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. 2023 Sep 29;14:1255819. doi: 10.3389/fpls.2023.1255819

Figure 4.

Figure 4

CRISPR/Cas9-mediated editing of the exogenous GmCPR5 gene in soybean protoplasts using electro-transfection. (A) GmCPR5 locus, location of target sites (T1, T3, and T5), and their gRNA sequences. (B–D) Results of T7E1 endonuclease assay for target sites T1, T3 and T5. Lane M: a DNA ladder. Lane WT: non- transfected wild type (control). Lane Cas9: transfected with SpCas9 only. Lanes T1–T5: electro-transfected with RNPs at 700 V, 1,000 V, and 1,300 V. Red arrows indicate the T7E1-mediated cleaved bands. The mutation patterns observed by targeted deep sequencing for the corresponding target sites of T1–T5 at GmCPR5 loci by electro-transfection at 1,300 V are shown on the right panel. Wild-type (WT) nuclease target sequences are in bold and underlined. PAM sites are denoted in red. RNPs, ribonucleoproteins; gRNA, guide RNA.