Cytosine base editors optimized for genome editing in potato protoplasts
In this study, we generated and compared three cytidine base editors (CBEs) tailor-made for potato (Solanum tuberosum), which conferred up to 43% C-to-T conversion of all alleles in the protoplast pool. Earlier, gene-edited potato plants were successfully generated by polyethylene glycol-mediated CR...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-08-01
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| Series: | Frontiers in Genome Editing |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fgeed.2023.1247702/full |
| _version_ | 1827855499058479104 |
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| author | Ida Westberg Frida Meijer Carlsen Ida Elisabeth Johansen Bent Larsen Petersen |
| author_facet | Ida Westberg Frida Meijer Carlsen Ida Elisabeth Johansen Bent Larsen Petersen |
| author_sort | Ida Westberg |
| collection | DOAJ |
| description | In this study, we generated and compared three cytidine base editors (CBEs) tailor-made for potato (Solanum tuberosum), which conferred up to 43% C-to-T conversion of all alleles in the protoplast pool. Earlier, gene-edited potato plants were successfully generated by polyethylene glycol-mediated CRISPR/Cas9 transformation of protoplasts followed by explant regeneration. In one study, a 3–4-fold increase in editing efficiency was obtained by replacing the standard Arabidopsis thaliana AtU6-1 promotor with endogenous potato StU6 promotors driving the expression of the gRNA. Here, we used this optimized construct (SpCas9/StU6-1::gRNA1, target gRNA sequence GGTC4C5TTGGAGC12AAAAC17TGG) for the generation of CBEs tailor-made for potato and tested for C-to-T base editing in the granule-bound starch synthase 1 gene in the cultivar Desiree. First, the Streptococcus pyogenes Cas9 was converted into a (D10A) nickase (nCas9). Next, one of three cytosine deaminases from human hAPOBEC3A (A3A), rat (evo_rAPOBEC1) (rA1), or sea lamprey (evo_PmCDA1) (CDA1) was C-terminally fused to nCas9 and a uracil-DNA glycosylase inhibitor, with each module interspaced with flexible linkers. The CBEs were overall highly efficient, with A3A having the best overall base editing activity, with an average 34.5%, 34.5%, and 27% C-to-T conversion at C4, C5, and C12, respectively, whereas CDA1 showed an average base editing activity of 34.5%, 34%, and 14.25% C-to-T conversion at C4, C5, and C12, respectively. rA1 exhibited an average base editing activity of 18.75% and 19% at C4 and C5 and was the only base editor to show no C-to-T conversion at C12. |
| first_indexed | 2024-03-12T11:51:57Z |
| format | Article |
| id | doaj.art-ce5e5bb089304dba835732085ceba216 |
| institution | Directory Open Access Journal |
| issn | 2673-3439 |
| language | English |
| last_indexed | 2024-03-12T11:51:57Z |
| publishDate | 2023-08-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Genome Editing |
| spelling | doaj.art-ce5e5bb089304dba835732085ceba2162023-08-31T06:46:50ZengFrontiers Media S.A.Frontiers in Genome Editing2673-34392023-08-01510.3389/fgeed.2023.12477021247702Cytosine base editors optimized for genome editing in potato protoplastsIda WestbergFrida Meijer CarlsenIda Elisabeth JohansenBent Larsen PetersenIn this study, we generated and compared three cytidine base editors (CBEs) tailor-made for potato (Solanum tuberosum), which conferred up to 43% C-to-T conversion of all alleles in the protoplast pool. Earlier, gene-edited potato plants were successfully generated by polyethylene glycol-mediated CRISPR/Cas9 transformation of protoplasts followed by explant regeneration. In one study, a 3–4-fold increase in editing efficiency was obtained by replacing the standard Arabidopsis thaliana AtU6-1 promotor with endogenous potato StU6 promotors driving the expression of the gRNA. Here, we used this optimized construct (SpCas9/StU6-1::gRNA1, target gRNA sequence GGTC4C5TTGGAGC12AAAAC17TGG) for the generation of CBEs tailor-made for potato and tested for C-to-T base editing in the granule-bound starch synthase 1 gene in the cultivar Desiree. First, the Streptococcus pyogenes Cas9 was converted into a (D10A) nickase (nCas9). Next, one of three cytosine deaminases from human hAPOBEC3A (A3A), rat (evo_rAPOBEC1) (rA1), or sea lamprey (evo_PmCDA1) (CDA1) was C-terminally fused to nCas9 and a uracil-DNA glycosylase inhibitor, with each module interspaced with flexible linkers. The CBEs were overall highly efficient, with A3A having the best overall base editing activity, with an average 34.5%, 34.5%, and 27% C-to-T conversion at C4, C5, and C12, respectively, whereas CDA1 showed an average base editing activity of 34.5%, 34%, and 14.25% C-to-T conversion at C4, C5, and C12, respectively. rA1 exhibited an average base editing activity of 18.75% and 19% at C4 and C5 and was the only base editor to show no C-to-T conversion at C12.https://www.frontiersin.org/articles/10.3389/fgeed.2023.1247702/fullcytosine base editorU6 promotornative promotorprotoplastpotatogenome editing |
| spellingShingle | Ida Westberg Frida Meijer Carlsen Ida Elisabeth Johansen Bent Larsen Petersen Cytosine base editors optimized for genome editing in potato protoplasts Frontiers in Genome Editing cytosine base editor U6 promotor native promotor protoplast potato genome editing |
| title | Cytosine base editors optimized for genome editing in potato protoplasts |
| title_full | Cytosine base editors optimized for genome editing in potato protoplasts |
| title_fullStr | Cytosine base editors optimized for genome editing in potato protoplasts |
| title_full_unstemmed | Cytosine base editors optimized for genome editing in potato protoplasts |
| title_short | Cytosine base editors optimized for genome editing in potato protoplasts |
| title_sort | cytosine base editors optimized for genome editing in potato protoplasts |
| topic | cytosine base editor U6 promotor native promotor protoplast potato genome editing |
| url | https://www.frontiersin.org/articles/10.3389/fgeed.2023.1247702/full |
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