus resistance to Xcc [26][27][28][29][30][31][32] . However, the citrus plants generated by transgenic overexpression and CRISPR genome editing approaches were all transgenic.…”