Cytosine base editors optimized for genome editing in potato protoplasts

Ida Westberg, Frida Meijer Carlsen, Ida Elisabeth Johansen, Bent Larsen Petersen*

*Corresponding author af dette arbejde

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Abstract

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.

OriginalsprogEngelsk
Artikelnummer1247702
TidsskriftFrontiers in Genome Editing
Vol/bind5
Antal sider8
DOI
StatusUdgivet - 2023

Bibliografisk note

Funding Information:
Independent Research Fund Denmark (1032-00399B) (2021) “Green CRISPR production platform for new starch qualities”; Novo Nordisk foundation (NNF19OC0057208); Grønt Udviklings og Demonstrations program (GUDP) (2020) “KRISPS Kartofler med Resistens og Innovativ Stivelse som Platform for Synergi egenskaber i kartoffel vha. DNA-fri CRISPR“; Kartoffelafgiftfonden (2022) “Reduceret miljø og klima aftryk em grøn og økonomisk bæredygtighed” (34009-19-1531); Kartoffelafgiftfonden (2021) “Samtidig introduktion af flere egenskaber gennem præcis forædling af skimmel resistens i stivelses kartofler” are acknowledged for funding support.

Publisher Copyright:
Copyright © 2023 Westberg, Carlsen, Johansen and Petersen.

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