Journal: Frontiers in Genome Editing
Article Title: Cytosine base editors optimized for genome editing in potato protoplasts
doi: 10.3389/fgeed.2023.1247702
Figure Lengend Snippet: C-to-T conversion derived from cytosine base editors A3A, rA1, and CDA1 optimized for potato. (A) Three cytosine base editor constructs comprising a cassette driving expression of the gRNA (target conferring part, GGTC 4 C 5 TTGGAGC 12 AAAAC 17 TGG) from the St U6-1 promotor , the Sp Cas9 nickase (nCas9) in fusion with one of the deaminases hAPOBEC3A (A3A) from human, evo_rAPOBEC1 (rA1) from rat, or sea lamprey evo_ Pm CDA1 (CDA1), followed by a uracil-DNA glycosylase inhibitor (UGI), interspaced by long flexible linkers are depicted. NLS, nuclear localization signal; serine (S) and glycine (G) linkers: SG and SGGS. (B) Chromatograms from direct sequencing of PCR products from the protoplast cell pool transformed with the base editing constructs with A3A showing 39% (C4), 43% (C5), and 36% (C12) editing (sample A3A replicate 4), rA1 showing 29% (C4) and 28% (C5) (sample rA1 replicate 3), and CDA1 showing 38% (C4), 38% (C5), and 21% (C12) (sample CDA1 replicate 2) C-to-T conversion in the target region (exon 1 of GBSS1 (cultivar Desiree)) when compared to the WT sequence. Target Cs in the gRNA and adjacent PAM site are shown in green and red, respectively. (C) C-to-T conversion (%) of Cs within (C4, C5, C12, and C17) and most closely adjacent (−C19, −C8 and C25) to the gRNA of protoplasts transformed with A3A, rA1, and CDA1 as evidenced by EditR analysis. Numbers 1–4 indicate replicates. (D) Average percentage of reads with C-to-T conversion (%) rates of protoplasts transformed with A3A, rA1, and CDA1 as shown for Cs within (C4, C5, C12, and C17) and most closely adjacent (−C19, −C8 and C25) to the gRNA. Data are shown as mean ± sd of four biological replicates.
Article Snippet: The basic construct, Sp Cas9/ St U6-1::sgRNA1, comprising the 35SPPDK:: Sp Cas9 cassette, driving the expression of the codon-optimized Streptococcus pyogenes Cas9 nuclease ( Sp Cas9) originally from the plasmid pHBT-pcoCas9 ( ) (Addgene plasmid #52254) and the St U6-1 promoter::sgRNA-1 cassette ( St U6-1 promoter (NCBI accession no. Z17290)) described in the work of ) was used as the basis for generation of the base editing constructs.
Techniques: Derivative Assay, Construct, Expressing, Sequencing, Transformation Assay
Addgene inc is a verified supplier 
