AUTHOR=Westberg Ida , Carlsen Frida Meijer , Johansen Ida Elisabeth , Petersen Bent Larsen 

TITLE=Cytosine base editors optimized for genome editing in potato protoplasts

JOURNAL=Frontiers in Genome Editing

VOLUME=5

YEAR=2023

URL=https://www.frontiersin.org/journals/genome-editing/articles/10.3389/fgeed.2023.1247702

DOI=10.3389/fgeed.2023.1247702

ISSN=2673-3439

ABSTRACT=<p>In this study, we generated and compared three cytidine base editors (CBEs) tailor-made for potato (<italic>Solanum tuberosum</italic>), 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 <italic>Arabidopsis thaliana At</italic>U6-1 promotor with endogenous potato <italic>St</italic>U6 promotors driving the expression of the gRNA. Here, we used this optimized construct (<italic>Sp</italic>Cas9/<italic>St</italic>U6-1::gRNA1, target gRNA sequence GGTC<sub>4</sub>C<sub>5</sub>TTGGAGC<sub>12</sub>AAAAC<sub>17</sub>TGG) 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 <italic>Streptococcus pyogenes</italic> 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_<italic>Pm</italic>CDA1) (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.</p>